Similarly, with chronic exposure to IL-1β, BK-induced Ca2+ mobilization was accentuated in cells from type 1 diabetics compared to the controls. Pretreatment with the protein synthesis inhibitor cycloheximide or the protein kinase C inhibitor calphostin C prior to the addition of TNF completely abrogated the TNF-induced increment in peak bradykinin response. Ca2+ transients induced by depleting endoplasmic reticulum (ER) Ca2+ with thapsigargin were also greater in TNF treated fibroblasts than in untreated cells, with greater increases in cells from diabetics. Exposing fibroblasts for 48 hours to 2 mM oleate also increased both the peak bradykinin response and the TNF-induced increment in peak response, which were significantly greater in diabetics than controls. These data indicate that cells from diabetic patients acquire elevated ER Ca2+ stores in response to both cytokines and free fatty acids,and thus exhibit greater sensitivity to environmental inflammatory stimuli and elevated lipids.The effects of cytokine and fatty acid treatment on signal transduction in dermal fibroblasts from type 1 diabetics and matched controls were compared. Chronic exposure to TNF, accentuated Ca The inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β) are implicated in both type 1 and type 2 diabetes 2 (PLA2), mitogen activated protein kinase, and sphingomyelinase/ceramide Despite abundant research, the etiology of type 1 diabetes remains unknown, however, there is support for a viral trigger for the disease 2+ homeostasis 2+-ATPase activity, expression of acetyl-CoA carboxylase, and peroxisome proliferation Although type 1 diabetic patients may also have elevated serum levels of free fatty acids (FFA) or triglyceride, much less is known about how this may contribute to diabetic pathology than is known about the hyperglycemia-related pathologies. Even in non-ketotic states, type 1 diabetics have dyslipidemia, or elevated levels of FFA in serum 2Inflammatory cytokines also affect lipid synthesis and metabolism. In rat liver, TNF increases hepatic fatty acid synthesis and lipid secretion 3) cascade and subsequent release of Ca2+ from internal stores 2 production in fibroblasts 2+.Bradykinin (BK) is a vasodilator that plays a role in the inflammatory process, mediating acute responses to injury such as vasodilation, edema, and pain. Binding of BK to the G-protein coupled B2 receptor subtype leads to the activation of the phospholipase-C/inositol 1,4,5-trisphosphate , our research was exempt from the Boston University Institutional Review Board (H25457). The Coriell Institute ensured compliance with DHHS regulations for the protection of human subjects (45CFR Part 46). Human tissue was handled solely by the authors of this paper in our country of residence.Dermal fibroblasts were obtained from the Coriell Institute for Medical Research, Camden, NJ. Fibroblasts were obtained from 7 apparently normal donors, 10 donors identified as type 1 diabetics, and 3 non-diabetic siblings of the diabetic donors. Diabetic donors were matched to controls based on age, gender and race.Cells were grown in Minimal Essential Medium (MEM) with Earle’s salts, 2X concentration of essential and non-essential amino acids for MEM, 1X MEM vitamin solution, 1X antibiotic/antimycotic, and 20% fetal bovine serum (FBS) from Hyclone Laboratories, Inc. . All other cell culture solutions were purchased from Gibco Life Technologies . Upon confluence, cells were removed from culture flasks by incubating for 1 minute in 0.7 mM EDTA in Dulbecco’s phosphate buffered saline , and then for 2–5 minutes in 0.25% trypsin. Cells were then washed with PBS/EDTA and used for experiments, or passaged in a ratio appropriate to the culture’s growth rate.2+ measurement, which was performed in the absence of cytokines. Cells were preincubated for 30 minutes with 20 µM indomethacin  and 100 nM Calphostin C  prior to the addition of TNF. Cycloheximide  was preincubated for 3 hours before the addition of cytokines. Glucose  and oleic acid  were preincubated for 24 hours before cytokine addition. Oleic acid (free acid) was prepared as a stock solution of 0.1 M in NaOH (pH 9), and complexed to 2% fatty acid free bovine serum albumin  at a final concentration of 2 mM.All experiments were performed in serum free media. Unless otherwise noted, the concentrations of TNF and IL-1β  were 10 ng/ml (0.6 nM) and 1 ng/ml (60 fM), respectively. The exposure to cytokine treatment was 24 hours (unless otherwise indicated) before cells were removed for CaAs all fibroblast cultures were grown and maintained in a medium containing 20% FBS, and all TNF treatments were done in serum free medium, a preliminary experiment was performed in order to confirm that our TNF solution was not simply replacing a serum component in otherwise serum-starved cells. The addition of FBS did not cause a significant change in peak bradykinin response above those not treated with FBS.2+ was determined from changes in the excitation signals of the fluorescent indicator fura-2 at 340 and 380 nm, measuring emission at 510 nm, using a Hitachi F-2000 fluorescence spectrophotometer, as described previously 3, 2 mM CaCl2, 1 mM MgCl2, 1 mM Na2HPO4, 10 mM glucose, 10 mM HEPES, 0.05% BSA, and 10 µM sulfinpyrazone (to block active extrusion of the fura), pH 7.4. The maximum Ca2+/fura-2 and minimum free fura-2 signals were determined after addition of Triton X-100 to equilibrate Ca2+ across the plasma membrane. The minimum Ca2+/fura-2 and maximum free fura-2 were determined after addition of EGTA (plus Tris buffer to maintain pH). The cytosolic free Ca2+ concentration was calculated by measuring the 2 fura signals as a percentage of the maxima where the Kd is 225 nM as described previously 2+ transients were measured in suspensions of confluent fibroblasts between passages 7 and 30; no consistent passage-dependent variation in the responsiveness of the cells was observed over this range.Cytosolic free Ca3, 20 mM HEPES, 1 mM MgCl2, 6 mM KH2PO4, 4 mM MgATP, 12 mM creatine plus creatine phosphate, 50 µg/ml creatine phosphokinase, and 1 µM fura-2 free acid 2+ uptake. Saponin (60 µg/ml) was added when indicated to permeabilize the cells. The Ca2+ concentration in the buffer was measured by the fluorescence of free fura-2 (1 µM).After detaching with trypsin/EDTA as described, cells were resuspended in a buffer containing 100 mM KCl, 22 mM NaCl, 5 mM KHCOTo determine statistical differences between groups, analysis of variance (ANOVA) was used with Tukey’s post-hoc test for comparison of independent groups when appropriate (unless otherwise noted). Error bars on the figures represent the standard error of the mean.2+ levels nor on BK-induced Ca2+ mobilization (data not shown). 2+ mobilization (compare heavy line (treated) with fine line (untreated)). This figure shows representative traces from control and diabetic donors. In both control and diabetic donors, TNF treatment augmented BK-induced Ca2+ mobilization, although to a much greater extent in the diabetic donor (right panel). Similar sensitivity of diabetic donors were seen with 24 hours of IL-1β treatment (60 fM). TNF treatment had neither an acute effect on human fibroblasts on CaTo determine the optimal time required for TNF to induce the peak BK response, time course series were performed in fibroblasts from 3 different donors, in which the cells were treated with 0.6 nM TNFfor 1, 2, 4, 12, 24, or 48 hr. The observation that time was required for TNF treatment to induce a change in peak BK response in fibroblasts  suggesteEvaluation of the concentration dependence of the stimulatory effect of TNF on the BK response  indicateThe concentration for maximal BK responses was tested between 0 and 10 µM and data are summarized in A major difference between control and diabetic cells can be seen clearly from the average TNF-induced increment in peak BK response that exceeded 100 nM in all subjects . However2+ response, the effects of TNF treatment on the increment in steady state, or final equilibrium, caused by BK were examined . This intermediate Ca2+ response to bradykinin from siblings of diabetics suggests that Ca2+ mobilization can be altered even in the presence of apparently healthy insulin response to glucose.The pathogenesis of type 1 diabetes indicates that there is a strong genetic component to the disease 2+ was determined in fibroblasts from 7 control and 10 diabetic donors (a mean of 96 to 105 separate determinations). In control donors, TNF did not significantly affect basal Ca2+ concentrations (71±2 nM pre- vs 74±2 nM post- 24 h TNF). In donors with diabetes, basal Ca2+ was lower inititially and was increased by TNF from 60±2 nM to 70±3 nM (p<0.001). Before treatment differences between fibroblasts from donors with and without diabetes were statistically signficiant (p<0.001).The effect of TNF treatment on basal Ca2+ from intracellular stores or extracellular Ca2+ uptake, the Ca2+ chelator EGTA was added to the extracellular media immediately prior to stimulation with BK. The peak response to bradykinin was unaltered in the presence of EGTA chelation of extracellular Ca2+ in both untreated cells and TNF-treated cells  compared to the controls (127±20 p = 0.001).Thapsigargin, an irreversible inhibitor of the ER Calic Ca2+ . In fibr+ stores . As withlization , the dif2+-ATPase regulates Ca2+ entry and, consequently, the size of the ER Ca2+ pools. To determine if the activity of the ER Ca2+-ATPase was affected by TNF treatment, fibroblasts were suspended in a buffer mimicking intracellular ion concentrations with no added Ca2+ and 1 µM fura-2 free acid (a fluorescent dye that fluoresces when it binds free Ca2+), and permeabilized with the detergent, saponin. The inhibitors oligomycin and antimycin A were added in order to inhibit the transport of Ca2+ into the mitochondria and limit the uptake of Ca2+ to the ER. As expected, the addition of saponin resulted in permeabilization of the cells and consequent rapid uptake of Ca2+ into the stores .Changes in calreticulin binding have been shown to modulate the responses of Ca2+ mobilization. To examine the effects of FFA on BK-induced Ca2+ mobilization and on the TNF-induced increment, fibroblasts from 4 diabetic and 3 control donors were treated with 2 mM oleate for 48 hours. The fibroblasts from control donors were specifically selected from our collection of fibroblasts because they had responded to TNF treatment with only modest increases in peak BK response in previous experiments. TNF and oleate both augmented Ca2+ mobilization; simultaneous treatment of TNF and oleate led to the greatest effect. The diabetic phenotype is associated with elevated blood glucose and lipid concentrations A summary of the results of experiments performed in 3 control and 4 diabetic donors is shown in 2+ following stimulation with BK (p<0.001), as shown in The fatty acid-containing media had a significant effect on the sustained plateau in cytosolic Ca2+ responses to BK and fatty acids. They also have a small but significantly lower basal Ca2+ that normalizes in response to cytokines. These features join several published studies documenting other differences between control and type 1 diabetic fibroblasts Human skin fibroblasts from type 1 diabetic subjects exhibit several distinguishing features that differentiate them from control fibroblasts. These include an altered sensitivity to cytokines that results in greatly enhanced Ca2+ response and steady state Ca2+ level following stimulation with BK is not known. These findings could have many separate causes, but also can be linked together in a simple speculative model (2+ uptake by the ER Ca2+-ATPase