ALBERT

Description: Background: Pain is one of the major comorbidities of sickle cell disease (SCD), which largely remains reliant on opioid use for analgesia. Side effects of opioids including, but not limited to fear of addiction, constipation, pruritus and opioid-induced hyperalgesia warrant the need for analgesic therapies devoid of side effects. Non-pharmacological strategies including acupuncture have been effective in pain treatment. A retrospective analysis (n=24 patients) showed that acupuncture reduced pain in a majority (75%) of SCD patients (Lu K et al., Clin J Pain. 2014). In a mouse model of SCD, electroacupuncture (EA) on conscious free-moving mice led to variable analgesic response ranging from high- (nociceptive threshold increase 〉200%), moderate- (threshold between 100~200%) to non-responders (threshold increase ≤100%) (Wang Y et al., Sci Rep. 2016). Substance P (SP), a proinflammatory vasoactive neuropeptide in the periphery and centrally and spinal activated p38 mitogen activated protein kinase (MAPK), critical mediators of chronic pain were significantly increased in sickle mice with moderate or no response to EA analgesia. Increased circulating SP has been reported in SCD patients at steady state and during chronic pain. We hypothesize that chronic pain in moderate- and non-responders is due to central sensitization mediated by SP-induced p38 MAPK phosphorylation; and that inhibiting the effect of SP and/or downstream p38 MAPK signaling would improve response to EA in moderate and non-responsive sickle mice. Methods: HbSS-BERK sickle mice expressing human sickle hemoglobin without any treatment and those showing moderate- (threshold between 100~200%) and no-response (threshold increase ≤100%); and HbAA-BERK control mice that express normal human hemoglobin A were used. All groups included mice of both genders at 5-7 months of age and were treated daily with 10 mg/kg, i.p. netupitant (antagonist of neurokinin 1 receptor, a receptor for SP), or SB203580, a p38MAPK inhibitor, with or without four sequential EA treatments (every 3rd day, frequency: 4 or 10 Hz, pulse width: 100 microsecond, duration: 30 min) at acupoint GB30. Hyperalgesia was evaluated daily before starting the inhibitor/EA treatment (baseline, BL) and after treatments throughout 12 days by determining the sensitivity to mechanical-, thermal- and deep tissue-stimuli using von Frey filaments, Hargreaves test, cold plate and grip force, respectively. Results: Sickle mice showing no- or moderate responsive to EA did not demonstrate a significant effect of netupitant or SB203580 without EA on hyperalgesia. However, co-treatment with netupitant and EA reduced mechanical, thermal and deep tissue hyperalgesia through the entire treatment, reaching significance at day 9 and/or day 12. Co-treatment with netupitant enhanced analgesia of EA by significantly decreasing mechanical hyperalgesia (p

Description: Sickle cell disease (SCD) is characterized by chronic hemolysis, inflammation, vascular dysfunction, and pain. Earlier we showed that mast cell activation contributes to neuroinflammation and pain and is accompanied by increased toll-like receptor 4 (TLR4) expression on mast cells (Vincent et al., Blood 2013), and that genetic deletion of TLR4 ameliorates neurogenic inflammation and hyperalgesia in HbSS-BERK sickle mice. Several other studies have shown increased TLR4 expression in peripheral system and its involvement in sickle pathobiology. We propose that free heme, due to hemolysis, activates TLR4 in the central nervous system in addition to peripheral activation, which further exacerbates neuroinflammation and hyperalgesia. Spinal cords of HbSS-BERK sickle mice show 3-fold mRNA transcripts for TLR4 and a 2-fold increase in hemin as compared to the spinal cords of HbAA-BERK control mice. Therefore, targeting TLR4 with pharmacological inhibitors may provide a therapeutic approach