In contrast to the enormous progress made in mapping out postsynaptic contributions of receptors, scaffolding structures and receptor trafficking to synaptic transmission and plasticity, the small size of nerve terminals has largely precluded direct analyses of presynaptic modulation of excitability and transmitter release in central synapses. Recent studies performed in accessible synapses such as the calyx of Held, a giant axosomatic synapse in the sound localization circuit of the auditory brainstem, have provided tremendous insights into how central synapses regulate the dynamic gain range of synaptic transmission. This review will highlight experimental evidence that resolves several long-standing issues with respect to intricate interplays between the waveform of action potentials, Ca Binding of Ca2+ to the release sensor (i.e. synaptotagmin) triggers membrane fusion between synaptic vesicles (SVs) and presynaptic active zones (AZs) via engagement of SNAREs. Upon opening of the fusion pore, neurotransmitters are unloaded into the synaptic cleft and activate postsynaptic receptors to generate excitatory or inhibitory postsynaptic potentials (EPSPs or IPSPs), ultimately affecting postsynaptic spike initiation and firing patterns that encode information.Information transfer between nerve cells is largely mediated by fast chemical neurotransmission across their junctions or synapses. Electric pulses or action potentials (APs or spikes) are initiated near the soma of presynaptic neurons and propagated along the axon to the nerve terminal where voltage-gated Ca2+ influx and quantal output is primarily based on classical work in invertebrate synapses from squid, crayfish and Aplysia n, where n denotes Ca2+ cooperativity (3~5) m) for immature and mature synapses o, 1~2 mM), and provided compelling evidence that microdomain and nanodomain coupling modalities are likely distinct physical entities, and may operate in the same or different central synapses depending on their functionalities or developmental stages.Collectively, these observations demonstrate that, at the calyx of Held synapse, there is a developmental transformation from "microdomain," the coupling modality involving cooperative action of many loosely associated N- and P/Q-type VGCCs, to "nanodomain", the coupling modality in which opening of fewer tightly coupled P/Q-type VGCCs effectively gates a fusion event. Incidentally, a recent study by Bucurenciu et al. (2008) showed nanodomain coupling modality also operates at the hippocampal basket cell-granule cell inhibitory synapses . Unlike 2+ imaging and flash photolysis of caged Ca2+ indicators (i.e. DM-nitrophen) with paired patch-clamp recordings to directly measure the sensitivity of the Ca2+ sensors in the developing calyx of Held synapse  in a spatially homogeneous manner in the terminal. Such increases bypass the dynamic coupling process of Ca2+ entry through VGCCs to the release machinery during an AP or a voltage-step, and directly trigger transmitter release correlated with a known [Ca2+]i. With appropriate experimental paradigms [2+]i in individual synapses are estimated. When release rates are plotted against [Ca2+]i, their relationship can be fitted with Ca2+ binding-release models similar to the five-site kinetic model proposed by Schneggenburger and Neher (2000). Using this approach, the sensitivity of the release machinery, or the Ca2+ sensor(s), to Ca2+ can be estimated. Surprisingly, experimental data showed that Ca2+ sensitivity of the release sensor in fact decreases with synaptic maturation while molecular cooperativity (n) remains unchanged. KD values increase from ~81 (P9-P11) to ~123 μM (P16-P19)  nanodomains, promoting highly synchronized release with minimal synaptic delay, temporal jitter, and asynchronous release during transmission at mature calyx synapses. Short Ca2+ transients will also reduce the likelihood of presynaptic Ca2+ overload that can occur during prolonged high-frequency transmission, lowering the energy required for Ca2+ clearance via ion pumps and exchangers o, and the number of VGCCs per AZ, prevents establishment of a realistic model for release site topography. Nevertheless, measurements of the Ca2+ channel/domain cooperativity (m) and the EGTA induced attenuation of transmitter release provide sufficient constraints for establishing theoretical models to recapitulate the nature of Ca2+-secretion coupling at the developing calyx of Held synapse.With accumulating evidence from the developing calyx of Held synapse, it is important to further conceptualize the spatial reorganization of VGCCs and SVs in AZs to interpret experimental data. However, lack of precise knowledge of the subsynaptic distribution patterns of VGCCs, their single-channel conductance at physiological concentrations of [Ca2+ diffusion model of Naraghi and Neher (1997) allows for the calculation of the [Ca2+] at any distance from the mouth of a single VGCC  from any desired arrangement of VGCCs simply by summating their individual contributions. This freedom allows us to assume any distribution of VGCCs around a SV while being able to determine the [Ca2+] at that point. This provides a constraint governing the peak [Ca2+] experienced by the release sensor for a particular set of buffer conditions.The linearized buffered Cand Neher 997 allowis known . A parti2+] at the release sensor, we selected a simple ring of VGCCs, at equal r from the SV, as a simplified topography. It is well established that, at least in the immature synapse, VGCCs are most likely at variable distances from their SV, located in clusters or some other heterogeneous spacing arrangement [2+] arising from multiple VGCCs and sufficiently address whether VGCC-SV coupling differences can account for developmental changes in SV release at this synapse.To determine the  transient in each case  [2+ channel/domain cooperativity values for this developmental stage is around 3 [r = 23 nm to the peak [Ca2+] reproduces our experimental findings well. These models represent a simplified interpretation of release site topography which allows for simplification of the calculations required to reproduce main experimental observations including developmental increases in quantal output and the extent of EGTA-induced attenuation of EPSCs in immature and mature synapses.Given these limits, we simulated a number of possible VGCC-SV separation distances with different numbers of VGCCs contributing to the i transients until the experimental release profiles of native synapses are accurately reproduced. Simulations performed from data recorded in the mouse calyx of Held, as summarized in Figure 2+]i "seen" by the Ca2+ sensors increases from 35 to 56 μM and the SV release rate increases from ~600 SVs/ms to 1000 SVs/ms despite smaller and briefer ICa [2+]i, were also derived from the developing rat calyx of Held synapse [The rise and collapse of local Ca(but see ). Howeveeriments . Ca2+ trH) model  as the iefer ICa . Similar synapse .2+]i values (10~300 μM) described in previous work  gradients are assumed to be at steady-state around an open VGCC and buffer saturation is small, the differential equations describing buffered Ca2+ diffusion can be linearized, considerably simplifying the mathematical description of the system  carried by three Ca2+ buffers can be described by the following;At steady state, the  at any distance r from a single VGCC for N described buffer species.In these equations, B2+] as a function of r for various buffer compositions . Note that the hypothesized endogenous mobile buffer properties, recently uncovered at the immature calyx of Held, have not been included here [Using the above equations, we have shown the [Cas Figure . In addided here . This as


Changes in [Ca2+]i and ROS production were analyzed employing fluorescence techniques after loading cells with fura-2 or CM-H2DCFDA, respectively.A significant percentage of patients with pancreatitis often presents a history of excessive alcohol consumption. Nevertheless, the patho-physiological effect of ethanol on pancreatitis remains poorly understood. In the present study, we have investigated the early effects of acute ethanol exposure on CCK-8-evoked Ca2+]i. In addition, ethanol evoked reactive oxygen species generation (ROS) production. Stimulation of cells with 1 nM or 20 pM CCK-8, respectively led to a transient change and oscillations in [Ca2+]i. In the presence of ethanol a transformation of 20 pM CCK-8-evoked physiological oscillations into a single transient increase in [Ca2+]i in the majority of cells was observed. Whereas, in response to 1 nM CCK-8, the total Ca2+ mobilization was significantly increased by ethanol pre-treatment. Preincubation of cells with 1 mM 4-MP, an inhibitor of alcohol dehydrogenase, or 10 μM of the antioxidant cinnamtannin B-1, reverted the effect of ethanol on total Ca2+ mobilization evoked by 1 nM CCK-8. Cinnamtannin B-1 blocked ethanol-evoked ROS production.Ethanol, in the concentration range from 1 to 50 mM, evoked an oscillatory pattern in [Ca2+ mobilization compared to normal conditions. The actions of ethanol on CCK-8-stimulation of cells create a situation potentially leading to Ca2+ overload, which is a common pathological precursor that mediates pancreatitis.ethanol may lead, either directly or through ROS generation, to an over stimulation of pancreatic acinar cells in response to CCK-8, resulting in a higher Ca IP3 in turn releases calcium (Ca2+) from cytoplasmic stores leading to an increase in cytosolic free calcium concentration ([Ca2+]i) [2+ extrusion across the plasma membrane [2+ uptake into intracellular organelles [2+ signals.Cholecystokinin stimulates the activity of pancreatic acinar cells via generation of different second messengers in the signal cascades . The actalcium Ca+ from cymembrane  as well ganelles  contribu2+]i is an important early signal by which physiological secretagogues elicit the release of digestive enzymes from pancreatic acinar cells, being