and increases Ca2+ stores 2+ stores. Fatty acids, by direct conversion to LC-CoA, also increase cytosolic long chain acyl CoA levels, increased Ca2+ stores, and enhanced BK response potentially by the same mechanism.The mechanism by which both TNF and a high fatty acid environment increase the peak Cave model  centered2+ stores via a direct effect on Ca2+-ATPase of the ER LC-CoA also has other modulatory roles in signal transduction including activation of PKC isoforms. PKC is known to play a role in TNF-induced signal transduction 2+ increment greater than 200 nM. This artificial separation into three groups, according to the response of the cells to TNF treatment, suggests a genetic or epigenetic component. Studies currently underway will rederive the type 1 diabetic fibroblasts to determine if the characteristics are retained or lost following removal of the epigenetic changes all exhibited greater effects of TNF than matched control donors. A trait present in 100% of such a small population may also occur with some frequency in the general population. If the cause of diabetes requires a combination of different factors, this can explain why two siblings who both carry a “diabetes gene” can be discordant for the disease. On the other hand, a person who does not carry the TNF-hypersensitivity trait may not become diabetic, whether or not exogenous stimuli such as a systemic viral infection occurs.Analysis of the TNF-induced increment in peak BK response in all the donors surveyed  showed t2+ signal transduction could lead to improved outcomes.The data presented here indicate that fibroblasts from patients with type 1 diabetic display an altered response to BK in the presence of TNF and fatty acid. Together with data documenting elevated cytokines and free fatty acids in people with type 1 diabetes


The shift towards significantly larger populations of antigen-specific B cells relative to the non-specific B cell pool is further corroborated by our previous findings in FcRn Tg mice. Consequently, our FcRn Tg rabbits have the potential to offer substantial qualitative and quantitative improvements for the production of rATG and other polyclonal or monoclonal antibodies.Immune suppression with rabbit anti-thymocyte globulin (rATG) is a well-established therapeutic concept for preventing host rejection of transplanted organs and graft versus host disease. Increasing the efficiency of rATG production by reducing the number of animals would be highly beneficial to lower cost and to improve quality standards. We have developed transgenic (Tg) mice and rabbits that overexpress the neonatal Fc receptor (FcRn) and have shown an augmented humoral immune response in these animals. To test whether our FcRn Tg rabbits produced rATG more efficiently, we immunized them and their New Zealand White controls with live Jurkat cells. By day 21 after immunization, Tg animals produced significantly, 1.5 times higher amount of total IgG compared to their wt littermates. Also, the binding efficiency of Tg sera to Jurkat cells and their complement-mediated cytotoxicity was significantly higher. The purified Tg IgG preparation contained 2.6 the amount of Jurkat specific IgG as the wt preparation analyzed by complement-mediated lysis, suggesting greater antigen-specific B cell activation in the Tg rabbits. To test this hypothesis, immunization with ovalbumin and human α Polyclonal ATG - the purified IgG fraction of serum from rabbits, horses, or less commonly goats immunized with human thymocytes or the Jurkat T-lymphoblastic cell line - contains antibodies with a wide range of specificities against antigens expressed on various normal and malignant hematopoietic cells including T-, B-, NK, and dendritic cells Currently, there are three commercially available preparations of ATG. ATG-Fresenius® (Fresenius-Biotech GmBH) is produced by immunization of New Zealand White (NZW) rabbits with the Jurkat human T-lymphoblastic cell line. Thymoglobulin® (Genzyme) is produced by immunizing NZW rabbits with human thymocytes while ATGAM® (Pharmacia Upjohn) is produced by immunizing horses with human thymocytes. Due to differences in manufacturing procedures, the different types of ATGs contain variable specificities and amounts of antibodies explaining the wide variability in doses used in the clinical setting. For Thymoglobulin, a total recommended dose is 4.5 to 8 mg/kg body weight for matched unrelated donor allogeneic hematopoetic stem cell transplantation; for ATG-Fresenius the recommended dose for the same indication is about ten times as high Increasing the efficiency of rabbit (r)ATG production and reducing the number of animals would be highly beneficial to maintain consistent quality standards. It would also lower costs and reduce the ethical concerns related to keeping large animal colonies for rATG production.The use of genetically modified animals is widely accepted for the production of therapeutic monoclonal antibodies and remains under investigation for the production of polyclonal antibodies Our data indicate that the sera of the Tg rabbits have greater binding and cytotoxic activity and that their purified IgGs contain proportionally higher amount of antigen-specific IgG. The higher specific activity of IgG harvested from FcRn overexpressing animals is highly valuable as lower doses of purified IgG are needed for therapy. In addition, they are harvestable from fewer animals and therefore reduce consistency problems and exposure to non-antigen specific rabbit IgG.Our previous studies with Tg mice that overexpress FcRn indicated that these animals produce greater number of antigen specific B cells as compared to their wt controls. Thus, we decided to analyze the number of antigen-specific B-cells of these FcRn Tg rabbits by immunizing them with soluble proteins and analyzing the number of antigen-specific B-cells with ELISPOT assay. The results of these studies confirmed that FcRn overexpression in these Tg rabbits enhances the expansion of antigen-specific B cells and plasma cells, similarly to what we observed in the immunized bFcRn Tg mice.This study shows that Tg rabbits that overexpress FcRn produce 1.5 times as much total IgG after immunization with Jurkat cells as their wild type controls. The proportion of rATG in the Jurkat-induced antibody pool is 2.6 times than that of the control rabbits. These findings offer major improvements for production of rATG in animals.Tg rabbits that have enhanced FcRn activity because they carry and express one extra copy of the rabbit FcRn α-chain encoding gene (rabbit FCGRT) in addition to the endogenous rabbit FCGRT gene on New Zealand White rabbit genetic background are coded as NZW Tg1 rabbit FCGRT (78) wherein 78 refers to the founder line The treatments of rabbits in this study were carried out in strict accordance with the recommendations in the Guide of the Institutional Animal Care and Ethics Committee at ImmunoGenes Kft that was approved with permissions 22.1/601/000/2009 and XIV-I-001/2086-4/2012 issued by the Food Chain Safety and Animal Health Directorate of the Government Office of Pest County, Hungary.8 cells per ml. Ten Tg and 10 littermate wt rabbits received two successive intravenous injections 14 days apart of 5×108 living Jurkat T cells, as was suggested in earlier reports, indicating that longer courses of injection usually yields less effective antisera Jurkat E6.1 T cells obtained from the European Collection of Cell Cultures  were used as antigen. Before immunization, Jurkat cells were harvested, washed twice with phosphate buffered saline , and used as a suspension at a concentration of 1×101-antitrypsin (hA1AT) immunization: 10 Tg and 4 wt rabbits were immunized subcutaneously with 300 µg hA1AT  in CFA and challenged 21, 42 and 63 days later with 150 µg antigen in IFA.Ovalbumin (OVA) immunization: 7 Tg and 7 wt rabbits were immunized subcutaneously with 300 µg OVA  in complete Freund's Adjuvant (CFA) and challenged 21 days later with 150 µg OVA in incomplete Freund's Adjuvant (IFA). Human αSerum samples of Tg and wt rabbits were pooled, diluted with binding buffer  and bound to Protein G agarose beads  by incubation for 30 minutes at room temperature with slow agitation. IgG was eluted using 0.1 M Na-citrate buffer (pH 2.9), dialyzed against PBS, aliquoted and stored at −20°C until use.Flow cytometry measurements were carried out using fluorescence-activated cell sorting (FACS) on a FACSCalibur cytometer . Results were analyzed using FCS Express Version 3 software .5 Jurkat cells per sample were washed with FACS buffer, then 100 µl diluted immune serum was added and incubated on ice for 30 minutes. After washing with FACS buffer, cell bound antibodies were detected by incubation with 30 µl of 1000-times diluted DyLight™ 649-conjugated AffiniPure goat anti-rabbit IgG  for 30 minutes on ice. Results were visualized by plotting the mean fluorescence intensities (MFI), which were measured at the same instrumental setting for all samples. Serial dilutions of the purified IgG samples were applied and antibody cell binding was determined by GraphPad Prism version 5 for Windows  using nonlinear regression - one site binding (hyperbola) algorithm.Dilutions of the individual sera and Protein G purified pooled IgG from the Jurkat-immunized Tg and wt rabbits were prepared in FACS buffer . 5×106 Jurkat cells per sample were washed with FACS buffer and incubated for 30 minutes at 37°C with 100 µl of diluted heat inactivated individual immune sera or total IgG purified from pooled Tg and wt immune sera, and 10 µl normal rabbit serum as complement source. Cytotoxicity was determined by propidium iodide (PI) incorporation. The number of lysed cells from the cytotoxicity of the immune serum samples were determined by comparison with the negative control samples (with added complement source without immune serum). ATG-Fresenius S was used as reference and all samples were measured at the same instrumental setting. Serial dilutions of purified IgG samples were applied and complement mediated cytotoxicity was determined by GraphPad Prism version 5 for Windows  using nonlinear regression - one site binding (hyperbola) algorithm.Complement-mediated cytotoxicity was performed as follows: 1×10Single-cell suspensions from OVA and hA1AT immunized rabbit spleens were isolated and incubated with FITC-coupled mouse anti-rabbit T lymphocyte (AbD Serotec) and RPE-coupled goat anti-rabbit IgM (Southern Biotech) antibodies using standard protocol. Isotype controls were obtained from BD Pharmingen. Proportion of granulocytes was estimated based on forward scatter/side scatter dotplots.High binding ELISA plates  were coated with 5 µg/ml OVA, 5 µg/ml hA1AT or 1 µg/ml unlabeled goat anti-rabbit IgG (H+L)  in 0.1 M sodium carbonate-bicarbonate buffer (pH 9.6) for 2 hours at room temperature (RT) or overnight a 4°C, washed with 0.1 M phosphate-buffered saline  containing 0.05% Tween-20 (PBS-T) and blocked with PBS containing 1% BSA for 1 hour at RT. Diluted serum samples were added to the wells (in independently diluted triplicates) and incubated for 1 hour at RT. In case of total IgG measurement each plate included standard controls of serially diluted, purified rabbit IgG (in duplicates). After washing, bound serum antibodies were detected by horseradish peroxidase labeled goat anti-rabbit IgG  using tetramethyl-benzidine  as chromogen. Blank corrected optical density at 450 nm was measured with a Multiscan ELISA Plate Reader  and interpreted using ScanIt Software 2.5.1 Research Edition for Multiscan FC . For data analysis, GraphPad Prism Version 5 for Windows software  was used. Serum IgG concentrations