to attenuate peripheral and central inflammation and hyperalgesia. In the present study we examined the potential of pharmacological inhibition of mast cell activation, neuroinflammation and hyperalgesia in HbSS-BERK sickle mice with TLR4 inhibitor, TAK242. Sickle mice were administrated intravenously with TLR4 inhibitor TAK242 (1 mg/kg body weight/day) for 5 days. Sensory testing was performed at baseline at recruitment and periodically during the 5-day treatment and for another 8 days after concluding the treatment to evaluate mechanical hyperalgesia with von Frey filaments, thermal hyperalgesia in response to heat/cold and grip force for musculoskeletal/deep tissue hyperalgesia. Following the 5-day treatment with TAK242, release of cytokines, tryptase (marker of mast cell activation) and substance P released from skin biopsies and spinal cords were analyzed by ELISA. TAK242 significantly decreased the release of tryptase (TAK242: 5.178 ± 0.7613 pg/ml vs vehicle: 8.801 ± 0.9403 pg/ml, p = 0.0181), substance P (TAK242: 11.56 ± 1.945 pg/ml vs vehicle: 25.51 ± 4.283 pg/ml, p = 0.018), and IL-6 (TAK242: 15.59 ± 0.4541 pg/ml vs vehicle: 29.74 ± 0.8249 pg/ml, p = 0.0045) from skin biopsies, suggesting that TAK242 reduced SCD-induced mast cell activation and inflammation. TAK242 also significantly decreased substance P (TAK242: 0.7198 ± 0.0587 pg/mg vs vehicle: 0.931 ± 0.0676 pg/mg, p = 0.0462) and phosphorylation of p38/MAPK (p = 0.0184) in the spinal cord, as well as dorsal cutaneous blood flow (TAK242: 6.392 ± 0.3857 PU vs vehicle: 12.32 ± 0.5575 PU, p 〈 0.0001), indicating that TAK242 ameliorated SCD-evoked central and peripheral activation of inflammation and nociceptive mechanisms. Furthermore, TAK242 administration gradually reduced the mechanical, deep tissue, and thermal hyperalgesia upto 5-day treatment (p 〈 0.01, vs vehicle HbSS). However, discontinuation of treatment led to a gradual increase in hyperalgesia observed upto day-8 post-treatment. TAK242 also significantly decreased acute pain induced by hypoxia/reoxygenation and accelerated recovery from injury of hypoxia/reoxygenation. These data reveal the significant therapeutic effect of pharmacological inhibition of TLR4 on inflammation and hyperalgesia in sickle mice. Therapies targeting TLR4 inhibition may be potentially beneficial in ameliorating sickle pathobiology and pain. Disclosures No relevant conflicts of interest to declare.

Description: Sickle cell disease (SCD) is associated with pain, which remains a major challenge to treat. Earlier, we showed that peripheral mechanisms including mast cell activation in the skin contribute to pain in sickle mice (Vincent et al., Blood 2013). Mast cell activation in sickle mice was accompanied by a significant increase in toll-like receptor 4 (TLR4) as compared to mast cells from control mice. Since peripheral as well as central mechanisms are involved in nociception, we examined the central mechanisms underlying pain in SCD. TLR4 signaling is involved in inflammatory and neuropathic pain (Wang et al., FASEB 2013 and Hutchinson et al., Eur J Neurosci 2008). Microglial cells, the “macrophages” of the central nervous system in the spinal cord are critically involved in the development and maintenance of pain. Binding of an endogenous ligand to TLR4 is an important step in the regulation of microglial activity in pain facilitation. We hypothesized that heme, released during hemolysis in SCD, is a ligand for TLR4 expressed on spinal microglia. Methods. We isolated microglial cells from the spinal cords of HbSS-BERK (sickle) and HbAA-BERK (control) mice. To assess mitochondrial activity, we analyzed reactive oxygen species (ROS) and ATP, since increased ROS and decreased ATP are suggestive of mitochondrial dysfunction, which in turn is influenced by endoplasmic reticulum (ER) stress. ROS in the microglial cells was determined by utilizing the cell permeable