the spatiotemporal pattern of agonist-induced Ca2+ signals of critical importance for exocytosis of enzymes i has been proposed to be a shared phenomenon in acute pancreatitis that could induce trypsin premature activation i via non-oxidative metabolites i. In the present study, we have investigated the early effects of acute ethanol exposure on CCK-8-evoked Ca2+ signals in mouse pancreatic acinar cells. Our objective was to shed more light onto the effects of ethanol on the physiological action of CCK-8. The findings will contribute to a better understanding of the mechanisms by which ethanol causes pancreatic disorders.We have previously hypothesized that ethanol might exert its deleterious effects in pancreatic acinar cells, at least in part, via generation of ROS, although it was not directly demonstrated ,21. In a2+, led to a transient increase in [Ca2+]i. Ca2+ mobilization consisted of an initial increase followed by a decrease of [Ca2+]i towards a value close to the prestimulation level , 10 mM (n = 4 experiments/27 cells studied) and 50 mM ethanol (n = 5 experiments/24 cells studied), and were also observed in the absence of external Ca2+ (data not shown). The effects of ethanol on [Ca2+]i did not depict a concentration-dependent effect. In addition, it was not possible to define a pattern for Ca2+ mobilization induced by ethanol, because it varied in terms of frequency and amplitude of oscillations, independently of the concentration employed and even at the same concentration of ethanol.Perfusion of pancreatic acinar cells with ethanol in the presence of Ca2+ transport mechanisms and amylase secretion in response to CCK-8 in pancreatic acinar cells i as we have shown above. The amplitude of oscillations progressively decreased after 15 minutes in the presence of the peptide. Under these conditions, inclusion of 50 mM ethanol in the perfusion medium induced a potentiation of CCK-8-evoked oscillations in [Ca2+]i, observed as an increase in the amplitude of Ca2+ spikes , perfusion of cells with 20 pM CCK-8 evoked an oscillatory patter of [Ca2+]i over an elevated level in the 18.27% of cells . The majority of cells (81.73%) showed a single transient change in [Ca2+]i, similar to that evoked by the supramaximal concentration of 1 nM CCK-8 . [Ca2+]i reached a steady-state value of 136.30 ± 7.75 nM (n = 17), calculated 5 minutes after stimulation with the peptide. The total Ca2+ mobilization after the stimulation was estimated to be 27930 ± 2801 nM (n = 17), whereas the decay constant of [Ca2+]i to the basal value was 0.0093 ± 0.0014 Δnmol/s (n = 17).Stimulation of cells with 1 nM CCK-8 in the presence of extracellular Ca2+]i observed was higher compared to that obtained with CCK-8 alone . In the presence of ethanol, [Ca2+]i reached a significantly higher steady-state value compared to that found in the presence of CCK-8 alone .When pancreatic acinar cells were stimulated with CCK-8 (1 nM) after 1 minute preincubation in the presence of 50 mM ethanol, the peak of i to the basal value was slowed down in the presence of ethanol, compared to decay observed after application of CCK-8 alone .The total Ca2+ responses between the control experiments with CCK-8 alone and those performed in the presence of ethanol, it was of interest to investigate the changes in [Ca2+]i after inhibition of ADH. These experiments were performed on cell suspensions.Alcohol dehydrogenase (ADH) plays an important role in the metabolism of alcohol. The following set of experiments was carried out in order to evaluate whether the effects of ethanol are direct or not. In order to characterize the differences in CaUnder these experimental conditions, cells were preincubated for 30 minutes in the presence of the ADH inhibitor 4-methylpyrazole (4-MP) -25.2+]i response and the total Ca2+ mobilization were significantly increased compared to the values obtained in the presence of CCK-8 alone. However, the "steady-state level" and the rate of decay of [Ca2+]i to basal values did not differ statistically from the values obtained in the presence of CCK-8 alone . At the end of the experiment, 100 μM H2O2 was added to the cells as a control to show increased fluorescence following additional oxidation of the probe by exogenous H2O2.It has been shown that ethanol leads to ROS production in mouse pancreatic acinar cells . To confIn another set of experiments, cells were preincubated in the presence of 10 μM of the antioxidant cinnamtannin B-1. This substance is a naturally occurring A-type proanthocyanidin, which belongs to a class of polyphenols that is widely distributed throughout the plant kingdom. These compounds have long been investigated due to the antioxidant functions, which have been shown to involve radical scavenging, quenching, and enzyme-inhibiting actions .2DCFDA-derived fluorescence by a 89.43%, although it failed to completely abolish ethanol response on ROS production , did not significantly change the CCK-8-evoked peak i, evoked by a physiological concentration of CCK-8, into a sustained response usually evoked by supraphysiological concentrations of the agonist. Our results support previously reported works, which propose that ethanol-induced sensitization of the pancreatic acinar cell results in pancreatitis responses with low doses of CCK-8, that by itself does not cause pancreatitis i response evoked by CCK-8 was not blocked by the inhibition of ethanol metabolization by ADH; conversely, it was slightly increased. However, the "steady-state level" and the rate of decay of [Ca2+]i to basal values after CCK-8 stimulation in the presence of ethanol were significantly changed, i.e., [Ca2+]i recovered faster in the presence of ADH inhibition.Our results show that the effect of ethanol on the peak i recovery mechanisms. We think that ethanol itself sensitizes the pancreas to the effects of other stimuli such as the physiological agonist CCK-8, acting on CCK-8-receptor or on the Ca2+ releasing channels. This could initially have an effect on the Ca2+ releasing mechanisms, and therefore would result in a bigger Ca2+ response of the tissue. This is reflected as a transformation of the physiological oscillations in [Ca2+]i into a supraphysiological sustained change in [Ca2+]i, and by the higher peak of [Ca2+]i observed in the presence of ethanol.Our results could be explained on the basis of a direct action of ethanol onto the Ca2+]i is mainly carried out by the sarco-endoplasmic reticulum Ca2+-ATPase and the plasma membrane Ca2+-ATPase, the pumps that actively transport Ca2+ into the endoplasmic reticulum and towards the extracellular medium respectively. A delay or decreased extrusion of Ca2+ from the cytosol will lead to an increased mobilization of Ca2+ compared to normal conditions. This can potentially lead to a cytosolic Ca2+ overload. Thus, t decrease in the generation of ethanol metabolites by inhibition of its metabolization would allow the Ca2+ transport mechanisms to effectively recover resting [Ca2+]i, as we have shown in the present study.On the other hand, the recovery of i in response to CCK-8. This further supports the hypothesis that ethanol might be having direct effects on the Ca2+-releasing mechanisms in response to CCK-8, as suggested above. However, in the presence of the antioxidant, the "steady-state level" achieved after stimulation of cells with the hormone was lowered compared to that observed in the presence of ethanol, and the rate of decay of [Ca2+]i to basal values after CCK-8 stimulation was faster than in the presence of ethanol, reaching values similar to those observed after stimulation of cells with CCK-8 alone. This supports the hypothesis that ethanol metabolization could lead to ROS generation which, in turn, would affect Ca2+ transport mechanisms.On the other side, preincubation of pancreatic acinar cells in the presence of this antioxidant did not block ethanol-induced increase in the peak of [Ca2+]i both by a direct and an indirect action, the latter mediated through ROS generation. Our findings are in agreement with others which show that ethanol can induce several cellular reactions which result in a modification of cellular red-ox status, leading to overproduction of ROS i following stimulation of cells with CCK-8. Ethanol will consequently lead to Ca2+ accumulation within the cytosol, creating a situation potentially leading to cytosolic Ca2+ overload, which is a common pathological precursor that mediates pancreatitis.In summary, our findings show that ethanol has dual effects on the physiology of intracellular Ca2+ mobilization compared to normal conditions. The actions of ethanol on CCK-8-stimulation of cells create a situation potentially leading to Ca2+ overload, which is a common pathological precursor that mediates pancreatitis.Ethanol may lead, either directly or through ROS generation, to an over stimulation of pancreatic acinar cells in response to CCK-8, resulting in a higher CaSwiss mice were used for this study. Mice were humanely handled and sacrificed in accordance to the institutional Bioethical Committee. Fura-2/AM was obtained from Invitrogen (Spain) and cinnamtannin B-1 from Alexis Corporation (Switzerland). All other materials used were obtained from Sigma Chemicals Co. (Spain).Adult male A suspension of mouse pancreatic single cells and small acini (2-4 cell clusters) was prepared by collagenase treatment following a previously described method . Briefly2, 1 mM MgCl2, 1.2 mM KH2PO4, 10 mM glucose, 10 mM Hepes, 0.01% trypsin inhibitor (soybean) and 0.2% bovine serum albumin (pH = 7.4 adjusted with NaOH). During fluorescence determinations, trypsin inhibitor and bovine serum albumin were not included in the medium. In experiments where Ca2+ free medium is indicated, Ca2+ was omitted from the extracellular solution and 250 μM EGTA was added.Throughout the preparation procedure, as well as during dye loading, we employed a physiological solution containing: 130 mM NaCl, 4.7 mM KCl, 1.3 mM CaClCell viability was not significantly