were interpolated from the linear portion of the standard curve, based on the blank-corrected absorbance values using the one site binding hyperbola function of non-linear regression curve fit. IgG titers are given as half-maximal values  or as dilutions at an optical density of 0.05 (endpoint titer).5 cells/well) of spleen lymphocytes harvested on day 26 of OVA and day 70 of hA1AT immunizations were added to the wells. The plates were incubated at 37°C with 5% CO2 overnight and washed with PBS-T; HRP-conjugated goat anti-rabbit IgM and IgG  was then added to each well. After 1 h incubation at room temperature, the plates were washed with PBS-T. The plates were then incubated in the presence of a chromogen, 3-amino-9-ethylcarbazole (Sigma-Aldrich), and H2O2 as substrate at room temperature, and the reaction was terminated by a water wash. The spots were counted in the ImmunoScan ELISPOT reader (Cellular Technology) and evaluated by ImmunoSpot software version 3.2 (Cellular Technology).MultiScreen HTS plates  were coated with 100 µg/ml OVA, or hA1AT, respectively in PBS, at room temperature for 3 h. The plates were then washed with PBS and blocked with RPMI 1640 medium containing 5% FCS and mercaptoethanol (50 mM) for 30 min at room temperature. Serial dilutions . As observed in our previous experiments, Tg rabbits had slightly, though not statistically significantly, higher total IgG levels even before immunization. By day 21, Tg animals had significantly, 1.5 times higher (P = 0.0016) total IgG as compared to their wt littermates  (Antisera were raised by immunizing Tg rabbits and their wild type siblings with two successive intravenous injections 14 days apart of a single cell suspension of 5×10P = 0.0216) compared to the wt animals  Thus the IgG preparation of the Tg rabbits contains 60% more Jurkat specific IgGs compared to the IgG preparation from wt animals Figure 3The immunosuppressive activity of ATG has been thought to result primarily from the depletion of peripheral T lymphocytes from the circulating pool through complement-dependent lysis or activation associated apoptosis. In our experiments, we analyzed the efficiency of the pooled serum samples and purified IgG preparations by complement-mediated cytolysis. The Tg-pool of immune sera was more efficient in killing Jurkat cells at all dilutions in mediating cytotoxicity as compared to its wt control -specific B cell development in these animals, a feature that we observed in our FcRn Tg mice In both cases, FcRn Tg rabbits produced higher titers of antigen-specific IgM and IgG, showed larger spleen and produced many more antigen-specific B-cells than their wt controls Figure 5ATG is widely used as a state of the art treatment for various clinical conditions including prevention or rescue treatment of acute rejection in organ transplantation There is an increasing incidence of organ transplantations, and associated with this is a growing need for immune suppressive therapy with ATG. Hundreds of thousands of rabbits, kept in cost intensive and labor intensive specified pathogen free (SPF) conditions, are required to secure market demand. We were interested to assess if using our recently developed genetically modified rabbits that overexpress the rabbit neonatal Fc receptor (FcRn) and that generate an augmented humoral immune response FcRn is known to be involved in transporting IgGs within and across cells of diverse origin, and in doing so, they regulate concentration and transport of IgG and albumin throughout the body Rabbits are important sources of polyclonal and monoclonal antibodies. We have recently reported on the development of Tg rabbits on New Zealand White genetic background that carry and express an extra copy of rabbit FcRn (when they are heterozygous) and that generate a boosted immune response when immunized with ovalbumin, TNP-KLH and KLH-conjugated peptide The presented study was undertaken to test our FcRn Tg rabbits with another type of antigen, i.e. live cells. We have chosen the human lymphoblast T-cell line (Jurkat) that is used to immunize rabbits for the generation of ATG. As mentioned before, the quantities of ATG that need to be produced at consistent qualities creates a challenge for this kind of manufacturing process. In this study, we immunized FcRn overexpressing Tg rabbits and their wild type littermates with live Jurkat cells and analyzed their immune sera with binding and complement mediated cytotoxic assays to compare their ATG production characteristics., , , We could show that FcRn overexpression in Tg rabbits increases total IgG level by 60% and more significantly the antigen-specific antibody production against Jurkat cells , , , 4This is the first published data set that shows an increased number of antigen-specific B cell in FcRn Tg rabbits post immunization. Moreover our previous data revealed that these FcRn Tg rabbits have improved serum IgG protection For human therapy, especially for long-term administration, fully human form of ATG is required. Such humanized forms of ATG may be obtained from Tg animals that have disrupted endogenous immunoglobulin production and, produce human immunoglobulins. Rabbits have a short generation time, produce large numbers of offspring and can be raised under SPF condition. They have long been an important source of polyclonal and, more recently also, monoclonal antibodies The responsiveness of these animals to given antigens, and subsequent production of sufficient quantities of polyclonal antibodies is a key prerequisite for the viability of these humanized systems for commercial use. Human IgG injected into the maternal circulation is transported well to the rabbit fetus The results of this study show the usefulness of the technology in a medically relevant application, i.e. to overcome the limitations in production of ATG and create the opportunity for a more consistent and more potent product with a significantly improved yield per animal. Our data show that the quantity of harvestable polyclonal Jurkat-specific rATG per Tg animal is approximately 3.9 times higher , when using our specified methods, and thus one could estimate that the number of rabbits required to produce any given quantity of polyclonal Jurkat-specific ATG could be reduced by approximately 80%. This reduction in the size of the animal colony would enable a significant improvement in inter-animal variability and would significantly reduce complexities of manufacturing-specific QC programs.These data provide the rationale for introducing FcRn overexpression as a major improvement into the process of animal (rabbit)-produced ATG and other polyclonal or monoclonal antibodies.


Gene expression profiling has shown its ability to identify with high accuracy low cytogenetic risk acute myeloid leukemia such as acute promyelocytic leukemia and leukemias with t or inv(16). The aim of this gene expression profiling study was to evaluate to what extent suboptimal samples with low leukemic blast load  and/or poor quality control criteria could also be correctly identified.Specific signatures were first defined so that all 71 acute promyelocytic leukemia, leukemia with t or inv(16)-AML as well as cytogenetically normal acute myeloid leukemia samples with at least 60% blasts and good quality control criteria were correctly classified (training set). The classifiers were then evaluated for their ability to assign to the expected class 111 samples considered as suboptimal because of a low leukemic blast load (n = 101) and/or poor quality control criteria (n = 10) (test set).With 10-marker classifiers, all training set samples as well as 97 of the 101 test samples with a low blast load, and all 10 samples with poor quality control criteria were correctly classified. Regarding test set samples, the overall error rate of the class prediction was below 4 percent, even though the leukemic blast load was as low as 2%. Sensitivity, specificity, negative and positive predictive values of the class assignments ranged from 91% to 100%. Of note, for acute promyelocytic leukemia and leukemias with t or inv(16), the confidence level of the class assignment was influenced by the leukemic blast load.Gene expression profiling and a supervised method requiring 10-marker classifiers enable the identification of favorable cytogenetic risk acute myeloid leukemia even when samples contain low leukemic blast loads or display poor quality control criterion. PML/RAR-alpha fusion gene with reciprocal translocation t for APLs, AML1/ETO  fusion gene for t-AMLs, and CBFB/MYH11 fusion gene for AMLs with either inv(16) or balanced reciprocal translocation t. Of note, in up to 15 percent of APLs, no translocation t is detected by conventional cytogenetics at diagnosis, despite PML/RARA fusion gene is detected using molecular assays [Prognostic evaluation is a critical step in newly diagnosed patients with acute myeloid leukemia (AML) in order to identify those at high risk of relapse. For AML patients, cytogenetic abnormalities as well as gene mutations and/or hyper-expressions detected at diagnosis are the main prognostic factors guiding the initial treatment strategy in a risk-oriented manner -4. Hypercation t1;17r assays .With samples containing a high leukemic blast load, microarray-based gene expression profiling (GEP) and class prediction analyses have demonstrated their ability to assign AML samples to one of these three well characterized favorable cytogenetic risk AML subtypes, with high accuracy and low error rates -18. One To our knowledge, the impact of the leukemic blast load and/or of the sample quality on microarray-derived class prediction results has not been specifically studied in AMLs. For centers wishing to integrate microarray-based GEP in a routine prognostic workflow for newly diagnosed AML patients, one critical issue is the ability to perform accurate class prediction analysis with suboptimal samples, i.e., containing as low as 20 percent blasts and even below, and/or not fulfilling all quality control criteria along their process.In this study, GEP was first used to define a limited set of markers allowing to correctly classify 71 patients with either APL, t-AML, inv(16)-AML or cytogenetically normal AML (NK-AML) based on samples containing at least 60 percent of leukemic blasts and characterized by good quality control criteria . The classifiers derived from this first supervised analysis were then evaluated for their ability to assign to the correct class 111 suboptimal samples with low leukemic blast load (from 2 to 59 percent) and/or poor quality control criteria , as well as duplicates of three AML cell lines.in situ hybridization, and RT-PCR analysis of fusion gene transcripts were used to identify patients with APL, t-AML or inv(16)-AML, as previously reported [NPM1, FLT3 and CEBPA for mutations was also performed for all patients [A total of 182 bone marrow or peripheral blood samples from 97 AML patients, diagnosed with either APL (n = 18), t-AML n = 19), inv(16)-AML (n = 29), or NK-AML (n = 31), followed at Angers University Hospital (n = 72) or Reims University Hospital (n = 25), were analyzed, as well as duplicated samples of NB4, Kasumi-1, and ME-1 cell lines (n = 6 samples), which are derived from patients with APL, t-AML and inv(16)-AML, respectively. In addition, 18 samples of unique (n = 9) or pooled (n = 9) normal bone marrows obtained from 12 healthy volunteers were included in the study. The main characteristics of AML patients and samples are summarized in Tables , inv(16)patients -23. All The gene expression dataset, generated from 206 samples , was divided into a Training Set (n = 89 samples) and a Test