reagent 2’,7’-dichlorofluorescein, which is oxidized by ROS to form a fluorescent compound, with the max excitation and emission spectra of 495 nm and 529 nm, respectively. ATP production was measured by a luminescence based assay from PerkinElmer (ATPlite). Results. Stimulation of microglia from control and sickle mice with hemin in vitro led to a several-fold increase in TLR4 gene transcripts in a time-dependent manner. Additionally, hemin induced the production of proinflammatory cytokines, TNF-α and IL-6, and ROS compared to vehicle-treated microglial cells from both sickle and control mice (p

Description: Mast cell activation in the skin contributes to inflammation, neurogenic inflammation and hyperalgesia in sickle mice (Vincent et al., Blood 2013). However, the underlying cause of mast cell activation and challenges in inhibiting mast cell activation remain unclear. Hemolysis in sickle cell disease leads to excess free heme, which contributes to endothelial activation and neutrophil extracellular trap (NET) formation. We therefore hypothesized that free heme activates mast cells and extracellular trap formation in a sickle microenvironment. We examined hemin-induced activation in mast cells from the skin of HbAA-BERK control and HbSS-BERK sickle mice. Mast cells in culture were incubated with vehicle, or I ng/ml TNF-α for 4 hours, or 40 μM hemin for 2 hours, or with hemin for 2 hours after 2 hours of priming with TNF-α to create an inflammatory sickle microenvironment. Cells were stained with cell impermeable DNA dye SYTOX orange and cell permeable dye SYTO13 to visualize the extracellular DNA fibers. Incubation with TNF-α or hemin did not show significant eruption of DNA from the cell body. However, sickle mast cells incubated with both TNF-α and hemin showed distinct DNA containing fibers exploding from the cell body with the appearance of spider web like formation similar to NETs. The length of majority of individual fibers was more than 50 μm stretching to more than 500 μM, indicative of TRAP formation. This response was blunted in mast cells from control mice. Thus, mast cells in a sickle microenvironment are primed and activated, and further priming with cytokines such as TNF-α leads to hemin-induced mast cell extracellular trap (MET) formation. Sickle mast cells express significantly higher TLR4 and FcεRI as compared to control mice (Vincent et al., Blood 2013). Therefore, we examined if silencing of FcεRI and/or inhibition of TLR4 attenuated TNF-α primed/hemin-induced MET formation. TAK242 at a dose of 1 μM but not at 0.5 μM inhibited TNF-α primed/hemin-induced MET formation, but silencing of FcεRI had no effect. Since cannabinoids, imatinib, palmitoylethanolamide (PEA) and cromolyn are known mast cell inhibitors, we examined their effect on TNF-α/hemin-induced METs. Imatinib (30 - 100 μM), a known mast cell inhibitor, showed no significant effect, while cromolyn (100 μM) led to a modest decrease in MET formation. Cannabinoid CP 55,940 and PEA at a relatively low dose of 30 μM completely blocked MET formation. Functionally, imatinib, PEA and CP 55,940 reduced the release of cytokines TNF-α and RANTES from mast cells incubated with TNF-α/hemin for 24 hours. Hemin in the presence of TNF-α stimulated sustained activation of NLRP3 inflammasome signaling. Thus, mast cells in a sickle microenvironment are activated due to a rich inflammatory cytokine milieu, via activation of the inflammasome signaling upon stimulation with hemin. The unique phenomenon of MET formation is orchestrated by a combined action of inflammatory milieu and hemin, imparting resistance to common mast cell inhibitors such as cromolyn and imatinib. This new phenomenon of MET formation identified by us may in part contribute to challenges of treating sickle cell disease and other mast cell associated pathological conditions. Disclosures No relevant conflicts of interest to declare.