changed by the experimental treatments as assayed by trypan blue exclusion test and was greater than 95%. After loading with fluorescent dyes, cells were kept at 4°C until use and the experiments were performed within the next 4 hours.2+-dependent fluorescence signals was carried out by placing small aliquots of cell suspensions into a coverslip mounted on an experimental perfusion chamber, and placed on the stage of an epifluorescence inverted microscope . The cells were continuously superfused with Na-Hepes buffer. For fluorescence change determination, an image acquisition and analysis system for video microscopy was employed . Cells were alternatively excited with light from a xenon arc lamp passed through a high-speed monochromator  at 340/380 nm. Fluorescence emission at 505 nm was detected using a cooled digital CCD camera  and recorded using dedicated software . All fluorescence measurements were made from areas considered individual cells. All stimuli were dissolved in the extracellular Na-Hepes buffer and applied directly to the cells in the perfusion chamber. Experiments were performed at room temperature (23-25°C).Freshly isolated mouse pancreatic acinar cells were loaded with fura-2- acetoxymethyl ester (4 μM) at room temperature (23-25°C) for 40 min as previously established . In sing6 cells/ml) of dye-loaded pancreatic acinar cells into a cuvette in a fluorescence spectrophotometer . Cells were continuously stirred and experiments were performed at 37°C. All stimuli were dissolved in the extracellular Na-Hepes buffer, with or without Ca2+, and were directly added into the cuvette to yield the final concentration required.In cell suspensions experiments, monitorization of changes in fluorescence signals was performed by placing 2 ml aliquots  the peak [Ca2+]i response, which was calculated at the maximal value achieved after stimulation of cells with the agonist; (2) a "steady-state level", calculated 5 minutes after application of the stimulus; (3) the total Ca2+ mobilization after the stimulus was estimated as the integral of the rise in [Ca2+]i over the basal values for 8 minutes after addition of CCK-8; (4) and, finally, the rate of decay of [Ca2+]i to basal values, after application of the stimulus, which was calculated using the constant of the exponential decay. Traces were fitted to the equation Y = A × e-KX + plateau, where k is the constant of the exponential decay, X is the time and A is the span.For comparisons of the effects of CCK-8 on [Ca2+]i. In our experimental conditions, the concentration of ethanol employed did not influence fura-2-derived fluorescence. Furthermore, no apparent morphological changes of pancreatic cells were observed that could introduce errors in Ca2+ measurements in our experimental conditions (data not shown).We can discard an effect of ethanol on fura-2- emission fluorescence that could induce artefacts in the determination of [Ca2+]i are expressed in nM ± SEM (n), where n is the number of independent experiments, following the calibration method proposed by Grynkiewicz et al. i: cytosolic free Ca2+ concentration; CCK-8: cholecystokinin octapeptide; CM-H2DCFDA: 5-(and-6)-chloromethyl-20,70-dichlorodihydrofluorescein diacetate acetyl ester; EGTA: ethylene glycol-bis(2-aminoethylether)-N,N,N'N'-tetraacetic acid; ER: endoplasmic reticulum; Fura-2/AM: fura-2 acetoxymethyl ester; IP3: inositol 1,4,5-trisphosphate; 4-MP: 4-methylpyrazole; ROS: reactive oxygen species.[Ca2+]i determinations in single cell. ACV carried out the experiments for [Ca2+]i and ROS determinations in cells suspensions. AG conceived the study, its design and wrote the manuscript. GMS collaborated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.MFS carried out the experiments for [Ca


Whereas the involvement of IP3-induced Ca2+ signalling (IICS) in the physiological activity of numerous cell types is well documented, the role of IICS in cancer cells is still largely unknown. Our purpose was to characterize the role of IICS in the control of growth of the estrogen-dependent human breast cancer epithelial cell line MCF-7 and its potential regulation by 17β-estradiol (E2).Ca3 receptor (IP3R) inhibitors caffeine, 2-APB and xestospongin C (XeC) inhibited the growth of MCF-7 stimulated by 5% foetal calf serum or 10 nM E2. Furthermore, Ca2+ imaging experiments showed that serum and E2 were able to trigger, in a Ca2+-free medium, an elevation of internal Ca2+ in a 2-APB and XeC-sensitive manner. Moreover, the phospholipase C (PLC) inhibitor U-73122 was able to prevent intracellular Ca2+ elevation in response to serum, whereas the inactive analogue U-73343 was ineffective. Western-blotting experiments revealed that the 3 types of IP3Rs are expressed in MCF-7 cells and that a 48 hours treatment with 10 nM E2 elevated IP3R3 protein expression level in an ICI-182,780 (a specific estrogen receptor antagonist)-dependent manner. Furthermore, IP3R3 silencing by the use of specific small interfering RNA was responsible for a drastic modification of the temporal feature of IICS, independently of a modification of the sensitivity of the Ca2+ release process and acted to counteract the proliferative effect of 10 nM E2.Our results show that the IP3R3 expression by E2 is involved in this effect.Altogether, our results are in favour of a role of IICS in MCF-7 cell growth, and we hypothesize that the regulation of IP IP3R protein subtypes  are encoded by three different genes in mammals but share high similarity in their primary sequences and are expressed to varying degrees in various cell types [2+ signals when expressed alone [2+ signal determine its intracellular function [2+ signal [Call death -3. Numeray ensue . In the osphates  and antaosphates  or Ca2+ + influx  arrest t+ influx . Furtherus cells -11. Freell types . Even thed alone ,14. Tempfunction . For exa+ signal ,17.2) is a key growth regulator involved in normal breast development where it stimulates growth of the ductal system. However, clinical and experimental data have clearly established that exposure to estrogens is the leading cause of sporadic female breast cancer [2 have traditionally been considered to be based on its interaction with intracellular estrogen receptors [2 has also been shown to be involved in cellular responses that do not require the stimulation of estrogen receptors  [3-induced Ca2+ signalling (IICS) in the physiological activity of numerous cell types is well documented, the role of IICS in cancer cells is still largely unknown. In the case of breast cancer cells, only a few studies have described the Ca2+ release mechanisms [2 and anti-estrogens [The steroid hormone 17β-estradiol (Et cancer . The preeceptors . These aeceptors -23. Furtpathway) ,25. Wherchanisms  and theistrogens -30.2 in regulating IICS in the estrogen-dependent MCF-7 cell line. We show that the expression level of IP3R3 is controlled by E2 in an estrogen receptor-dependent manner and that the growth of MCF-7 cells induced by E2 is sensitive to pharmacological inhibitors of IP3Rs. Furthermore, IP3R3 gene silencing using specific siRNA diminishes E2-induced cell growth and changed the temporal feature of ATP-induced intracellular Ca2+ signals. We conclude that IICS is involved in E2-induced MCF-7 cell growth and that the regulation of IP3R3 expression could explain this effect.In this study, we investigate the potential involvement of E2+ and these cells are able to elicit intracellular Ca2+ signals in response of multiple ligands [2+-free medium, serum was able to trigger Ca2+ release  by the known IP3R antagonist 2-APB  similarly inhibited serum-triggered IICS by 64.7 ± 11.7% . The latter completely suppressed the serum-induced intracellular Ca2+ release  evoked Ca2+ signals  were tested on 5-FCS- and E2-induced cell growth. As could be expected, cell counting using the trypan-blue exclusion method showed that 5-FCS was able to increase MCF-7 cell growth compared to a starvation culture medium . Figure 2 . The cell growth in the presence of caffeine alone was not statistically different from that in 0-FCS . The effect of 2-APB could not be adequately tested as it triggered even under control conditions a significant elevation in cell death (>55%). Furthermore, as caffeine and 2-APB have been described as non-specific IP3R antagonists, we performed experiments using XeC, another IP3R inhibitor, in order to increase the weight of evidence in support of a role for IICS in stimulating cell proliferation. A concentration of 10 μM XeC was chosen in order to limit potential side effects of the compound during a long-term treatment; this concentration was verified to be sufficient to inhibit E2-induced Ca2+ release (data not shown). Figure 2 and 5-FCS, respectively.In order to verify the possible involvement of IICS in MCF-7 cell growth, pharmacological inhibitors of IPm Figure . Interesh Figure . We test2-induced cell growth. Addition of caffeine (500 μM) at the beginning of the experiment or at intermediate time  at 36 h and 47.9 ± 8.4%  at 72 h  higher compared to the condition in which caffeine was still present in the culture medium  for IP3R2 and 140.2 ± 11.3%  for IP3R3  or a control siRNA (siC). At 24, 48 and 72 h post-transfection, siR3 reduced the IP3R3 mRNA by 68.7 ± 7.4% , 69.8 ± 6.3%  and 66.2 ± 5.7% , respectively. Also at the protein level, siR3 induced a similar decrease in IP3R3 expression , 88.6 ± 6.3%  and 75.4 ± 10.8%  at 24, 48 and 72 h respectively .In order to further investigate the involvement of IPn Figure . The expy Figure . Treatme2-induced MCF-7 cell growth. Cells were transfected with either siC or siR3 and seeded in 5-FCS for 18 hours. After that, cells were starved for 6 h before being stimulated with 10 nM E2 for 48 hours. Whereas siR3 did not significantly modify the growth of MCF-7 cells in the absence of E2 , it appeared that siR3 was able to inhibit E2-induced increase in cell number by 63.2 ± 6.7%  in siC-transfected cells and only by 32.9 ± 12.6%  in siR3-transfected