Set (n = 117 samples). The Training Set, used to identify specific class markers (classifiers), included 18 samples of unique or pooled normal bone marrows (see details about the pools below) as well as 71 AML samples obtained from patients  newly diagnosed with APL (n = 14 patients), t-AML (n = 14 patients), inv(16)-AML (n = 15 patients) or NK-AML (n = 28 patients), which contained at least 60 percent blasts. The class prediction analysis was performed on a Test Set including 111 suboptimal AML samples , i.e., with a low leukemic blast load (n = 101 samples containing from 2 to 56 percent blasts) and/or poor quality control criteria (n = 10 samples) as well as duplicated samples of NB4, Kasumi-1, and ME-1 cell lines (n = 6 samples). Among test samples with optimal quality control criteria, 22 originally contained less than 60% blasts , whereas 79 were high blast load ones artificially diluted within one of the five pools of normal bone marrows . Total RNA quantification was performed using the Nanodrop ND-1000 spectrophotometer  according to manufacturer recommendations. Integrity of the extracted RNAs was assessed with the Bioanalyzer 2100 and the RNA6000 Nano kit . A RNA integrity number (RIN) greater or equal to 7.00 was achieved for 203 samples (RNA degradation observed for 3 AML samples). No sign of DNA contamination was detected in any of the 206 samples analyzed. The starting amount of total RNA used for the reactions was 200 nanograms per sample, for all samples. The Illumina Total Prep RNA Amplification Kit  was used to generate biotinylated, amplified cRNA according to the manufacturer recommendations . Isolated cell samples were then immediately cryopreserved. Total RNAs were extracted from 10XIST transcript in males, and CYorf15B transcript in females); and maximal value for probe signal intensity of 50 000 across all samples. Among the 48 803 probes assessed on HumanHT-12 v3 Expression BeadChips, 11 779 satisfied these low stringency filtering criteria. Filtered data were then log-transformed and exported to appropriate softwares for the analyses. Regarding the Test Set samples processing, only data concerning the markers identified during the first analysis, restricted to the Training Set, were used. These raw data were log-transformed and merged to the one of the Training Set for class prediction analyses. The Class Prediction Module of ArrayMiner 5.3.3 , which uses a proprietary method based on grouping genetic algorithms, was used for all class prediction analyses  was used for principal-component analyses. S-Plus® 8.0 Enterprise Developer software  was used for all other statistical analyses. The two datasets (Training Set and Test Set) discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE34823 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE34823. The dataset associated with the Training Set is also linked to the SubSeries accession number GSE34577. The dataset associated with the Test Set is also linked to the SubSeries accession number GSE34714.GenomeStudio 2010.3 software  and its Gene Expression Analysis Module (version 1.8.0) were used for signal extraction and normalization (for the Training Set only). Briefly, the Invariant Rank normalization method was applied to the primary probe data obtained from the Training Set samples. Processed probe data were then filtered according to the following criteria: minimal signal intensity fold change of 1.50 across all samples; minimal probe signal intensity absolute change of 150 across all samples -AML, inv(16)-AML, NK-AML classes were defined based on 71 samples containing at least 60% of leukemic blasts and characterized by good quality control criteria . At this stage, unique and pooled normal bone marrow samples were also included in the model as the objective was to enhance the predictive capacity of the classifiers, especially for Test Set samples containing a majority of residual normal cells and a low leukemic blast load. Evaluating classifiers which included from 1 to 100 markers per class, all Training Set samples were assigned to the correct class when selecting from 3 to 46 markers per class , whether the source of leukemic cells was bone marrow or peripheral blood. The best model fitness was obtained with 8, 9 or 10 markers per classifier. To select the optimal model among these three, a principal-component analysis was performed. The highest cumulative percent of variance accounted for by the first three components was obtained with the 10-marker classifiers -AML and inv(16)-AML classes, the blast load significantly influenced the confidence level of the class assignment -AML Test Set samples were correctly classified, even when containing as low as 2 percent blasts  , and for each class, sensitivity and specificity, negative and positive predictive values of the class assignments ranged from 91 to 100 percent . Finally, APL, t-AML, and inv(16)-AML samples containing as low as 2 percent blasts could be correctly classified, whether the classifiers mainly relied on a single marker, such as for inv(16)-AMLs and t-AMLs, or on the overall set of 10 markers defining the classifier, as for APL samples. However, regarding APL, t-AML and inv(16)-AML samples, the confidence level of the class assignment was correlated to the percentage of leukemic blasts within the studied samples. For NK-AML samples, such a finding was not observed, probably because NK-AMLs represent a more heterogeneous group of leukemias as compared to t-AMLs, inv(16)-AMLs or APLs in term of oncogenic processes. This heterogeneity likely led to a higher variance of gene expression profiles within the NK-AML class, to the detriment of the interclass variance, hence influencing the class prediction confidence level more than the leukemic blast load. This hypothesis was supported by the fact that, within the NK-AML class, the best predictive capacity of the GGA was achieved for utations -31.CBFB/MYH11 fusion gene was confirmed by FISH analysis, all contained less than 20 percent blasts and two were obtained at the time of relapse - from patients UPN7 and UPN37 -. Regarding these two samples, which respectively contained 5 and 14 percent blasts, they were characterized by a low expression level of most markers defining the inv(16)-AML specific signature; especially MYH11, which expression level was within the estimated background signal range. It is noteworthy that for one of those two patients, UPN37, the bone marrow sample obtained at diagnosis was correctly classified, even when diluted at 50 and 75 percent . For this patient, a clonal evolution of the disease was suspected as CD2, CD13, CD34 and CD117 expression values, assessed by flow cytometry on gated leukemic cells, and MYH11 expression level (microarray data) were significantly lower in the relapse sample as compare to the ones observed in the diagnostic sample. Verhaak et al in their study using Affymetrix GeneChips reported that MYH11 expression level was sufficient to identify inv(16)-AMLs [MYH11 as the sole marker for the inv(16)-AML class could lead to unstable results in a class prediction model, especially in low leukemic blast load samples or in case of a clonal evolution at relapse. Of note, the inv(16)-AML signature developed on the AMLProfiler™ kit http://www.skyline-diagnostics.com, which uses the Affymetrix™ platform, also includes a set of 19 markers [Unexpectedly, the worst results of the class prediction analysis were achieved for inv(16)-AML Test set samples . Among the three misclassified inv(16)-AML samples and for which the presence of  markers .As reported by Kohlmann et al., sub-optimal AML samples with poor quality control criteria, mainly because of low amounts of hybridized cRNAs, were all correctly classified, even though half of these contained less than 60 percent blasts (down to 9 percent for UPN90) . StudyinAlthough, in most cases, these favorable prognostic chromosomal abnormalities and/or their related gene products can be detected by karyotype, FISH or PCR assay, they represent main targets for a microarray-based class prediction analysis to be identified before considering GEP as a useful tool in a routine workflow for prognostic assessment of AML patients. The fact that the AMLProfiler, a microarray-derived kit based on the Affymetrix™ platform, has been recently commercialized with the aim of achieving a molecular diagnosis for APLs, t-AMLs and inv(16)-AMLs, respectively using 27, 31 and 19 markers, emphasizes this assumption .CERCAM, COL23A1, LOC643201, LOXL4, SLC39A11), t-AMLs , and inv(16)-AMLs .Finally, regarding the classifiers associated with APLs, t8;21)-AMLs or inv(16)-AMLs, 18 of the 28 markers identified in this study (64 percent) had already been reported in previous studies using various microarray technologies . - Generation of low leukemic blast load AML samples by a "dilution/mixture" approach. - Hybridization on Illumina HumanHT-12 v3 Expression BeadChips, staining and detection of cRNAs on microarrays using an I-Scan system. Additional table S1 - Citations in previous studies of the identified markers. Additional figures S1 to S7. Figure S1. Three-dimensional projection of the 3 principal components in a principal-components analysis of APL, t-AML, inv(16)-AML, NK-AML, and Normal Bone Marrow samples belonging to the Training Set, with the use of the 10-marker classifiers. Figure S2. Per class median confidence level of the assignment for APL, t-AML, inv(16)-AML, NK-AML and normal bone marrow samples belonging to the Training Set according to the number of markers per class (from 1 to 100 markers per class). Figure S3. Median confidence level of the class assignments for the NK-AML samples belonging to the Training Set according to FLT3 and NPM1 mutational status and the number of markers per class (from 1 to 100 markers per class). Figure S4. Relationship between the percentage of leukemic blasts within the 101 Test Set samples (X axis) and the confidence level of their class assignments (Y axis) . Figure S5. Confidence level of the class assignments according to the percentage of leukemic blasts including comparisons between diluted and not diluted samples. Figure S6. Results of the class assignment for the 10 AML Test Set samples with suboptimal quality control criteria based on the 10-marker classifiers characterizing the APL, t-AML, inv(16)-AML and NK-AML classes. Figure S7. Sensitivity, specificity, negative and positive predictive values of the prediction model for the class assignment of the 111 Test Set samples  to the APL, t-AML, inv(16)-AML and NK-AML classes with 10-marker classifiers.Click here for file