Description: SerpinA3N, a serine protease inhibitor, has been shown to ameliorate neuropathic pain by inhibiting leukocyte elastase activity (Vicuña et al., Nature Med 2015, 21:518-523). Patients with sickle cell disease (SCD) have activated leukocytes and experience neuropathic pain. We observed that SerpinA3N protein expression is significantly reduced in the dorsal root ganglion (DRG) of 5 month old homozygous HbSS-BERK sickle mice, compared to age-matched HbAA-BERK control mice expressing normal human hemoglobin A (p 〈 0.05). Since HbSS-BERK sickle mice demonstrate tonic hyperalgesia (Kohli et al., Blood 2010, 116:456-465), we hypothesize that decreased expression of SerpinA3N contributes to hyperalgesia by increasing elastase activity in these mice. We examined our hypothesis by using both genders of HbSS-BERK sickle and HbAA-BERK control mice, treated intravenously with and without sivelestat, a small molecule inhibitor of elastase. Mechanical, thermal and musculoskeletal/deep tissue hyperalgesia were analyzed before (considered baseline, BL) and after treatments. Elastase activity was determined using a fluorescent elastase substrate MeOSuc-AAPV-AMC (EMD Milipore). We observed that elastase activity is significantly increased in the DRG and lungs of male and female sickle mice compared to gender and age-matched control mice (p 〈 0.05 and 〈 0.005 for female and male DRG, respectively; p 〈 0.001 and 〈 0.05 for female and male lungs, respectively). Thus, decreased SerpinA3N expression perhaps contributes to increased elastase activity in sickle mice. Treatment of sickle mice with a single dose of 2 mg/Kg sivelestat led to a significant decrease in mechanical, heat and cold hyperalgesia for up to 24 hours compared to BL (p 〈 0.05), but no effect was noted in deep tissue hyperalgesia. Increasing the single dose to 4.5 mg/Kg led to a significant decrease in mechanical and thermal as well as deep tissue hyperalgesia for up to 24 hours in both genders compared to BL or saline treated mice (p 〈 0.05). Continuation of treatment with sivelestat 4.5 mg/Kg/day for 3 days led to significantly reduced hyperalgesia for up to 6 days in both genders of mice. These findings suggest that elastase activity contributes to nociceptive mechanisms, since hyperalgesia remained reduced for up to 3 days even after discontinuation of sivelestat. Reduced hyperalgesia following 3 days of sivelestat (4.5 mg/Kg/day) treatment was accompanied by a significant decrease in elastase activity in the DRG (p 〈 0.005) and lungs (p 〈 0.05) of both genders of sickle mice compared to saline treatment, but no histopathological changes were observed in lungs, kidney, liver, spleen and heart. Activating transcription factor 3 (ATF3), a marker of neuropathic pain, is elevated in the DRG of sickle as compared to control mice (Vincent et al., Blood 2013, 122:1853-1862). Sivelestat treatment significantly reduced ATF3-immunoreactivity compared to saline treatment in the DRG of sickle mice (p 〈 0.05), suggesting a decrease in neuronal injury which underlies neuropathic pain. None of the treatments had any effect on control mice. Sivelestat treatment showed no significant effect on serum tryptase or cutaneous mast cell degranulation in sickle or control mice. Thus, sivelestat specifically inhibits elastase activity. However, both elastase and tryptase activate protease-activated receptor-2 (PAR2) in the peripheral nervous system, stimulating the release of pro-nociceptive neuropeptides and activation of transient receptor potential vanilloid (TRPV) channels, leading to hyperalgesia. Since PAR-2 and TRPV channels are activated in sickle mice, it is likely that sivelestat treatment has an inhibitory effect on these mechanisms. It has been suggested that leukocytes, including activated neutrophils, contribute to acute lung injury (a common complication of SCD) and the pathogenesis of SCD (Zhang et al., Blood 2016, 127:801-809). Our observations that decreased SerpinA3N protein expression leads to increased elastase activity in DRG and lung in sickle mice suggest that this mechanism may contribute to sickle pathobiology and pain. In Japan, sivelestat is already approved for treatment of acute lung injury. Therefore, pharmacological inhibition of elastase may offer the advantage of treating chronic pain while concomitantly ameliorating one of the pathogenic mechanisms of SCD. Disclosures No relevant conflicts of interest to declare.

