Characteristics and Potential Biomarkers for Chronic Pain in Patients with Sickle Cell Disease

Abstract Vaso-occlusive episodes (VOE) or pain crises are a hallmark of sickle cell disease (SCD), with increasing recognition that a significant portion of SCD patients develop chronic pain. In the landmark PiSCES study (Smith et al), patients reported pain on 55% days, with ~30% reporting pain on >90% days. Thus, the episodic, nociceptive pain (VOE) in younger patients, evolves into a chronic pain syndrome, with neuropathic and centralized components in some adults. Kutlar et al (Blood, 2014), reported on the association of different pain related phenotypes (pain diaries, frequency of hospitalizations/ED visits, pressure pain threshold) with polymorphisms in candidate genes in 167 SCD patients, providing evidence that multiple signaling pathways and mechanisms are likely involved. In this study, 12 SCD subjects with "chronic pain", defined by reported pain >50% of days in pain diaries collected over 6 months, were enrolled (SCD-CP). 17 SCD patients who did not have chronic pain (SCD-NCP), and 9 non-SCD African-Americans (C) were enrolled as controls. Informed consent was obtained. Age, gender, Hb F levels, HU usage, and pressure pain algometer readings were recorded from SCD subjects. 8 ml of blood (EDTA) was collected from subjects at "steady state" and from normal controls. Plasma was separated and kept at -80 C until the assay. Plasma tryptase and Substance P levels were assayed by ELISA using kits from Biomatik, Inc. (catalog # EKU07922) and Enzo Life Sciences (Catalog #ADI-900-018), respectively. SCD-CP patients were significantly older than SCD-NCP: mean age 41 vs 32.2 (p=0.033). The pressure pain algometer readings did not differ significantly between SCD-CP and SCD-NCP at three sites (trapezius, ulna, masseter, p= 0.67-0.74). There were 12/17 patients on HU (70.6%) among SCD-NCP, and 6/12 (50%) among SCD-CP (p=0.435). Similarly, Hb F levels were not significantly different (14.7% in SCD-CP, vs 11.7% in SCD-NCP, p=0.446). Opioid use (average morphine equivalent as mg/day) was significantly higher in the chronic pain group (11.45 mg/day, vs 2.92 mg/day, p=0.015). Plasma tryptase and substance-P levels are shown in the table: Table 1. Tryptase (pg/ml) Substance-P (pg/ml) SCD-CP 1388.6 ±519.8 7221.1±7742.7 SCD-NCP 1023.64±221.04 5983.1±3473.0 Control 768.9±416.16 3939.7±1350.1 The difference in substance-P levels did not reach significance across groups by ANOVA (p=0.337) or in pairwise comparison between groups. However, tryptase levels were significantly different across groups by ANOVA (p=0.00615). Pairwise comparisons between two groups showed that tryptase levels were significantly different between SCD-CP and controls (p=0.0053). These results highlight characteristics of SCD patients with chronic pain: they are older, have a higher use of opioids, and have significantly higher tryptase levels. These observations support previous findings that age is a significant factor in transition to chronic pain in SCD. Higher dose of opioid use in SCD-CP could result from dose escalation to control pain; conversely, it could be argued that higher opioid use itself could be a factor in development of chronic pain through opioid-induced hyperalgesia. To our knowledge, this is the first study of plasma tryptase levels in SCD, in relation to different pain phenotypes. Tryptase is released into plasma with degranulation of mast cells and leads to inflammation, anaphylaxis, urticaria, and neuropathic pain. It binds PAR2 (protease activated receptor 2), releasing inflammatory mediators and substance P, inducing neurogenic inflammation. Elevated tryptase levels are found in systemic mastocytosis, and the newly recognized Mast Cell Activation Syndrome (MCAS). Vincent et al (Blood, 2013) showed that mast cell activation played an important role in neurogenic inflammation and chronic pain in a mouse model of SCD. They also demonstrated that inhibition of mast cell activation, via c-kit knockout or with imatinib or cromolyn sodium improved neurogenic inflammation and chronic pain. Two recent case reports (Murphy et al, Stankovic et al) document significant improvement in pain in SCD patients who developed CML, during treatment with imatinib. These observations, and the findings of our pilot study, not only suggest a novel mechanism and biomarker for chronic pain in SCD, but also a potential therapeutic target by inhibition of mast cell activation via c-kit pathway, or stabilization with cromolyn. Disclosures No relevant conflicts of interest to declare.