Nociceptive pain is one of the most common types of pain that originates from an injury involving nociceptors. Approximately 60% of the knee joint innervations are classified as nociceptive. The specific biological mechanism underlying the regulation of nociceptors is relevant for the treatment of symptoms affecting the knee joint. Intra-articular administration of exogenous hyaluronic acid (HA) in patients with osteoarthritis (OA) appears to be particularly effective in reducing pain and improving patient function.in vitro study conducted in CHO cells that expressed a panel of opioid receptors and in primary rat dorsal root ganglion (DRG) neurons to determine if HA induces the activation of opioid peptide receptors (OPr) using both aequorin and the fluorescent dye Fura-2/AM.We performed an in vitro model followed by stimulation with HA. The results showed that HA induces stimulatory effects on the κ receptor (KOP). These effects of HA were also confirmed in rat DRG neurons, which express endogenously the OPr.Selective agonists and antagonists for each OPr expressed on CHO cells were used to test the efficacy of our 50 value of 7.57.HA activates the KOP receptor in a concentration dependent manner, with a pEC The word “pain” comes from the Latin “poena”, which mean a fine or a penalty. Pain is indeed an unpleasant stimulus that warns of an injury that either is immediately impending or has already occurred, such as touching a hot object. Pain experts have divided the physical causes of pain into two types: neuropathic and nociceptive pain. Whereas neuropathic pain refers to the dysfunction of either the peripheral  or central  nervous systems, nociceptive pain (the more common of the two) refers to the discomfort that results when a stimulus causes tissue damage to the muscles, bones, skin or internal organs The most common form of joint damage is OA, which is accompanied by significant joint pain The intra-articular injection of morphine and opioid compounds appears to be somewhat effective in the treatment of pain in various arthritides without producing major side effects. Studies by several independent groups indicated that the application of morphine and opioid compounds might function through their receptors (OPr) in the periphery Nociceptive sensitivity is modulated by a large variety of mediators in the extracellular space. These mediators activate a large number of receptor classes, which in turn activate a plethora of signaling cascades Recent studies have discovered the importance of the extracellular matrix (ECM) components in the modulation of nociception In light of these clinical considerations, we have investigated if HA induces the activation of opioid peptide receptors (OPr).To study this effect, CHO cell lines stably expressing the classical opioid receptor types DOP, KOP, and MOP as well as the nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor, were tested. OPr are Gi-coupled receptors that alter the concentration of cyclic AMP (cAMP) and, like many other G protein-coupled receptors, can undergo rapid desensitization and internalization following exposure to agonist 2) release from intracellular stores. To overcome this technical complication, we decided to couple the activation of OPr to Ca2+ signals using CHO cell lines expressing the promiscuous G protein Gαqi5, which can couple any GPCR to an increase in cytosolic Ca2+ concentration ([Ca2+]c) GPCRs that are coupled to cAMP require complex and less efficient screening methods such ligand binding assays or radioimmunoassay to measure activity compared to the GPCRs linked to phospholipase C activation and calcium : small MW: 6 kDa; medium MW: 200 kDa  at increasing concentrations comprised between 0,005 and 5 mg/ml. As selective agonists the following molecules were used: N/OFQ for the NOP line; -Enkephalin (DPDPE) for the MOP line, dermorphin for the DOP line, and dynorphin A for the KOP line. norbinaltorphimine (norBNI) has been used as KOP receptor antagonist. As negative control it was used the buffer (KRB) without agonist.Tissue culture media and supplements were from Invitrogen  and from Cambrex Bioscience . All other reagents were from Sigma Chemical .qi5 protein were generated as previously described CHO stable clones expressing DOP, KOP, MOP, NOP receptors and stably expressing the Gα2+-phosphate procedure. All experiments were performed 36 h post-transfection.For transfection, CHO cells were seeded 48 h prior to transfection onto different sized glass coverslips depending on the assay: 13 mm diameter for the aequorin experiments and 24 mm diameter for the Fura-2/AM measurements. Cells were allowed to grow to 50% confluence followed by transfection with a standard CaIsolation, dissociation plating and transfection of rat DRG neurons from adult rats (12 weeks) were performed as previously described All cells were cultured at 37°C in humidified air containing 5% carbon dioxide.2+ concentrations measurements were carried out as previously described 3PO4 1 mM, MgSO4 1 mM, glucose 5.5 mM, HEPES 20 mM, pH 7.4) supplemented with 5 μM coelenterazine. Then all measurements were performed using an automatized luminometer . KRB additioned of the different HA was then injected and luminescence was recorded for 60 s. To terminate the experiments and discharge the remaining aequorin for the normalization of the values obtained, a hypotonic solution containing 500 μM digitonin and 50 mM CaCl2 was injected. The results are expressed as % of probe discharged ± standard error (SE). This is possible because, when Ca2+ ions bind to three high-affinity sites (EF-hand type), aequorin undergoes an irreversible reaction, in which a photon is emitted and the resulting protein is inactive (discharged).Mitochondrial Ca2+] values, computer algorithm based on the Ca2+ response curve of aequorins has been used as previously described To convert the aequorin luminescence data into [Ca2+] were evaluated using the fluorescent Ca2+ indicator Fura-2/AM . Briefly, cells were incubated in medium supplemented with 2.5 μM Fura-2/AM for 30 min, washed with KRB to remove extracellular probe and placed in a thermostatic incubation chamber at 37°C on the stage of an inverted fluorescence microscope (Zeiss Axiovert 200). Dynamic video imaging was performed using MetaFluor software . [Ca2+] calculated as previously described Cytosolic free  is the concentration of agonist, EC50 is the concentration of agonist producing a 50% maximal response, and n is the Hill coefficient of the concentration response curve to the agonist The data were analyzed by nonlinear curve fitting equation using Graph Pad 4.0 software. The data are expressed as the mean ± SEM of at least 6 experiments and were analyzed statistically using one-way analysis of variance followed by Dunnett's test for multiple comparisons. Agonist potencies are given as pEC2+ signals instead of their native physiological second messenger cAMP, the cells were co-expressed with the promiscuous G protein Gαqi5, which can couple any GPCR to Ca2+ changes 2+ release from the intracellular stores and an influx from the extracellular medium–this causes a transient rise in cytoplasmic [Ca2+] ([Ca2+]c) 2+]c using the fluorescent cytosolic Ca2+ dye Fura-2/AM To determine if HA is able to activate the OPr, we utilized CHO cells that expressed different opioid receptor types . To couple the activation of these receptors to Ca2+]c in the presence of their respective selective agonist (The following molecules were used as selective agonists: N/OFQ peptide (Nociceptin) for the NOP line; -Enkephalin (DPDPE) for the DOP line, dermorphin for the MOP line and dynorphin A for the KOP line. All of the cell lines demonstrated a well-defined increase in c cause a rapid increase in the [Ca2+] within the mitochondrial matrix ([Ca2+]m) where the probe is located, which usually exceeds the increase in the cytosol Though there are several methodologies to monitor changes in intracellular Ca2+ response upon binding with their respective receptors compared to the negative control (buffer KRB without agonist). The mitochondrial Ca2+ responses were expressed as either a percentage of probe discharged during the stimulation . The maximal effects induced by HA were approximately half of those induced by dynorphin A; thus, HA appears to behave as a partial agonist at KOP receptors.The same cell clones were stimulated with two types of HA (low molecular weight (MW) at 6 kDa and medium MW at 200 kDa) at concentrations ranging from 0,005 to 5 mg/ml. Though the smaller MW HA was unable to induce any significant light emission, the larger HA induced a significant detectable light emission only in KOP-expressing CHO cells . This stWe then assessed the sensitivity of the KOP receptor antagonist norbinaltorphimine (norBNI) on the stimulatory effects of both dynorphin A and HA. As shown in 2+]c were investigated with the Fura-2/AM method in KOP cells. The stimulation of KOP-expressing CHO cells with the specific agonist dynorphin A (1 μM) resulted in an expected transient increase in [Ca2+]c .As shown in 2+ in rat DRG neurons after HA stimulation. In these experiments (2+] (with EGTA 100 µM), under those conditions the increase of [Ca2+] was due only to the release of Ca2+ from the intracellular stores. The following addition of Ca2+ to the extracellular medium caused a second smaller [Ca2+] rise due to the influx through the plasma membrane channels induced by the Ca2+-depletion of the intracellular Ca2+ stores.Finally, we investigated the contribution of intracellular and extracellular Caeriments , the celNative HA is a large glycosaminoglycan with repeating disaccharide subunits of N-acetylglucosamine and D-glucuronic acid VS is an approved treatment modality for OA. Administration of HA preparations to the joint synovial fluid restores the biological properties of normal hyaluronic acid in the ECM, providing pain relief and increasing knee joint mobility in vitro model. We decided to couple the activation of OPr to Ca2+ signals using CHO cell lines expressing the promiscuous G protein Gαqi5, which can couple any GPCR to an increase in [Ca2+]c2+]c results in a rapid and transient increase in the [Ca2+]m that exceeds the concentration in the cytosol by at least one order of magnitude The aim of the present study was to determine the activity of HA on OPr on 2+ indicators: the light emitted does not required any excitation, which eliminates issues related to auto-luminescence (and auto-fluorescence), and background measurements of aequorin are close to zero because mammalian cells are not naturally endowed with chemiluminescent proteins Aequorin-based assays have been successfully applied for screening of agonists, antagonists and allosteric modulators for different families of GPCRs. This is because aequorin has a number of advantages over other Ca2+] increases that occur in some cell types. The use of aequorin is now widely accepted as a method to identify agents able to induce GPCR activation 2+ homeostasis in different pathophysiological contexts 2+ dye) to identify if OPr are activated by HA.The wide dynamic range (between 50 nM and 500 µM), together with the low buffering effect, makes the aequorin probe the tool of choice when quantitatively estimating large [Ca2+ response in cells endogenously expressing KOP receptors such as the rat DRG neurons.Experiments performed in CHO cells expressing OPr clearly demonstrated that HA with a medium MW  is able to selectively activate the KOP receptor acting as partial agonist. Antagonist experiments performed with norBNI displayed superimposable potency values against HA and dynorphin A, which supports the hypothesis that the effects of HA are due to the selective activation of the KOP protein Considering that no interesting data were obtained regarding the DOP, MOP and NOP receptors along with the fact that KOP receptors are expressed on fibroblast-like synoviocytes In conclusion, the present results indicate that HA is able to activate the KOP receptor. The approaches presented in this study are the canonical procedures used for the identification of new ligands for specific receptors as well as in receptor deorphanization programs. Future experiments that involve testing peripheral terminal function such as the inhibition of neuropeptide release from peripheral and central terminals  of primary sensory neurons (nociceptors) will reveal the physiological relevance of this study. Moreover, by using opioid receptor knockout animals, it will be possible confirm that HA selectively affects KOP, as shown in this study through the use of the KOP receptor antagonist norbinaltorphimine.Figure S1Representative traces of (TIF)Click here for additional data file.