Description: Background: Inflammation, neurogenic inflammation and pain remain challenging to treat in sickle cell disease (SCD). Alternative therapies including acupuncture have been used for centuries to reduce pain and ameliorate underlying pathobiology of many disorders. We examined the mechanisms underlying acupuncture therapy in sickle mice. To prevent the influence of anesthetics and constraint on the pathobiology we developed electroacupuncture (EA) treatment for awake/conscious freely moving mice to simulate treatment conditions in patients, and then examined the peripheral and central mechanisms of neuroinflammation and nociception. Methods: HbSS-BERK sickle and HbAA-BERK control mice were treated with four EA treatments (every 3rd day, frequency: 4 or 10 Hz, pulse width: 100 microsecond, duration: 30 min) at acupoint GB30. Untreated and sham-EA treated (acupuncture without electrical stimulation) were used as controls. Hyperalgesia was evaluated daily by determining mechanical threshold, deep tissue hyperalgesia and thermal hyperalgesia using von Frey filaments, grip force, and cold plate, respectively. Blood and tissues were collected for analysis after four sessions of treatment. Skin biopsies were incubated overnight and culture medium was analyzed for mast cell activation marker tryptase, and neuromodulatory marker substance P. Results: Varied analgesic response to EA treatment was observed in sickle mice. About 86% treated mice equally showed positive (〉50% pain relief) or moderate (20-30% pain relief) response and 14% were non-responsive (

Description: Cannabidiol (CBD), one of the 2 major active components of cannabis, has been shown to have beneficial effects on pain, anxiety, cognition, inflammation, oxidative stress and ischemia/reperfusion injury. Unlike tetrahydrocannabinol (THC) containing preparations, CBD does not induce psychoactive effects or catalepsy. THC leads to increased anxiety, dysphoria, and physical and mental sedation in patients when compared to CBD.The lack of psychoactive and sensorimotor effects of CBD make it a desirable therapeutic candidate with potential for pain therapy. Many unregulated over the counter preparations of CBD are available, but their purity and reliability are unknown. Epidiolex, a 99% pure extract of CBD, is approved by the FDA for treatment of seizures associated with Lennox-Gastaut syndrome (LGS) and Dravet syndrome (DS). It remains to be known whether CBD also has therapeutic potential in the treatment of sickle cell disease (SCD) which is characterized by acute and chronic pain as well as severe inflammation, oxidative stress, and organ damage. Thus, we performed a randomized double-blind placebo-controlled trial using Epidiolex to examine the effect of CBD on acute and chronic pain in homozygous HbSS BERK (sickle) and HbAA BERK (control) mice, expressing 〉99% human sickle hemoglobin and normal human hemoglobin A, respectively. For acute pain assessment, 2.2 month old male and female mice were treated daily with 100 mg/kg of Epidiolex via gavage for 19 days and underwent hypoxia/reoxygenation (H/R) treatment to simulate acute pain due to VOC on days 6, 7, and 13. Sickle mice at 2.2 months do not show increased hyperalgesia compared to control mice. Mechanical-, cold-, and musculoskeletal/deep hyperalgesia were assessed at baseline (before treatment) and one hour post-gavage on days 1, 4, 6, 7, 13, and 19, as well as post H/R treatment on days 6, 7, and 13. For assessment of chronic pain, ~ 6 month-old male mice were treated daily with 100 mg/kg of Epidiolex via gavage for 9 days, and mechanical-, thermal- (heat and cold), and deep hyperalgesia were assessed at baseline (before treatment) and 1-hour post-gavage on days 1, 4, 6, and 9. A 2-way repeated measures ANOVA with Tukey's correction was used for comparisons between time points and treatments. For acute pain, we observed an