cells. Taken together, this strongly suggests that the Ca2+ signal resulting from the activation of IP3R3 is at least partly involved in the proliferative effect of E2.Subsequently, we tested the effect of siR3 on the E3R3 silencing on the intracellular Ca2+ signals in response to ATP. ATP delivers very reproducible, standardized Ca2+ signals that could be easily analyzed and quantified; furthermore, ATP is also able to stimulate MCF-7 proliferation [2+-free medium after 72 hours of transfection with either siC or siR3 are depicted in Figure 3R3 levels provoked a drastic change in the characteristics of the ATP-induced Ca2+ signal. Indeed, Ca2+ signals changed from a plateau-type of response to a sinusoidal oscillatory-shaped signal. Statistical analysis revealed that at 24, 48 and 72 h post-transfection, the number of oscillating cells in response to ATP was much higher in siR3-transfected cells compared to siC-transfected cells  for each trace. Figure 2+ signals measured at 72 h post-transfection in both conditions after perfusion with 5 μM ATP in a Ca2+-free medium. Superimposition of both traces clearly suggests that despite the pattern of the Ca2+ signal was changed, the global amount of Ca2+ released into the cell remained the same. This latter point was confirmed following statistical analysis. Indeed, the mean AUC values for Ca2+ signals elicited in siC-transfected cells versus siR3-transfected cells were not statistically different  is the same for both types of cells. Furthermore, the percentage of responding cells is unchanged in siC vs siR3-transfected cells  is significantly diminished by 18.4 ± 5.7%  [3R3 functions as an anti Ca2+-oscillatory unit in DT-40 cells [3R3 levels in MCF-7 cells drastically changed the characteristics of the Ca2+ signals. Importantly, no changes occurred in the sensitivity of the Ca2+ signals to ATP after down-regulation of IP3R3, which is probably due to the expression of IP3R1 and IP3R2 which have a higher affinity for IP3 [2+ release was observed, but the largest difference was the profound increase  in the number of cells displaying a pattern of sinusoidal Ca2+ oscillations instead of a plateau phase. This fully supports the hypothesis that E2 partly controls MCF-7 cell growth by encoding specific Ca2+ signals through the IP3R3. A relation between Ca2+ oscillation frequency and transcription factors has already been shown [3R3 silencing could be related to the modification of the temporal feature of the Ca2+ signal.We showed in this study that MCF-7 cells express the 3 IPferation . In the feration  and mousferation . It has mination . In the poptosis ,30 and ppoptosis . Interesferation . As the e and E2 -43 or gle and E2 . Furthercle cell . Other sllatory) . For exa40 cells  and in H40 cells . In full for IP3 . At maxien shown ,17. It c3R3 silencing show that basal MCF-7 proliferation in serum-deprived medium is not affected. This is due to the fact that in those conditions there is no factor present that can trigger internal Ca2+ release. We have previously observed [2+-dependent K+ channel BK, could impair the proliferation induced by E2 but not in basal conditions (0 FCS). Indeed, in the latter condition, basal Ca2+ activity is probably too low to ensure the activation of the BK channels while after induction with E2, the internal Ca2+ level is sufficiently elevated to activate these channels and therefore to uncover the sensitivity to iberiotoxin.Our results obtained following IPobserved  a simila3R3 expression and cellular proliferation, though IP3R3 has also been previously implicated in cell death [3Rs and IICS can convey and/or enhance cell death signals by allowing for an efficient Ca2+ shuttling between ER and mitochondria leading to mitochondrial Ca2+ overload  a stimulation of cell metabolism and ATP production by a low to intermediate flux of Ca2+ to the mitochondria, large enough to stimulate the Ca2+-sensitive mitochondrial dehydrogenases but not to cause detrimental effects and (2) Ca2+ signals with a temporal pattern able to activate more efficiently transcription factors acting on the expression of genes involved in proliferation.These various mechanisms may explain why, even though IP2 is sensitive to pharmacological inhibitors of IP3Rs. Moreover, E2 treatment induced an upregulation of IP3R3 in an estrogen receptor-dependent manner while IP3R3 gene silencing affected both intracellular Ca2+ signalling and cellular proliferation. Taken together, these results are suggestive in MCF-7 cells for a regulation of cell growth by specific Ca2+ signals, but further work is needed to elucidate the precise mechanism(s) involved.In conclusion, our observations indicate that the growth of MCF-7 human breast cancer cells induced by E® HTB-22™, LGC Promochem) and cells were used for a maximum of 10 passages after receipt or resuscitation. Cells were grown in an atmosphere saturated with humidity at 37°C and 5% CO2 in Eagle's Minimal Essential Medium supplemented with 2 mM L-glutamine, 0.06% HEPES Buffer and a mixture of penicillin (50 UI/ml)/streptomycin (50 μg/ml). In addition, the culture medium was either supplemented with 5% FCS (5-FCS) or not supplemented with FCS (0-FCS) and was renewed every two days.The MCF-7 cell line was purchased from the American Type Culture Collection (ATCC2 (10 nM) or 5-FCS, alone or in association with caffeine (500 μM) or 2-APB (75 μM). After 2 days of treatment, the cell number was determined by trypan blue exclusion method. The counts were replicated six times and the experiments were repeated at least three times. Alternatively, cell viability was measured by the use of the 3--2,5-diphenyltetrazolium bromide reduction assay (MTT-assay) [4 cells per well and allowed to grow for 48 h. Cells underwent a 24 h starvation in 0-FCS and were then stimulated with 10 nM E2 in the presence or the absence of caffeine (500 μM) for 36 h and 72 h. On the day of assay, treatment medium was replaced with medium containing 0.5 mg/ml of MTT and incubated for 1 h at 37°C. Medium was then aspirated off and 800 μl of DMSO was added to solubilise crystals. The optical density of each sample was read on a microplate reader  at 570 nm against a blank prepared from cell-free wells.For cell growth assays, 75,000 MCF-7 cells were seeded in Petri dishes (diameter 60 mm) in 5-FCS. After 48 h, cells were incubated in a phenol-red-free 0-FCS for a 24 h starvation period. Cells were then washed and incubated with ET-assay) . In brie4 cells per dish on glass cover slips and cells were loaded for 1 h with Fura-2/AM  at 37°C in a CO2 incubator and subsequently washed three times with the dye-free recording solution. The cover slip was then transferred into a perfusion chamber of a Zeiss inverted microscope equipped for fluorescence. Fluorescence was excited at 340 and 380 nm alternately, using a monochromator , and captured by a Cool SNAP HQ camera (Princeton Instruments) after filtration through a long-pass filter (510 nm). Background fluorescence was determined at 340 and 380 nm from an area of the cover slip free of cells. These values were routinely subtracted. Metafluor software  was used for acquisition and analysis. All recordings were carried out at room temperature . The cells were continuously perfused with the saline solution, and chemicals were added via the perfusion system. The flow rate of the whole-chamber perfusion system was set at 10 ml/min, and the chamber volume was 1 ml. Recording solution had the following composition (in mM): NaCl 145, KCl 5, CaCl2 2, MgCl2 1, and Hepes 10 at pH 7.4 (NaOH). In experiments where Ca2+-free solution was used, Ca2+ was omitted and EGTA (1 mM) was added to the solution. The "Area Under Curve" (AUC) was calculated using OriginPro v.8 and permitted to measure the global amount of Ca2+ released into the cells following stimulation.MCF-7 cells were cultured at 5.102 on the expression level of the various IP3R isoforms, microsomal preparations from MCF-7 cells were performed according to an earlier published procedure [3R1  [3R2  [3R3 . Following primary antibody probing, the membranes were washed three times with TBS-T and incubated for 1 h at RT with the respective secondary horseradish-peroxidase-conjugated antibodies (Santa Cruz): anti-mouse  was used for the detection of IP3R3, anti-rabbit  for IP3R1 and IP3R2 and anti-goat  was used for the detection of β-actin. Proteins were visualized using the enhanced chemiluminescence system (Amersham) on a Chemidoc Apparatus and quantification was realized using Quantity One software.Cells were washed twice with phosphate-buffered saline (PBS) and lysed by addition of RIPA buffer (200 μl/60 mm dish) containing protease inhibitor cocktail . After 30-45 min incubation on ice, the cell lysates were scraped off the Petri dish and transferred to 1.5 ml tubes. The extracts were then centrifuged at 10,000 × g for 10 min at 4°C in a table-top centrifuge and the supernatants were saved for analysis. For the determination of the effect of Erocedure . Protein1/1,000) ; rabbit 2, 1/30) ; purifie3Rs on β-actin reference gene.Total RNA from MCF-7 cells was extracted by the Trizol-phenol-chloroform (Sigma Aldrich) procedure, including DNAse I treatment . Total RNA was then reverse-transcribed into cDNA using oligodT primers and MultiScribe™ reverse transcriptase (Applied Biosystems). PCR experiments were carried out on an iCycler thermal cycler (Bio-rad) using Taq DNA polymerase (Invitrogen). PCR products were analysed by electrophoresis on 1.5% agarose gel and visualized by ethidium bromide staining. Finally, PCR products were quantified using Quantity One software and expressed as the ratio of IP® apparatus according to the manufacturer's instructions. Briefly, 2.106 cells were transfected with 2 μg siRNA directed against the human IP3R3 mRNA sequence  or control siRNA . After the electroporation (program E-14), 500 μl of prewarmed culture medium were added and cells were transferred to a 1.5 ml tube and placed at 37°C for 15 min in a CO2 incubator. After that, cells were seeded in Petri dishes (diameter 60 mm). 18 h later, cells were treated for 6 h in 0-FCS and were then stimulated with E2 (10 nM) for 48 h.MCF-7 cells were collected after trypinization and submitted to electroporation using a Gene PulserResults were expressed as mean ± S.E.M. Experiments were repeated at least three times. The Student's t-test was used to compare treatment means with control means. Statistical significance is indicated in the figures .All the products were from Sigma (France) unless otherwise stated. Final concentrations were obtained by appropriate dilution of stock solutions so that the solvent never exceeded 1/1,000.3: inositol 1,4,5-trisphosphate; IP3R: IP3 receptor; IICS: IP3-induced Ca2+ signalling; E2: 17β-estradiol; 2-APB: 2-aminoethoxydiphenyl borate; XeC: xestospongin C; FCS: foetal calf serum; 5-FCS: culture medium containing 5% FCS; 0-FCS: serum-deprived culture medium; ER: endoplasmic reticulum.IPThe authors declare that they have no competing interests.CS performed experiments and analysed the data. JBP and HOA participated in the design of the study and helped to draft the manuscript. FM conceived and performed experiments, analysed the data and drafted the article. All authors read and approved the paper.