In the present study, [Ca2+]i transient, reactive oxygen species (ROS) and mitochondrial activity were measured by the high-speed TILLvisION digital imaging system using the indicators of Fura 2-am, DCFDA and rhodamine-123, respectively. We found that glibenclamide, pinacidil and other unselective K+ channel blockers had no effect on the resting [Ca2+]i of Raw 264.7 cells. Extracellular ATP (100 µM) induced [Ca2+]i transient elevation independent of extracellular Ca2+. The transient elevation was inhibited by an ROS scavenger (tiron) and mitochondria inhibitor (rotenone). Glibenclamide and 5-hydroxydecanoate (5-HD) also decreased ATP-induced [Ca2+]i transient elevation, but pinacidil and other unselective K+ channel blockers had no effect. Glibenclamide also decreased the peak of [Ca2+]i transient induced by extracellular thapsigargin . Furthermore, glibenclamide decreased intracellular ROS and mitochondrial activity. When pretreated with tiron and rotenone, glibenclamide could not decrease ATP, and Tg induced maximal [Ca2+]i transient further. We conclude that glibenclamide may inhibit ATP-induced [Ca2+]i transient elevation by blocking mitochondria KATP channels, resulting in decreased ROS generation and mitochondrial activity in Raw 264.7 macrophages.Increasing evidence has revealed that glibenclamide has a wide range of anti-inflammatory effects. However, it is unclear whether glibenclamide can affect the resting and adenosine triphosphate (ATP)-induced intracellular calcium ([Ca The emission wavelength was 510 nm. The background intensity was subtracted from the fluorescent intensity changes, and the resulting [Ca2+]i values were normalized as the differences between the fluorescence intensities with different agents and the intensity in standard bath solution (BS) by averaging the values of at least 50 cells/treatment.Calcium imaging was performed as we described previously 2DCFDA) as a fluorescent dye. The cells were trypsinized, and the cell suspension was treated with H2DCFDA at a final concentration of 10 µM in the recording solution for 30 min at 37°C. H2DCFDA is oxidized to the fluorescent dichlorofluorescein (DCF), which is monitored at excitation and emission wavelengths of 488 and 510 nm, respectively, using a TILLvisION digital imaging system. After incubating cells with different reagents, the ROS levels were determined by comparing the changes in fluorescence intensity with that in the standard extracellular recording solution. The fluorescence values were determined by averaging the fluorescence values of at least 50 cells/treatment.The production of intracellular ROS was monitored by dichlorodihydrofluorescein diacetate  fluorescent dye imaging. A Raw 264.7 cell suspension was loaded with 10 µg/ml Rh-123 at room temperature for 15 min. After loading, the cells were continuously perfused with recording solution. Rh-123 fluorescence images were captured using the method described above. The fluorescence was excited at 490 nm and filtered at 530 nm. The fluorescence values were determined by averaging the fluorescence values of at least 50 cells/treatment.2, 1.5 CaCl2, 10 HEPES and 10 glucose titrated to pH 7.4 with NaOH. The BS was then adjusted to an osmotic pressure of 290 mOsm with D-mannitol. The recording BS lacked Ca2+. When indicated, 1.5 mM CaCl2 was added back to the BS. Stock solutions of glibenclamide, pinacidil, diazoxide, diphenyleneiodonium (DPI), H2DCFDA, and Rh-123 were prepared in dimethylsulfoxide (DMSO). 5-hydroxydecanoate (5-HD), tiron, 4-aminopyridine (4-AP), and tetraethylammonium (TEA) were dissolved in distilled H2O. All chemicals were obtained from Sigma  and diluted on the day of the experiment.The standard BS comprised the following (in mM): 130 NaCl, 10 CsCl, 1.2 MgClt-test and one-way ANOVA were used for statistical analysis where appropriate. P<0.05 was considered statistically significant.The data were expressed as means ± standard errors. The unpaired Student's 2+]i of Raw 264.7 cells with or without Ca2+ in extracellular solution  and other unselective potassium channel blockers  did not change the resting [Ca2+]i  had no effect on the resting i transient elevation in Raw 264.7 cells , ATP could not induce [Ca2+]i transient elevation again . 2-APB (100 µM) was able to block the [Ca2+]i transient elevation induced by both ATP and Tg  induced i was very modestly increased, which then completely recovered to baseline levels quickly (When Raw 264.7 cells were treated with ATP (100 µM) or Tg (1 µM) in Ca quickly , B.2+]i transient elevation induced by extracellular ATP (100 µM)  also decreased the maximal [Ca2+]i  decreased the peak of the i . The mit P<0.05, . When si P>0.05, .2+]i transient elevation induced by extracellular Tg (1 µM) .Glibenclamide (100 µM) or 5-HD (100 µM) also decreased the peak of the [Ca2+]i induced by extracellular ATP  inhibitor DPI (10 µM) did not decrease the maximal i transient elevation - or Tg (1 µM)-induced maximal i of Raw 264.7 macrophages; 2) extracellular ATP induced the [Ca2+]i transient elevation in Raw 264.7 macrophages by depleting [Ca2+]i stores; 3) intracellular ROS can regulate the ATP-induced [Ca2+]i transient; and 4) glibenclamide decreased the ATP-induced [Ca2+]i transient elevation by inhibiting ROS that were mainly generated from the mitochondria in macrophages.In the present study, for the first time, we have demonstrated the following: 1) glibenclamide cannot affect the resting [Ca2+ was considered to be an important second messenger to activate immune cells 2+]i2+ elevations are required for diverse immune cellular functions, including differentiation, effector function, and gene transcription 2+]i in macrophages. In the present study, we found that glibenclamide did not affect the resting [Ca2+]i of Raw 264.7 macrophages, which was very different than the effect of glibenclamide on excitable cells, such as smooth muscle cells ATP, leading to membrane depolarization, which in turn opens voltage-gated calcium channels and increases [Ca2+]i via Ca2+ influx.Cytosolic Ca2+. In the present study, we found that ATP induced [Ca2+]i transient elevation independent of extracellular Ca2+ in Raw 264.7 cells. We also found this phenomena in THP-1 cells, whereas other studies found that ATP induced [Ca2+]i independent of extracellular Ca2+ in Sertoli cells 2+]i transient independent of extracellular Ca2+ in a manner that was not specific to macrophages.At sites of inflammation, macrophages are exposed to various chemical mediators, such as nucleotides, prostanoids, and oxygen radicals 2+]i transient again. The Ca2+ influx was very modest and recovered to the baseline level quickly when 1.5 mM Ca2+ was reintroduced to the extracellular buffer following pretreatment with ATP or Tg in Ca2+-free buffer. Mikulski Z et al2+-release from intracellular stores. In murine J774 macrophages, low concentrations (10 µM) of ATP evoked Ca2+ transient in a phospholipase C (PLC)-dependent manner 2+ influx resulting from the depletion of Ca2+ stores was modest and transient in Raw 264.7 cells and was covered by ATP- or Tg-induced [Ca2+]i release. Furthermore, we found that 2-APB, a membrane permeable IP3 receptor antagonist 2+]i elevation induced by ATP. Many studies found that ATP induced Ca2+ release from the endoplasmic reticulum (ER) through inositol 1,4,5- trisphosphate (IP3) receptors (IP3Rs) 2+]i transient elevation by releasing Ca2+ from the intracellular Ca2+ stores in Raw 264.7 macrophages.When the calcium stores were pre-released by thapsigargin, ATP could not induce [Ca2+]i transient elevation is worthwhile. For the first time, we found that glibenclamide decreased the maximal [Ca2+]i transient elevation induced by extracellular ATP or Tg. However, a membrane KATP opener (pinacidil) and other unselective potassium channel blockers (TEA and 4-AP) did not change the maximal [Ca2+]i transient. The mitochondrial KATP blocker (5-HD) also decreased the maximal [Ca2+]i transient. Glibenclamide and 5-HD showed no additive effect on decreasing the maximal [Ca2+]i transient. Because glibenclamide has been shown to have an inhibitory effect on mitochondria KATP channels 2+]i transient elevation by blocking mitochondrial KATP channels.Because previous studies showed that glibenclamide has a wide range of anti-inflammatory effects ATP channels and ATP-induced intracellular calcium transient is not clear. Recently, Hänninen SL et al2+ stores in cardiomyocytes. Thus, we hypothesized that ROS mainly from the mitochondria participated in the regulation of ATP-induced [Ca2+]i transient and that glibenclamide might inhibit this transient by inhibiting ROS generation by blocking mitochondrial KATP channels.However, the link between mitochondrial K2+]i transient elevation. The NOX inhibitor DPI did not decrease the maximal [Ca2+]i transient induced by extracellular ATP or Tg. An ROS scavenger (tiron) and a mitochondrial inhibitor (rotenone) inhibited the extracellular ATP- or Tg- induced maximal [Ca2+]i. Mitochondria depolarization was previously found to increase the generation of mitochondria-derived ROS, which stimulated Ca2+ sparks in cerebral artery smooth muscle cells et al2+]i and contraction in the pulmonary artery smooth muscle cells. These results suggested that ROS mainly from the mitochondria participated in the regulation of the ATP-induced intracellular calcium in Raw 264.7 macrophages.First, we used different ROS inhibitors to detect which sources of ROS affected the ATP-induced i transient. Additionally, glibenclamide had no effect on [Ca2+]i transient in Raw 264.7 macrophages pretreated with 5-HD. Because mitochondrial KATP channels play an important role in ROS production 2+]i transient elevation might be mediated by inhibiting ROS and blocking mitochondrial KATP channels.Finally, when pretreated with tiron and rotenone, glibenclamide and 5-HD could not further decrease the maximal ATP- or Tg-induced i, intracellular ROS, and mitochondrial activity. However, further studies should be performed to reveal the effect of glibenclamide on other physiological functions of macrophage cells by regulating [Ca2+]i homeostasis.In conclusion, extracellular ATP induced the intracellular calcium transient elevation by depleting calcium stores in Raw 264.7 macrophages, which could be regulated by ROS mainly from mitochondria. Glibenclamide might inhibit this transient activity by blocking mitochondrial KFigure S12+]i transient elevation in THP-1 cells.Extracellular ATP induced a i was modestly increased and then completely recovered to the baseline levels quickly.(TIF)Click here for additional data file.