increase in mechanical hyperalgesia in male sickle mice compared to baseline following the first H/R on day 6 which was maintained through the third H/R on day 13 and continued to increase until day 19 (last period of observation), suggesting that repeated episodes of VOC may lead to sustained chronic pain. Continued treatment with Epidiolex, however, led to a decrease in mechanical hyperalgesia back to baseline levels, which was significantly lower compared to vehicle treated mice (P 〈 0.05). We also observed a decrease in cold hyperalgesia at day 19 compared to baseline in male sickle mice, which was not statistically significant. No changes were observed with Epidiolex treatment for deep hyperalgesia in males, or for mechanical-, cold-, or deep-hyperalgesia in female sickle mice. Our data demonstrate that repeated episodes of acute pain lead to transition to chronic pain and that Epidiolex has a preventive effect in the transition from acute to chronic pain in male sickle mice. It is likely that a higher dose of Epidiolex may be required for acute pain in females, which may also be influenced by disturbed estrus cycle. Since male mice showed responsiveness to Epidiolex in acute pain, we also examined its effect on chronic pain in males. Epidiolex significantly reduced, [a] mechanical hyperalgesia starting at day 4, which was maintained through day 9 (P 〈 0.01 compared to baseline), [b] cold hyperalgesia starting at day 1 and maintained through day 9 (P 〈 0.01 compared to baseline), [c] heat hyperalgesia at day 9 of treatment (P 〈 0.05 compared to baseline). These results suggest that Epidiolex has a sustained time-dependent analgesic effect on different characteristics of chronic hyperalgesia without causing tolerance in sickle mice. Our findings demonstrate therapeutic potential of pure and reliable CBD preparations for treating and potentially preventing chronic pain in SCD. We speculate high translational potential for CBD for treating pain in SCD following clinical trials. Disclosures Gupta: 1910 Genetics: Research Funding.

Description: Sickle cell disease (SCD) presents with multiple comorbidities including pain and organ damage. Transcriptomic analysis of dorsal root ganglion (DRG) revealed a significant decrease in the small proline rich protein 1a (Sprr1a) in HbSS-BERK sickle mice compared to control HbAA-BERK at an early age of ~2 months (Paul et al., Nature Sci Data 2017). Sprr1a is associated with axonal regeneration and cornification, which prevent nerve damage and evaporation from the skin, exposure to infections and mechanical stress, all of which occur in SCD. HbSS-BERK mice show thinner skin, nerve damage and increased sensitivity to mechanical and thermal stimuli (Kohli et al., Blood 2010). We hypothesized that Sprr1a downregulation in sickle mice leads to cutaneous alterations, thus increasing sensitivity to noxious stimuli leading to hyperalgesia and that restoring Sprr1a expression would reduce hyperalgesia. Utilizing Sprr1a-knockout (Sprr1a-KO) mice, we examined the gain and/or loss of skin and neuronal function with Sprr1a deletion. Compared to wild-type (WT) C57BL/6 mice, Sprr1a-KO mice showed a ~30% decrease in epidermal skin thickness (p

Description: Sickle cell disease (SCD) is complex with disabling and life-threatening consequences including pain and organ damage. We hypothesized that calpain-1, a calcium-dependent protease may contribute to both organ damage and pain in SCD because deletion of calpain-1 in HbSS Townes mice ameliorated hyperalgesia (Nwankwo et al., Haematologica 2016), and BDA-410, a selective calpain-1 inhibitor, significantly improved age-associated organ damage in a mouse model of aging (Nabeshima et al., Sci Reports 2014). We performed a randomized double-blind placebo-controlled trial in homozygous female HbSS BERK (sickle) mice to examine the effect of BDA-410 on pain and organ pathology. Mice