We found that these domains of localized Ca2+ entry become dispersed by long-term (72 hr), but not by acute (2 hr), exposure to palmitate. Importantly, the release competence of the granules was not affected by palmitate. Thus, the location rather than the magnitude of the Ca2+ increase determines its capacity to evoke exocytosis. In both mouse and human islets, the palmitate-induced secretion defect was reversed when the β cell action potential was pharmacologically prolonged.Long-term (72 hr) exposure of pancreatic islets to palmitate inhibited glucose-induced insulin secretion by >50% with first- and second-phase secretion being equally suppressed. This inhibition correlated with the selective impairment of exocytosis evoked by brief  depolarizations, whereas that evoked by long (∼250 ms) stimuli was unaffected. Under normal conditions, Ca Type 2 diabetes mellitus is characterized by insufficient insulin secretion in response to elevations of plasma glucose . This de+ channels (KATP channels). This causes membrane depolarization and the opening of voltage-dependent Ca2+ channels. The resultant increase in [Ca2+]i triggers the exocytosis of insulin-containing granules. This secretory response consists of a rapid but transient first phase followed a slowly developing second phase  an increased cytoplasmic ATP/ADP ratio and closure of ATP-regulated Knd phase . Exocytochannels . In a prchannels , we repoThe effects of long-term palmitate treatment on the amounts of mRNA encoding key proteins involved in granule priming and exocytosis were evaluated by quantitative PCR  palmitate . Pancreatic β cells isolated from islets incubated for 72 hr in the presence or absence of palmitate were labeled with the 14-carbon lipids fluorescently tagged with individual BODIPY fluorophores. No palmitate-induced changes in membrane fluidity were detected by this method .2+]i close to the voltage-gated Ca2+-channels  of the Ca2+ signal in the entire cell footprint; a greater spatial heterogeneity will produce a higher CV. This analysis revealed that depolarization-evoked [Ca2+]i (labeled “0”) is significantly less compartmentalized in palmitate-treated β cells than in control cells (2+]i prior to depolarization (labeled “−70”) was not different between control and palmitate-treated cells. The latter finding argues that the observed effect on depolarization-evoked Ca2+ influx cannot be attributed to differences in plasma membrane adherence to the coverslip or dye infusion. We ascertained that the amount of Ca2+-sensitive dye infused into the cell did not differ between the two conditions . The redistribution of [Ca2+]i was only observed in response to long-term palmitate exposure and in β cells that were first cultured under control conditions for 72 hr and then exposed to palmitate for 2 hr, the measured CV remained the same as in control cells both at −70 and 0 mV.Exocytosis of insulin granules depends on localized increases in i and ΔCm in β cells expressing a fluorescently tagged variant of the granule protein IAPP  and 432 ± 82 fF (n = 8) in control cells and palmitate-treated cells, respectively. In the fluorescent imaging experiments, 20%–25% of the docked granules were released during the train in both control and palmitate-treated cells  of granules that are molecularly similar to the RRP but geographically separated from sites of Ca2+ entry  in these regions were compared to that of adjacent stationary granules (purple circles). In control cells, granules that underwent exocytosis had an ∼45% larger increase in local [Ca2+]i than those that were not released  and SRP (further away from the Ca2+ channels) granules, whereas palmitate-treated cells contain only SRP granules. Exocytosis of these two pools of granules should be differentially influenced by Ca2+ buffering; exocytosis of granules situated far away from the Ca2+ channels should be inhibited by the slow Ca2+ buffer EGTA, whereas granules residing in close proximity to the Ca2+ channels should be less affected  beyond that attained during the first 50 ms that reflects the intracellular diffusion of Ca2+. By contrast, the microdomains did not expand with pulse duration when the cells were infused with 10 mM EGTA . We conclude that the time- and diffusion-dependent expansion of the [Ca2+]i domain explains why palmitate does not inhibit exocytosis elicited by longer depolarizations at low intracellular Ca2+ buffering.In cells containing 50 μM EGTA, exocytosis evoked by 500 ms depolarizations was unaffected by palmitate, whereas the response to the 50 ms depolarization was reduced by 68% after palmitate treatment A. When i2+]i produced by photoliberation of “caged” Ca2+ from Ca2+—NP-EGTA in control and palmitate-treated cells (2+]i within the cell (cf. 2+ elicited robust exocytotic responses. 2+ (flash). During the first three pulses, exocytosis was inhibited by >65%, whereas the responses elicited by uncaging of [Ca2+]i were almost identical. The increases in ΔCm elicited by photorelease could be reasonably fit by double exponentials with time constants of 23 ± 5 ms and 497 ± 213 ms (n = 10) in control cells and 26 ± 4 ms and 920 ± 290 ms (n = 11) in palmitate-treated cells. The corresponding increases in ΔCm for the rapid and slow components of exocytosis averaged 85 ± 5 fF and 130 ± 38 fF in control cells and 91 ± 3 and 112 ± 39 fF in palmitate-treated cells. We conclude that pretreatment with palmitate inhibits insulin exocytosis by affecting the location of Ca2+ influx rather than the Ca2+ sensitivity of exocytosis.We tested this idea by comparing exocytosis evoked by brief voltage-clamp pulses with that elicited by a global increase in i microdomains should rescue insulin secretion. To test this idea, we used the K+ channel blocker tetraethylammonium (TEA), which markedly prolongs the β cell action potential  of its control value. Basal secretion in 1 mM glucose was marginally stimulated in palmitate-treated cells compared to control islets, and TEA had only a weak effect under these conditions. Long-term exposure to palmitate also reduced glucose-induced insulin secretion from human islets  action potential-like depolarizations. Although the β cell contains a large number of release-competent granules, only those in close proximity to the voltage-gated Ca2+ channels are released during β cell electrical activity. We have previously proposed that Ca2+ channels are physically tethered to the insulin granules   . Once thgranules B. Importansients B. First-2+-dependent exocytosis, develops within 72 hr and occurs before the other functional abnormalities are detectable.We emphasize that our data do not exclude the possibility that other adverse effects  with the exocytotic core complex consisting of syntaxin-1, synaptotagmin, and SNAP-25. In mouse insulinoma MIN6 cells, long-term exposure to palmitate alters the expression of several proteins involved in β cell exocytosis  exposure to palmitate , 2.5 CaCl2, 0.5 mg/ml BSA, and D-glucose as indicated. The chamber temperature was kept at 37°C. Samples were collected in 96-deep-well format plates every 30 s and kept at −20°C pending insulin measurements.Islet perifusions were performed using an in-house-designed system with four temperature-controlled chambers. Fifty size-matched islets were transferred onto a 1 μm nylon mesh cut to the size of the chamber, which was filled and perfused continuously with extracellular medium containing (in mM) 120 NaCl, 4.7 KCl, 25 NaHCOExpression of IAPP-mCherry was achieved by adenoviral infection using an in-house vector modified to express the mCherry fluorophore. Homologous recombination of the vector into the adenovirus was performed by Vector BioLabs .2, 2.6 CaCl2, 5 HEPES, and 5 D-glucose at pH 7.4 (adjusted with NaOH). The intracellular solution consisted of (in mM) 125 Cs-glutamate, 10 CsCl, 10 NaCl, 1 MgCl2, 0.05 EGTA, 3 Mg-ATP, 0.1 cAMP, 5 HEPES (pH 7.2 using CsOH), and 0.03 Oregon Green 6F. In Figures 2+ buffering was increased by adding 10 mM EGTA (with iso-osmolar reduction of 10 Cs-glutamate).The standard extracellular medium for imaging and electrophysiological measurements consisted of (in mM) 138 NaCl, 5.6 KCl, 1.2 MgCl2+ was initiated using a JML-C2 flashlamp . The pipette solution consisted of (mM) 110 K-glutamate, 10 KCl, 20 NaCl, 1 MgCl2, 25 HEPES (pH 7.1 with KOH), 3 MgATP, 3 NP-EGTA , and 2 CaCl2. The low-affinity Ca2+ indicator Oregon Green 6F was included in this medium at a concentration of 0.03 mM.Photorelease of “caged” CaAll solutions were osmotically balanced to between 290 and 310 mOsm. Experiments were performed at +32°C (electrophysiology) or +37°C (hormone release).2+ dye Oregon Green 6F , and a 561 nm line of a steady-state diode