OF1 ATP synthase form a channel with properties identical to those of the mitochondrial megachannel (MMC), the electrophysiological equivalent of the PTP. Open questions remain, but there is now promise that the PTP can be studied by genetic methods to solve the large number of outstanding problems.The permeability transition (PT) denotes an increase of the mitochondrial inner membrane permeability to solutes with molecular masses up to about 1500 Da. It is presumed to be mediated by opening of a channel, the permeability transition pore (PTP), whose molecular nature remains a mystery. Here I briefly review the history of the PTP, discuss existing models, and present our new results indicating that reconstituted dimers of the F The occurrence of swelling in isolated mitochondria, its strict dependence on matrix Ca2+ had a physiological role in steroidogenesis. These Authors showed that Ca2+ induces a “transformation” of adrenal cortex mitochondria allowing diffusion of extramitochondrial pyridine nucleotides through the otherwise impermeable inner membrane, and that NADPH entering in this way supports the 11-β hydroxylation of deoxycorticosterone  whose enzymatic activity is blocked by CsA  A .TSPO is an 18 kDa highly hydrophobic protein, involved in cholesterol and protophorphyrin transport, which is located in the outer mitochondrial membrane  and Mg2+ (which inhibits it). Finally, and at striking variance with the pore, channel activity was inhibited by Pi and unaffected by ADP . CyPD is the mitochondrial receptor for CsA and modulates the PTP but is not a structural pore component  mitochondria are not null for the PTP.Ppif This consideration is important for the interpretation of results obtained with CsA because (similarly to the absence of CyPD) CsA can desensitize but not block the PTP; thus, lack of sensitivity to CsA does not necessarily mean that the PTP is not involved in the event being studied. Furthermore, expression of CyPD is modulated, e.g., by muscle denervation  is the rotary enzyme that synthesizes the vast majority of ATP in respiring cells . Monomers of ATP synthase were devoid of channel activity in spite of the same overall subunit composition as the dimers . Removal of OSCP, or CyPD binding to OSCP, would induce similar conformational effects leading to increased probability of PTP opening, a working hypothesis that awaits experimental verification.Bz-423 was characterized as an apoptosis-inducing agent acting through mitochondria  is not high enough for the PT to occur (panel A). Binding of CyPD, which is favored by Pi . The transition would essentially depend on replacement of Mg2+ with Ca2+, which in the absence of CyPD binding would require higher matrix Ca2+ loads (panel C). Accessibility of the metal binding site (and probability of occurrence of the PT at a given Ca2+ load) would be increased by thiol oxidation and counteracted by thiol reduction. Ion and solute permeation would then occur at the interface between the c rings . The PT can be fully reverted by Ca2+ chelation with EGTA  complex V dimers suffice to induce MMC activity and (2) CaBcl-2 family proteins, which are generally believed to locate to the outer membrane, affect the PTP  in Ca2+ homeostasis may be essential only for relatively large mitochondrial Ca2+ loads  in a variety of cell types  PT can occur in mitochondria from yeast strains that do not possess an energy-conserving complex I  channel openings are favored by Bz-423 and thiol oxidants; (3) channel openings are inhibited by adenine nucleotides and Mg2+; and (4) monomers lack any channel activity, all represent strong indications that the PTP forms from a specific, Ca2+-dependent conformation of the dimers. The FOF1 ATP synthase readily accommodates key pathophysiological effectors of the PT since Ca2+, Mg2+, adenine nucleotides, and Pi bind the catalytic core at F1; and the membrane potential and matrix pH, which are key PTP modulators  dimers of the ATP synthase treated with CaThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Here, we investigated how a 20% hypotonic shock modulates the ionic current in these cells. Polarized alveolar epithelial cells isolated from rat lungs were cultured on permeant filters and their electrophysiological properties recorded. A 20% bilateral hypotonic shock induced an immediate, but transient 52% rise in total transepithelial current and a 67% increase in the amiloride-sensitive current mediated by ENaC. Amiloride pre-treatment decreased the current rise after hypotonic shock, showing that ENaC current is involved in this response. Since Cl- transport is modulated by hypotonic shock, its contribution to the basal and hypotonic-induced transepithelial current was also assessed. Apical NPPB, a broad Cl- channel inhibitor and basolateral DIOA a potassium chloride co-transporter (KCC) inhibitor reduced the total and ENaC currents, showing that transcellular Cl- transport plays a major role in that process. During hypotonic shock, a basolateral Cl- influx, partly inhibited by NPPB is essential for the hypotonic-induced current rise. Hypotonic shock promoted apical ATP secretion and increased intracellular Ca2+. While apyrase, an ATP scavenger, did not inhibit the hypotonic shock current response, W7 a calmodulin antagonist completely prevented the hypotonic current rise. These results indicate that a basolateral Cl- influx as well as Ca2+/calmodulin, but not ATP, are involved in the acute transepithelial current rise elicited by hypotonic shock.Alveolar epithelial cells are involved in Na For al cells . As with p<0.05; . The kin p<0.05; . To detel buffer . While treatment . In addi2+/calmodulin antagonist, could influence the current rise elicited by hypotonic shock in alveolar epithelial cells. While 25 µM W7 pretreatment had no impact on basal and ENaC transepithelial current, it inhibited total and ENaC transepithelial current increment after 20% hypotonic shock  on the apical side of the monolayer peaking after 1 min (1.77 X 10-13 moles/106 cells above the baseline), followed by a rapid decrease within 5 min , ruling out a role of ATP in this process , before challenging them with 20% hypotonic shock. Apyrase treatment could not prevent the current rise elicited by hypotonic shock  could modulate total and amiloride-sensitive current in alveolar epithelial cells. We noted an acute but transient rise in total and amiloride-sensitive currents induced by 20% hypotonic shock. Moreover, we found that Cl- transport is important for the basal transepithelial current generated by alveolar epithelial cells and for the current rise induced by hypotonic shock.Transepithelial Nase cells ,23. Sinc+ uptake in response to different tonicity challenges in alveolar epithelial cells i in rat alveolar epithelial cells. As reported in 2+]i that reached a plateau after ~4.5 min. The response was different from that in A549 cells where hypotonic shock induced a strong but transient [Ca2+]i spike from thapsigargin-sensitive stores, followed by a sustained plateau from thapsigargin-insensitive stores i rise important for transepithelial current modulation by hypotonic shock? Ussing chamber experiments were repeated in physiological buffer devoid of Ca2+ or in presence of 50 µM BAPTA-AM to chelate cytosolic Ca2+. While the absence of extracellular Ca2+ had no impact on the basal and hypotonic current, 50 µM BAPTA decreased the basal and hypotonic current, and almost abolished ENaC hypo shock current  than the time needed to reach maximum [Ca2+]i. In fact, transepithelial current was already decreasing when [Ca2+]i reached its maximum, indicating that a low increase in [Ca2+]i is sufficient to modulate the current rise. At higher concentrations, Ca2+ could decrease ENaC activity. This hypothesis could be in agreement with several reports showing that [Ca2+]i in μM concentrations inhibits ENaC open probability [2+]i after 20% hypotonic shock, Ca2+ inhibition of ENaC could explain the transient nature of the current rise detected in alveolar epithelial cells.As with i, calmodulin does not modulate transepithelial current in alveolar epithelial cells. However, calmodulin activates key elements in the current rise when Ca2+ is mobilized by exposure to hypotonic shock. Calmodulin is a protein that is very abundant in cells  and has several Ca2+-binding domains with different affinities for calcium i and to impact ENaC, we investigated if exogenous ATP, in the absence of osmotic stress, could increase this current. We found that apical, but not basolateral ATP, reduced total transepithelial current by 25%. These results are similar to what was reported in airway epithelial cells [Isc was also recorded after hypotonic shock in the presence of apyrase, an ATP scavenger. Apyrase had no impact on the current rise elicited by hypotonic shock. Altogether, the data presented here ruled out a role for ATP in the rapid transepithelial current increase after hypotonic shock.20% hypotonic shock induced significant ATP release on the apical side of the monolayers 1 min after stimulation, while no ATP was detected on the basolateral side . The kinal cells , tracheaal cells ,83 and aal cells ,11,84,85+ current modulation is the result of a complex interplay between K+ and Cl- channels, the Na+/K+-ATPase and KCC in alveolar epithelial cells (+ transport by recycling K+ and allowing apical/basolateral transcellular Cl- transport. During hypotonic shock, the rapid transepithelial current rise is dependent on basolateral Cl- as well as Ca2+/calmodulin signaling. In the context of lung pathology, a better knowledge of the channels and transporters that are expressed in alveolar epithelial cells is important for the finding of new therapeutic targets to increase Na+ transport in cardiogenic or non-cardiogenic lung oedema. A comprehension of the interactions of Na+, Cl- and K+ current and transport involved in the cell volume regulation of these cells is also important to understand the repair process following epithelial injury and for the control of cell volume changes during cell growth and apoptosis.In conclusion, our results show that transepithelial Naal cells . KCC is


This sensitization has been shown to correlate with the remodeling of the endoplasmic reticulum into large ER patches, however the mechanisms involved are not clear. Here we show that IP3 receptors within ER patches have a higher sensitivity to IP3 than those in the neighboring reticular ER. The lateral diffusion rate of IP3 receptors in both ER domains is similar, and ER patches dynamically fuse with reticular ER, arguing that IP3 receptors exchange freely between the two ER compartments. These results suggest that increasing the density of IP3 receptors through ER remodeling is sufficient to sensitize IP3-dependent Ca2+ release. Mathematical modeling supports this concept of ‘geometric sensitization’ of IP3 receptors as a population, and argues that it depends on enhanced Ca2+-dependent cooperativity at sub-threshold IP3 concentrations. This represents a novel mechanism of tuning the sensitivity of IP3 receptors through ER remodeling during meiosis.The activation of vertebrate development at fertilization relies on IP Oocyte maturation is driven by multiple kinase cascades induced downstream of the hormonal signal that releases meiotic arrest 2+ signaling is fundamental to the developmental competence of the egg 2+ signals given the broad bandwidth inherent in their temporal and spatial features. This results in an information rich signal that instructs multiple cellular events at egg activation, including the block to polyspermy and completion of meiosis The egg-to-embryo transition marks the initiation of multi-cellular organismal development and is instigated by a series of cellular events following fertilization collectively referred to as egg activation 3 receptors in different cell types cluster in a hierarchical fashion leading to Ca2+ release events of varying sizes, referred to as blips for the smallest events, presumably due to the opening of a single IP3 receptor, or puffs for larger events 2+ release waves 3 or Ca2+ levels lead to a slow aggregation of IP3 receptors into large clusters independently of ER structure, presumably through protein-protein interactions 