were treated daily intraperitoneally with vehicle (50 μl DMSO in 950 μl sesame oil) and BDA-410 at 30 mg/kg or 100 mg/kg for 2 weeks. Mechanical-, thermal- (heat and cold), and deep hyperalgesia were assessed at baseline (before treatment), during treatment (1-hour post-injection, as well as on days 4, 8, 12), and after discontinuation of treatment (days 16, 20, 24, and 28). Using 2-way repeated measures ANOVA with Tukey's correction, we observed a significant decrease in mechanical, heat, and cold hyperalgesia in sickle mice treated with BDA-410. Compared to baseline, we observed a significant decrease in, [a] mechanical hyperalgesia, on day 8 with 30 mg/kg (p=0.0312), and days 8, 12, 16, 20, and 24 with 100 mg/kg dose (p=0.0012; p

Description: Mast cells are in close proximity to the vasculature and cause endothelial activation, plasma extravasation, and vascular dysfunction (Gupta & Harvima, Immunol Rev 2018). Vascular dysfunction in sickle cell disease (SCD) is accompanied by increased expression of P-selectin. Treatment with Crizanlizumab, an antibody against P-selectin, led to significantly less sickle cell-related pain crises (Ataga et al., NEJM 2017) highlighting the role of endothelial P-selectin in vasoocclusive crises (VOC). Earlier studies demonstrated that mast cell activation with morphine or ischemia/reperfusion stimulates endothelial E- and/or P-selectin expression. However, it is unknown how mast cells stimulate endothelial selectin expression in SCD. Endothelial dysfunction contributes significantly to the pathobiology of SCD including VOC and may play a critical role in increased blood-brain barrier (BBB) permeability, which may contribute to stroke, another major comorbidity of SCD. One of the known triggers of endothelial dysfunction, inflammation and oxidative stress is endoplasmic reticulum (ER) stress. We hypothesize that in a sickle microenvironment, mediators derived from activated mast cells stimulate endothelial P-selectin expression via ER stress leading to increased BBB permeability. We examined the ability of mast cells to stimulate P-selectin expression and BBB permeability via ER stress in a sickle microenvironment. We isolated MCs from HbAA-BERK and HbSS-BERK, control and sickle mice, respectively; incubated them in vitro and collected mast cell conditioned media (MCCM) from HbAA MCs and HbSS MCs. Normal mouse brain microvascular endothelial cells (mBMECs) were treated with unconditioned MCCM, HbAA MCCM, or HbSS MCCM to examine the effect of mast cell activation on endothelium. We observed increased mast cell activity in HbSS mice evinced by significantlyhigher plasma and skin histamine levels, compared to HbAA mice (p〈 0.02 for both). Mast cells from HbSS mouse skin showed significantly increased expression of histamine compared to HbAA skin mast cells (p〈 0.04). mBMECs incubated with HbAA and HBSS MCCM exhibited about 3- and 6-fold fold increases in P-selectin expression, compared to unconditioned culture medium, respectively (p〈 0.0001 for both). Therefore, mast cells in culture release substances that stimulate P-selectin expression which is further increased by mast cells from sickle (HbSS) microenvironment. Preincubation of mBMEC with 5 microM salubrinal, an inhibitor of dephosphorylation of elongation initiation factor-a, which reduces ER stress, significantly inhibited HbSS MCCM-induced P-selectin expression on mBMEC (p〈 0.0001) to the level induced by HbAA-MCCM. In contrast, salubrinal did not inhibit HbAA-MCCM-induced P-selectin expression on mBMEC, suggesting that in a sickle microenvironment mast cells contribute to P-selectin expression via ER stress. We next examined mast cell activity on endothelial permeability in vitro on mBMEC monolayers and in vivo in the brain of HbSS mice. mBMECs incubated with HbSS MCCM showed a significant increase in Evans blue leakage compared to unconditioned or HbAA MCCM (p