laser was used to excite IAPP-mCherry. Two excitation ports with separate focal pathways were used to make independent adjustments of each laser. The emission pathway for both fluorescent reporters was imaged simultaneously with an image splitter  creating side-by-side images of both emission wavelengths on the chip of a charge-coupled device (CCD) camera . All images were acquired using CellˆR software  and later analyzed with MetaMorph software version 7.53 . Images were acquired at 20–30 Hz with 10 ms excitation exposure. To correct for alignment between the two channels, live images were adjusted by hand to align the emission pathway to overlap in both channels by imaging 200 nm tetraspeck beads . This correction was done on a daily basis.Images were obtained with a customized Olympus IX-81 total internal reflection fluorescence (TIRF) microscope equipped with a 150×/1.45 Apo lens . The 488 nm line of an argon ion laser  was used to excite the Ca2SO4  in methanol. Total phospholipid and triglyceride fatty acids were eluted into hexane. Separation and quantification of the fatty acid methyl esters (FAMEs) from islet total phospholipids and triglyceride were achieved using gas chromatography  fitted with a 30 m × 0.53 mm (film thickness 1 μm) capillary column (RTX-Wax). Individual fatty acid peaks were identified by reference with known FAMEs. Fatty acid compositions  in these peaks were then determined.Total phospholipids and triglycerides were separated by spotting lipid extracts onto silica gel 60 (Merck) thin-layer chromatography plates using hexane:diethyl ether:acetic acid (85:15:1 vol/vol/vol) as the solvent . Lipid b13C] enrichment was determined by GC-mass spectrometry (GC-MS) using a 5890 GC coupled to a 5973N MSD . The GC was equipped with a 30 m × 0.25 mm (film thickness 0.25 μm) capillary column (rTX-5) and ions with mass-to-charge ratios (m/z) of 270  and 286  were determined by selected ion monitoring. Dwell time was 100 ms. Tracer-tracee ratios (TTRs) for [U-13C]palmitate (M+16)/(M+0) were multiplied by the corresponding PL or TG fatty acid concentrations to give islet tracer concentrations.[U-Measurements of the rate of exocytosis E as well2+ influx  around exocytosing granules were measured within 1 μm diameter circular regions centered on the location of all identified exocytotic events within a cell . These localized increases were then compared to increases of [Ca2+]i within adjacent, but nonoverlapping (>1.5 μm but <2 μm) regions around granules that did not undergo exocytosis. To correct for irregularities in the evanescent field and amount of membrane within a measured region, all measurements of [Ca2+]i increases (ΔF) were normalized to basal fluorescence (F) levels prior to depolarization for each region.For measurements of Ca+ influx , the staData are presented as mean values ± SEM for the indicated number of experiments (n). Statistical significance measurements were performed by Student's t test, with Bonferroni corrections in


We have identified a novel mAb, SG31, which recognizes the mouse integrin α4 subunit. Unlike the epitopes recognized by other anti-α4 antibodies, the SG31 epitope is expressed on subpopulations of thymocytes and peripheral T cells. After manganese ion, but not phorbol myristic acetate activation, the epitope is induced and expressed on the majority of peripheral T cells. These data suggest that the SG31 epitope is an activation epitope and that manganese ions activate α4 integrins by inducing a conformational change. Comparative flow cytometric analyses showed that the SG31 epitope as well as the epitope detected by other anti-α4 antibodies is expressed on all B lineage cells. In the T lineage, expression of the α4 integrins is down-regulated during thymocyte development. Although mature thymocytes still express the α4 integrins, they lose almost entirely the activation epitope recognized by SG31. In contrast, the most immature thymocytes express high levels of this epitope. In the periphery, SG31 epitope is expressed mostly by activated T cells, in contrast to the overall population of T cells that express the α4 integrins at homogenous levels. These results suggest that the activation of the α4 integrins is parallel to that of T cells.


Immunological contact urticaria is a hypersensitivity reaction that appears on the skin following contact with an eliciting substance. Recent advances in our understanding of the molecular mechanism and pathogenesis of this reaction have altered its classification, diagnosis, and treatment. We discuss classification, epidemiology, diagnosis, testing, and treatment options that are available to patients with contact urticaria. Contact urticaria (CU), first described by Fischer in 1973, is defined as the development of a wheal-and-flare reaction at a site where an external agent contacts the skin or mucosa.1 Symptoms4Contact urticaria is divided into two subtypes: Immunological CU and nonimmunological CU. Nonimmunological CU is an immediate reaction not requiring prior exposure to the substance, while immunological CU is a Type 1 IgE-mediated hypersensitivity reaction in which the patient's immune system has been previously sensitized to the eliciting substance. PreviousInitial presentation of the reaction appears within minutes to hours of exposure, affecting normal or eczematous skin with nonspecific symptoms. Local seNonimmunological contact urticaria is the most common type of CU, occurring without prior exposure to an eliciting substance as a direct result of urticants on blood vessels. SymptomsDue to the lack of response to antihistamines and the positive response to acetylsalicylic acid (ASA) and non-steroidal anti-inflammatory drugs (NSAIDS) in nonimmunological urticaria, it has been proposed that the pathophysiology involves prostaglandin release from the epidermis rather than histamine release from the mast cells, as previously assumed. In testiThe release of histamine is the major mechanism of action seen in immunological urticaria. The pathophysiology of immunological CU differs from nonimmunological CU, as it occurs due to the production of allergen-specific IgE following prior sensitization to a causative agent. After th119Many causes of immunological CU have been described, thus emphasizing the need for a thorough history as an initial step toward diagnosis. An individual presenting with urticaria should be questioned about: time and duration of onset, presence of nonspecific symptoms, wheal and angioedema distribution, family history, drug use, previous allergies and history of previous anaphylaxis, reaction to insect stings, stress, recent travel, vocation, hobbies, relationship to menstrual cycle, and effect of the urticaria on the quality of life. A study Eliciting factors for CU typically arise from occupational settings. A 12-year retrospective study in Australia reported that of the 151 patients diagnosed with CU, 143 (94.7%) were work-related. Addition5The most common cause of immunological CU by a single agent is said to be the presence of latex proteins in rubber agents such as gloves, condoms, and balloons.21 Thus, 19After a thorough history is taken, the physician should follow with a focused physical examination, ensuring that antihistamines have not been used within two days of performing the examination. Testing 3If the open testing is negative, occlusive patch testing may be performed. However, the prick test is usually performed next in the diagnosis of CU, and is considered the diagnostic method of choice if open testing is negative. The test substance is applied to the volar aspect of the forearm and pierced into the skin using a lancet. As only a small amount of allergen is introduced into the skin, there is a low risk of anaphylaxis. Reading of a prick test is usually performed in 30 minutes. Although not used as often, the scratch test is more useful for non-standardized allergens. The area of the skin is scratched with needles after the allergen has been applied. Similarly, this test is also read in 30minutes after contact with the allergen. Finally, the use test requires the patient to use the causative agent that resulted in contact allergy. For example, a person with latex-induced CU would wear latex gloves during testing.Emphasis for treatment of CU should be placed on prevention–thorough history taking and appropriate clinical testing will help determine the problematic materials. Once the diagnosis of contact urticaria is made, avoidance is almost always possible. In case of contact, patients should carry antihistamines and receive education on self-administration of subcutaneous epinephrine.Because contact with an eliciting substance may still occur, management is divided into first-, second-, and third-line therapies . In addiThese agents should infrequently be needed, as preventing further exposure remains the ideal approach.et al., described the increased effectiveness of second-generation drugs cetirizine, mizolastine, and ebastine over certain first-generation agents, in the treatment of generalized urticaria.[Slater rticaria. In additrticaria.Second-line therapies may be employed if a trial of antihistamines fails. UV radiation and photochemotherapy have been used successfully in chronic urticaria, and may be effective in CU.32 In oneTricyclic antidepressants, such as doxepin, often have potent H1 and H2 receptor antagonist activity. Doxepin 12The third-line therapy for urticaria involves the use of immunomodulatory agents such as cyclosporine and methotrexate. Long-term therapy is limited in these agents due to adverse side effects, including hypertension and renal toxicity. Kessel aWe emphasize that prevention (of further exposure) remains the desideratum of therapy. When extracutaneous organs are involved, this is especially important. Taken together, the diagnosis and prophylaxis should greatly lessen morbidity. A high priority research goal is to develop a reliable screening assay for new immunological contact urticaria.