3 receptor clustering allows for a continuum of the IP3-dependent Ca2+ release events that serve specific cellular needs.At steady state IP2+ signaling differentiation during oocyte meiosis that is conserved among different species, is the increased sensitivity of IP3-dependent Ca2+ release 2+ release through IP3 receptors is a major contributor to the fertilization-induced Ca2+ signal in vertebrates 3-dependent Ca2+ release is often described as a larger Ca2+ transient in eggs following store mobilization 3 receptors per se, since additional factors could contribute to the duration and amplitude of the Ca2+ release signal, including Ca2+ extrusion and influx, cytosolic buffering capacity, and Ca2+ reuptake into stores. A more accurate description of IP3 receptor sensitization is reflected as Ca2+ release in the egg at lower threshold IP3 concentrations that induce minimal or no release in the immature oocyte 2+ signaling pathways are modulated during meiosis, including inhibition of Ca2+ influx, internalization of the plasma membrane Ca2+-ATPase, and Ca2+ recycling between the cytoplasm and ER lumen due to prolonged opening of IP3 receptors 2+ signaling modules defines the dynamics of the fertilization-induced Ca2+ transient.An important aspect of Ca2+ release in the egg correlates with IP3 receptor phosphorylation, and is clearly dependent on the activation of the oocyte maturation kinase cascades 3 receptor phosphorylation in the sensitization of Ca2+ release 3 receptor sensitization is increased affinity of the IP3 receptor 3-dependent Ca2+ release sensitization during meiosis remains largely undefined. In addition, the number of functional IP3 receptors increases during Xenopus oocyte maturation following the disruption of annulate lamellae, which are specialized membranous structures where the activity of a sub-population of IP3 receptors is inhibited Enhanced Ca3-dependent Ca2+ release have been shown to correlate with ER remodeling in oocytes from several species Xenopus ER patches nucleate Ca2+ waves and patch appearance coincides with IP3-dependent Ca2+ release sensitization 2+ wave pacemaker localizes spatially with ER patches, and ER remodeling correlates with modulation of IP3-dependent Ca2+ release 3 in the mature egg 3-dependent Ca2+ release sensitization. IP3 receptors enriched in ER patches respond to sub-threshold IP3 concentrations that do not produce Ca2+ release in the neighboring reticular ER, showing that IP3-dependent Ca2+ release is sensitized in ER patches. We present evidence that this sensitization in ER patches is due to enhanced Ca2+-dependent cooperativity between receptors due to the increased density of IP3 receptors within an ER patch.Furthermore, alterations to IP3-dependent Ca2+ release events (Ca2+ puffs) cluster dramatically during oocyte maturation, and that this clustering is important for the mode and speed of Ca2+ wave propagation in the egg 2+ puffs in the oocyte are separate, they coalesce in the egg following maturation   . ER patc.087 µm2 , consist.087 µm2 . Further.087 µm2 , and the.087 µm2 .2+ puffs clustering during meiosis is not due to lateral diffusion of IP3 receptors, but rather to ER remodeling. This is supported by the similar width of ER patches and that of elementary Ca2+ release events (SREs) in eggs  demonstrate co-localization of individual SREs to ER patches . This results in a local Ca2+ rise over the reticular ER ROI  and the other over the reticular ER (Ret), and imaged Catrations . An init3 is uncaged at the same rate in both the patch and reticular ER ROIs, arguing that IP3 receptors in both ROIs are subject to similar IP3 concentrations over a prolonged period of time (10–20 sec). Nonetheless, only IP3 receptors in the patch ER ROI respond by releasing Ca2+. IP3 receptors in the reticular ER ROI respond only when a local Ca2+ rise occurs due to the diffusion of Ca2+ released from the ER patch ROI. This fits with Ca2+ acting as a co-agonist of the IP3 receptor, where at a constant IP3 concentration a Ca2+ rise below a certain threshold increases the probability of opening of IP3 receptors, since IP3 receptor gating exhibits a bell-shaped dependence on Ca2+3 receptors in ER patches are more sensitive when compared to those in the reticular ER. Note that the amount of Ca2+ released from the reticular ER ROI is comparable to that observed over the ER patch ROI , one over an ER patch and the other over a reticular ER region  , egg reticular ER (56.5+0.86) and egg ER patches (57.61+0.57) is also similar (n = 14–21). Given the percent immobile fraction it is possible that a sub-population of IP3 receptors localize differentially to ER compartments. This is unlikely though, because ER patches are continuously remodeling and fusing with reticular ER domains (3 receptors exists in ER patches and reticular ER domains.How can the sensitivity of IPg (FRAP) . The ratmodeling . This arcomplete  showing  domains , arguing3 receptors in the different ER sub-domains modulates their sensitivity as a population. The probability of opening (Po) of IP3 receptors exhibits a bell-shaped dependence on Ca2+3 receptors within a cluster 3 receptors in close physical proximity. Therefore, increased density of IP3 receptors within an ER patch could cooperatively increase their Po, leading to functional sensitization. To determine whether this is the case we modeled the time delay in Ca2+ release as a function of IP3 receptor cluster size, which shows an inverse relationship (3 receptors (large cluster) the shorter the time delay in responding to IP3 to gate the receptor open (3 receptor sensitization due to increased density. We then modeled the response of two IP3 receptor clusters of different sizes (20 or 1000 receptors)  . Simulat release , replica2+-dependent cooperativity within a large cluster of IP3 receptors modulates their sensitization. To alter Ca2+-dependent cooperativity we varied Ca2+ diffusion coefficient, reasoning that higher Ca2+ diffusion coefficients increase IP3 receptors cooperativity by enhancing cross-talk (Ca2+-dependent gating) between channels within a cluster. Changing the Ca2+ diffusion coefficient will not affect IP3 receptor affinity to its ligand IP3. A dose response relationship of IP3-dependent Ca2+ release as a function of IP3 concentrations shows a shift to the right as Ca2+ diffusion coefficient decreases (3-dependent Ca2+ release as the threshold IP3 concentration required to produce Ca2+ release (F/F0), in this case set as double the resting Ca2+  (2+ wave speed.To test the role of ER patches in Ca patches . Howeverhes (−4) , arguingXenopus and mouse oocytes the kinase cascade driving maturation plays an important role in the sensitization of IP3-dependent Ca2+ release Xenopus, sensitization of Ca2+ release correlates with the activation of MPF and hyper-activation of either the MAPK cascade or MPF is sufficient to sensitize Ca2+ release 3 receptor is phosphorylated at consensus MPF/MAPK sites during oocyte maturation 3 receptor affinity is a determinant of the changes observed in both elementary and global Ca2+ signaling during oocyte maturation 3 receptor phosphorylation to IP3 receptor sensitization. To address this issue, we correlated Ca2+ release sensitivity with the activation state of the kinase cascades and the formation of ER patches. To assess the sensitivity of IP3-dependent Ca2+ release a threshold uncaging pulse was empirically defined in oocytes and applied to eggs , are induced following sustained stimulation of Ca2+ signaling 3 receptor aggregations appear to be due to lateral diffusion of IP3 receptors and protein-protein interactions, and do not involve changes in ER structure 3 receptors observed during oocyte meiosis is due to restructuring of the ER. We show that ER remodeling during Xenopus oocyte meiosis requires MPF activation, consistent with results in mouse 2+ signaling differentiation during oocyte maturation The IP3-dependent Ca2+ release is sensitized and contributes to the generation of the specialized Ca2+ transient, which is essential for egg activation 3-dependent Ca2+ release 2+ waves in mouse, and as important for wave propagation in Xenopus2+ signaling remodeling during oocyte maturation suggests a has  localize to ER patches   . This isntration . We alteER patch . This iscular ER .3 receptors due to ER remodeling represents a physiological modulator of IP3 receptor function during meiosis. This mechanism could be applicable to other physiological situations as during mitosis where the ER restructures with the preponderance of the evidence arguing for a transition from a tubular to a cisternae-like structures in mitosis Therefore, ‘geometric sensitization’ of IP3 receptor function. During oocyte meiosis ER remodeling regulates both elementary and global aspects of Ca2+ signaling and is involved in sensitizing IP3 receptors. We argue that ER patch formation during meiosis increases the density of IP3 receptors thus enhancing Ca2+-dependent cooperativity. This leads to sensitization of IP3 receptors allowing them to gate open at sub-threshold IP3 concentrations. IP3 receptor sensitization is central to producing the fertilization specific Ca2+ transient, and as such for egg activation. Therefore, ER remodeling through geometric sensitization of IP3 receptors plays a central role in oocyte maturation.In summary we show that the physical structure of the ER has a profound effect on IPAll studies were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and in accordance with Qatari regulations regarding the use of animal subjects in research as outlined by the Supreme Council of Health. Studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Weill Cornell Medical College protocol #0806-759A.in vitro transcription, after linearization with NheI or NotI, capped RNAs were transcribed using T7 RNA polymerase with T7 mMESSAGE mMACHINE kit (Ambion). Xenopus oocytes were obtained as described previously 3 receptor and were kept at 18°C for 7–9 days depending on the batch of oocytes before analysis. For experiments aimed at modulating the kinase cascade differentially in the oocyte  with pinhole set at 1 airy unit (0.9 µm) unless otherwise specified. GFP and Oregon-Green signals were excited with 488 nm laser and emissions collected through a bandwidth of 492–558 nm, mCherry was excited at 561 nm and emission collected through a bandwidth of 572–699 nm. Cells were imaged in OR2 solution . Images were analyzed using ZEN 2008 and MetaMorph and figures compiled using Adobe Photoshop.For FRAP analyses pre-bleach and recovery images were scanned (pixel dwell time: 0.84 µs) at 561 nm with 1% laser power. A 25×25 pixels area was bleached at 60% laser power at reduced scanning speed (pixel dwell time: 5.31 µs). FRAP recovery curves were derived by comparison with reference unbleached area, subtracting the background, and fitting with a monoexponential decay function.2+ waves were analyzed in eggs expressing mCherry-KDEL and injected with caged-IP3  and 40 µM Oregon Green 488 BAPTA-1 hexapotassium salt (Invitrogen O6806). Cells were scanned in Ca2+-free OR2 solution using Plan-Neofluar 40×/1.3 oil DIC objective with 488 and 561 nm laser set at 2% intensity. The image size is 256×256 pixel (352×352 µm) with pinhole set at 4 µm. After 5 initial scans, an circular ROI (diameter 30 µm) was bleached 50 times with the 405 nm laser set at 20% power resulting in propagating Ca2+ wave that crossed the entire cell.CaPhospho-MAPK and phospho-Cdc2 Westerns were performed as described previously The details of the model used are included in Figure S1ER patch spatial distribution.A. ER patches form in GFP-KDEL expressing eggs. Planes at which confocal images were taken along z-stack are indicated by the matching number in the orthogonal z-section. B. Images from eggs expressing mCherry-KDEL at GVBD which were either untreated (Egg Con) or treated with Nocodazole (25 µg/ml) for 1 hr.(TIF)Click here for additional data file.Figure S2Low (A) and high (B) magnification transmission EM images of ER patches in a Xenopus egg. Patches in the low magnification image are highlight by the white contour.(TIF)Click here for additional data file.Appendix S1Computational Modeling.(DOCX)Click here for additional data file.