Wyoming (EIAVWY).Equine infectious anemia virus (EIAV), a lentivirus that infects horses, has been utilized as an animal model for the study of HIV. Furthermore, the disease associated with the equine lentivirus poses a significant challenge to veterinary medicine around the world. As with all lentiviruses, EIAV has been shown to have a high propensity for genomic sequence and antigenic variation, especially in its envelope (Env) proteins. Recent studies have demonstrated Env variation to be a major determinant of vaccine efficacy, emphasizing the importance of defining natural variation among field isolates of EIAV. To date, however, published EIAV sequences have been reported only for cell-adapted strains of virus, predominantly derived from a single primary virus isolate, EIAVPA) since the widely utilized and referenced EIAVWY strain. The data demonstrated that the level of EIAVPA Env amino acid sequence variation, approximately 40% as compared to EIAVWY, is much greater than current perceptions or published reports of natural EIAV variation between field isolates. This variation did not appear to give rise to changes in the predicted secondary structure of the proteins. While the EIAVPA Env was serologically cross reactive with the Env proteins of the cell-adapted reference strain, EIAVPV (derivative of EIAVWY), the two variant Envs were shown to lack any cross neutralization by immune serum from horses infected with the respective virus strains.We present here the first characterization of the Env protein of a natural primary isolate from Pennsylvania (EIAVTaking into account the significance of serum neutralization to universal vaccine efficacy, these findings are crucial considerations towards successful EIAV vaccine development and the potential inclusion of field isolate Envs in vaccine candidates. Equine Infectious Anemia Virus (EIAV), a macrophage-tropic lentivirus of the family Retroviridae, causes a persistent and potentially fatal infection in equids and a chronic disseminated disease that is of worldwide importance in veterinary medicine . Natural and experimental infection with EIAV results in a rapid and dynamic disease process that differs markedly from the slowly progressive degenerative diseases associated with other lentiviral infections including HIV-1 infection of humans. EIAV infection can be transmitted via iatrogenic sources such as contaminated syringe needles, but is predominantly spread by blood-feeding insect vectors (mainly horseflies and deerflies). Hence, disease is most problematic in regions with warmer climates , but theEIA disease in equids emerges as a vigorous progression through three stages: acute, chronic, and inapparent. The acute and chronic stages of EIA are defined by episodes of clinical disease that are triggered by waves of viremia and distinguished by fever, anemia, thrombocytopenia, edema, diarrhea, lethargy, and various wasting signs. By 8-12 months post-infection, horses typically progress to life-long (long-term) inapparent carriers, presumably due to the development of enduring protective host immunity . These iAmong virulent lentiviruses, EIAV is unique in that greater than 90% of infected horses progress from a chronic disease state to an inapparent carrier stage despite aggressive virus replication and associated rapid antigenic variation. However, the United States Department of Agriculture (USDA) along with state animal regulatory agencies require euthanasia or strict lifelong quarantine for EIAV seropositive horses. Within the US, each state drafts its own requirements with reference to EIAV and the movement of horses as well as changes in ownership of horses. All seropositive horses must be registered with the state veterinarians and the federal Animal and Plant Health Inspection Service (APHIS) office ,10. GiveStudies of EIAV variation during persistent infection in experimentally infected equids have clearly identified characteristic changes in envelope sequences that alter viral antigenic properties, evidently as a result of immune selection -14. The in vivo/in vitro derivatives of this strain . The variant nature of the antigenicity of EIAV which has thus far obstructed successful vaccine development mandates that a larger pool of viral strains be analyzed both for consideration of pathogenesis and determination of immune correlates of protection.Despite the worldwide prevalence of EIAV infections, experimental studies to date have centered on relatively few viral isolates. Analyses of viral pathogenesis have essentially focused on a strain of EIAV termed Wyoming (isolated in North America) and its derivatives while a minority of reported studies have utilized a Chinese variant. In fact, in the last thirty years, 97% of published studies on EIAV natural isolates have been based on the Wyoming isolate directly, or on PA Env compared to published EIAV isolates indicates that the current understanding of genomic divergence is greatly underestimated. Further, functional analyses of how the gp90 variation affected antigenic specificity demonstrated that the observed genomic alterations rendered the isolate neutralization distinct to immune sera from horses experimentally infected with a Wyoming-derived virus strain . The. ThePA E4 in ELISA assays against the EIAVPV reference strain, consistent with the seropositive results also observed in AGID diagnostic assays (data not shown). Quantitative RT-PCR analysis of plasma from the donor horse revealed a viral load in the periphery of approximately 5 × 103 copies of RNA/ml plasma. To characterize the viral population of the field isolate, EIAVPA, viral RNA was pelleted from the plasma. We designed consensus primers  intravenously. The recipient horse was monitored daily for clinical signs of EIA  and blood samples were taken at regular intervals for measurement of platelets, plasma virus, and EIAV-specific serum antibodies. At approximately 70 DPI the horse experienced an acute clinical EIA episode characterized by concurrent thrombocytopenia and fever accompanied by a viremic episode of 105 copies RNA/ml  was infected with EIAVPA, we isolated viral RNA from plasma taken during the acute episode, or 74 days post infection (DPI), in the recipient horse. The majority of EIAV genomic variation occurs in the 3' half of the viral RNA that encodes the envelope, rev, and the long terminal repeat (LTR) [To characterize the viral quasispecies of EIAVat (LTR) ,4,7. Thuenv gene, specifically the gp90 region. Three other prime regions of relevance, but not of primary significance for vaccine development, namely the env gp45, S2, and rev genes, as well as the LTR were also sequenced; and the results are included in the additional files PA sequences as compared to the widely utilized Wyoming-derivative EIAVPV and the published Chinese vaccine strain  from the Wyoming strain  and neutralization (homologous and heterologous) assays. We have previously characterized a complex and lengthy maturation of immune responses to viral envelope proteins during the first six to eight months post-infection that appears to be a distinctive feature of lentiviral infections as steady state infection and host immunity levels are established [PV as demonstrated in earlier analyses (c.f. section 3.1). Hence, we initially characterized the development of serum antibodies in horse #9807 by longitudinal analyses of serum end-point titers to the Env protein of the reference strain EIAVPV. The evolution of the end-point titer of EIAV-specific serum antibodies demonstrated a characteristic development of a mature response that gradually increased throughout the first 6 months of infection, at which time the end point titer reach a steady state of approximately 104  and the original Pennsylvanian inapparent carrier (two months post transfer to #9807) to neutralize EIAVPA and EIAVPV gp90 species, as presented on otherwise common proviral constructs  was assayed in parallel as a control. Interestingly, immune sera from the experimentally EIAVPA and the EIAVPV infected horses were able to neutralize only virus containing the homologous virus gp90; there was no detectable neutralization of the virus containing the heterologous gp90 species. In contrast, however, the immune serum from the naturally infected inapparent carrier displayed neutralization activity against both the EIAVPA (1:200 titer) and EIAVPV (1:55 titer) gp90 Env species. Two-way ANOVA analyses of the neutralization results indicate a significant difference between the ability of the inapparent carrier serum to neutralize the two different gp90 Env proteins (P < 0.0001).We have reported a moderately slow development of serum neutralizing antibodies over a several month period following experimental EIAV infection of horses, with average maximum neutralization titers averaging 1:300 ,27. To ects Fig. . The neuEIAV in addition to being an animal model for HIV/AIDS studies is a potentially fatal and economically significant infectious disease of equines found in populations of horses worldwide. We have thoroughly explored the evolution of Wyoming-derivative EIAV strains -14,29-35PA isolate represents the first characterization of the Env protein of a natural primary isolate. The data reveal a much greater extent of Env variation than previously deduced from published Env sequences from a limited number of reference viral strains, all cell-adapted. The observed variation of the EIAVPA inapparent carrier population was very similar to what we have recognized in inapparent carriers from experimental infections. The level of diversity was at the same average level (data not shown) and the included phenotypic changes of a similar nature to previously observed evolution in experimental infections. While the largest amount of variation previously reported among published Env sequences indicated a maximum divergence of up to 13% variation [PA gp90 sequence reveals a divergence of about 40% from EIAVPV and other published Env sequences. In addition to the presumed effects of this extent of Env variation on vaccine development, it is important to note that the EIAVPA gp90 sequence is derived from a primary virus isolate that has never been passaged in cell culture. Thus it may be assumed that the EIAVPA Env species may in fact be more representative of natural Env populations than the currently published Env species that are derived from virus isolated by cell culture. In this regard, the EIAVPA may be considered a better candidate for vaccine development compared to other cell adapted strains of EIAV.To address this problem of Env variation and vaccine efficacy, it is essential to develop a more detailed characterization of the natural level of variation in the primary protein conferring vaccine protection, gp90. The overall level of envelope divergence observed for other common lentiviruses such as the small ruminant lentiviruses (SRLV), FIV, SIV and HIV have averaged between 10-35% -51. Presariation ,12, the PA strain would present a substantial obstacle to the development of a broadly protective vaccine against EIA.Recently we published a report detailing the specific affects of envelope sequence variation on vaccine protection . In thatPA and EIAVPV Env would predict differences in immunogenicity and antigenicity, including neutralization sensitivity. The current data indeed indicated distinct neutralization specificities for the two variant Env species. However, the current immune assays also indicated a substantial amount of cross reactive serum antibody as measured by ELISA assays, indicating common antibody epitopes in the variant Env proteins, despite the 40% divergence in gp90 amino acid sequences  identified a naturally infected, clinically inapparent, EIAV-positive horse. The horse had been EIAV seropositive for approximately 15 years at the time of sampling, as determined by repeated serum testing in the present USDA reference AGID diagnostic assay . Per PenInitial attempts to amplify and clone EIAV from the plasma of the inapparent carrier yielded insufficient levels of viral genomic PCR products (data not shown), probably due to a very low level of viremia at the time of isolation . We havePA) field isolate by transferring 5 ml of infectious plasma from the identified naturally infected donor animal. The animal was monitored daily and maintained as described previously [An outbred, mixed-breed naïve horse #9807) was infected with the Pennsylvanian EIAV . Individual clones were screened by PCR. Positive colonies were consequently grown, plasmid DNA was extracted, and clones automatically sequenced  using internal EIAV primers  were generated (refer to 2.2.2), gel-purified using Qiagen's Qiaex , and cloned individually into the pCR2.1-TOPOPV and EIAVWSU5, and the Chinese vaccine strain. The amino acid divergence calculations were determined using the Kimura distance correction.Sequences were assembled and error checked using GeneJockey II . Nucleotide and deduced amino acid sequences from each clone were aligned using the ClustalW multiple sequence alignment program from the GCG Wisconsin software package and edited manually when necessary. Alignments were performed for each genomic region with the reference strains: Wyoming, Wyoming derivative strains EIAVDistance analyses were conducted using Distance software as implemented in the GCG Wisconsin software package . PhylogeGBQ855742-GBQ855758.All sequences have been submitted to GenBank. Nucleotide accession numbers are PA envelope to those of the EIAVPV, we generated a chimeric clone in which the gp90 of the representative predominant EIAVPA clone (associated with the first disease cycle) was substituted into the proviral backbone of our reference EIAVUK3 molecular clone [UK3 backbone, which had also been digested with BtsI and Pas I . Clones were screened by sequencing using internal EIAV primers. Chimeric virus (EIAVPAgp90) was produced by transfecting a 4-μg sample of purified DNA from the chimeric proviral clone into 105 fetal equine kidney (FEK) cells as specified by the manufacturer of the GenePorter Transfection kit . The number of infectious units per ml of supernatants from transfected FEK cell cultures was then determined in a standardized infectious-center assay that uses a cell-based enzyme-linked immunosorbent assay detection system to study FEK cells [To compare the neutralization properties of the EIAVar clone  utilizinar clone . BrieflyEK cells .®/EIA kit per the manufacturer's instructions . Serum samples were also evaluated for seroreactivity by the standard Coggins AGID diagnostic assay for EIA. Serum IgG antibody reactivity to EIAV envelope glycoproteins was assayed quantitatively (end point titer) using our standard concanavalin A (ConA) ELISA procedures [PV [PAgp90 (refer to 2.6) mediated by immune sera was assessed in an indirect cell-ELISA based infectious center assay using a constant amount of infectious virus and sequential 2-fold dilutions of serum [Detection of serum antibody reactivity to the EIAV capsid protein p26 was conducted using the ViraCHEKocedures . Virus nures [PV -24 and Eof serum ,58. StatThe authors declare that they have no competing interests.JKC participated in the design and directing of the study; isolated, cloned and analyzed the sequence of the viral strains; performed immunoassays and drafted the manuscript. SB performed the viral load analyses, serology and immunoassays. BZ constructed the chimeric virus clones for the immunoassays. SJC performed all procedures on the animals as well as the daily observations on the subjects. LH performed DNA isolations and provided assistance with sequencing and blood collection from field animal. CJI directed the animal studies. RCM conceived and participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.Table S1. This file is a table depicting the primers used to amplify and sequence the primary isolate.Click here for filePA LTR populationFigure S1. Genomic sequence of EIAV. The nucleotide sequences of the EIAVPA population and reference EIAV sequences were aligned in ClustalW to the EIAV Wyoming strain. Residues that are different from Wyoming are indicated. Transcription factor recognition sequences are boxed. Bases identical to Wyoming sequence are indicated with (white square). WYO, Wyoming; PV, EIAVPV; CHVax, Chinese vaccine stain; white square, absent base.Click here for filePA S2 populationFigure S2. Genomic sequence of EIAV. The deduced amino acid sequences of the EIAVPA population and reference EIAV sequences were aligned in ClustalW to the EIAV Wyoming strain. Residues that are different from Wyoming are indicated by their single amino acid designations. Reported predicted nucleoporin motif, SH3 binding motif, and nuclear localization signal are underlined in the Wyoming strain. Residues identical to Wyoming sequence are indicated with (white square). Predicted N-myristilation signal is in red text and boxed. Predicted CK2 phosphorylation site is in pink text and boxed. PKC phosphorylation sites are in blue text and boxes. Predicted β-sheet is indicated with (white arrow) and boxed in orange. Predicted alpha helix is indicated with (white cylinder) and boxed in yellow. All structural predictions were performed using PredictProtein . WYO, Wyoming; PV, EIAVPV; CHVax, Chinese vaccine stain; white square, absent residue.Click here for filePA Rev second exon populationFigure S3. Genomic sequence of EIAV. The deduced amino acid sequences of the EIAVPA population and reference EIAV sequences were aligned in ClustalW to the EIAV Wyoming strain. Residues that are different from Wyoming are indicated by their single amino acid designations. Reported activation domain, RNA binding site and nuclear exportation signal are underlined in the Wyoming sequence and are boxed in the EIAVPA population and reference EIAV sequences. Residues identical to Wyoming sequence are indicated with (white square). Glycosylation sites are colored orange. WYO, Wyoming; PV, EIAVPV; CHVax, Chinese vaccine stain; white square, absent residue.Click here for filePA Env gp45 populationFigure S4. Genomic sequence of EIAV. The deduced amino acid sequences of the EIAVPA population and reference EIAV sequences were aligned in ClustalW to the EIAV Wyoming strain. Residues that are different from Wyoming are indicated by their single amino acid designations. The transmembrane domain is boxed. Residues identical to Wyoming sequence are indicated with (white square). Glycosylation sites are colored orange. WYO, Wyoming; PV, EIAVPV; CHVax, Chinese vaccine stain; white square, absent residue.Click here for file