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Characteristics and Potential Biomarkers of Adult Sickle Cell Patients with Chronic Pain

Blood ◽

10.1182/blood-2018-99-115084 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 857-857 ◽

Cited By ~ 1

Author(s):

Camila Albo ◽

Sanjiv Kumar ◽

Michael Pope ◽

Kyle Michael Kidwell ◽

Niren Patel ◽

...

Keyword(s):

Chronic Pain ◽

Mast Cell ◽

Substance P ◽

Cell Activation ◽

Mast Cell Activation ◽

Opioid Use ◽

The Past ◽

Chronic Pain Patients ◽

Age Dependent ◽

Pain Patients

Abstract A paradigm shift is occurring in our understanding of pain in patients with sickle cell disease (SCD). Vaso-occlusive episodes (VOEs) are crises of acute nociceptive pain, and have long been recognized as a hallmark of SCD (Brandow et al., 2017). While patients with SCD are traditionally considered to be at "steady state" and pain free between VOEs, recent studies have shown that a significant number of adults with SCD (~30%) experience daily chronic pain (>50% of the time in the past 6 months) (Smith et al., 2008). Although the precise mechanisms underlying this evolution from acute episodic to chronic pain are not well known, some contributing factors include age, chronic inflammation, organ damage, and opioid induced hyperalgesia (Stoicea et al., 2015; Rees et al., 2010). A recent study in a mouse model of SCD showed that mast cell activation was an important contributor to neurogenic inflammation and chronic pain (Vincent et al., 2013). We previously reported that SCD patients with chronic pain were older and had higher levels of mast cell activation markers, plasma tryptase and substance P, compared to those without chronic pain (Kuei et al., 2015). Recently, nerve growth factor (NGF) has been implicated in pathogenesis of some chronic pain syndromes (osteoarthritis), and clinical trials with anti-NGF monoclonal antibodies have been shown to result in superior pain control compared to placebo, opioids and NSAIDs. Here we report the results of our extended study of the evolution of chronic pain in SCD. A total of 72 subjects (age 15-66) were enrolled: 10 in the 15-19 age group, 19 in 20-29, 21 in 30-39, and 22 in ≥ 40. Patients transfused within the past 3 months and those who had an ED visit or hospitalization within the past 2 weeks were excluded. Information on the frequency of VOEs, presence or absence of chronic pain, HU therapy, opioid use (as mg morphine equivalents within the past 6 months), other medications and routine laboratory data (CBC, retics, chemistry panel, HbF) were collected. 4 mL of EDTA blood was collected at steady state and the plasma was separated by centrifugation and kept at -80 C. Plasma tryptase, substance P, and NGF levels were assayed by ELISA using kits from Biomatik inc (catalog# EKU07922), Enzo Life Sciences (catalog# ADI-900-018), and R&D Systems (catalog# DY256), respectively. Pressure pain threshold (PPTh) was measured using a hand-held digital algometer (AlgoMed, Medoc, Israel) four times at each of the following anatomic muscle groups on the left side of the body and then averaged for analysis: masseter, trapezius, and ulna in this consecutive order. Cutaneous mechanical pain was assessed using a Von Frey monofilament on the back of the subject's left hand. A baseline of one stimulus and then two separate series of 10 repeated stimuli each were conducted. The subject was asked to rate the pain on a scale of 0 to 10 (MFB, MF1 and MF2). Overall, 34 patients had chronic pain and 38 did not; there was an age dependent increase in the frequency of chronic pain, VOE frequency, opioid use and Von Frey MF values. Similarly, QST showed significantly lower pressure pain thresholds in subjects with chronic pain at ulna and trapezius (p=0.026 and 0.024 respectively). As expected, opioid use (daily morphine equivalents) was significantly higher in the chronic pain patients (52.8 mg vs 6.94 mg, p=0.009), suggesting a correlation between opioid use and hyperalgesia. Tryptase and substance P levels were higher in chronic pain patients, though the difference did not reach statistical significance. NGF levels were significantly higher in the chronic pain group (1126 pg/ml vs 473 pg/ml, p=0.051). Our results confirm previous observations that there is an age dependent increase in the proportion of patients with chronic pain (Table 1, Fig. 1-3). The trend towards higher levels of tryptase and substance P is in support of mast cell activation and neurogenic inflammation as a contributing factor to chronic pain (Vincent et al., 2013). To our knowledge, this is the first study of NGF as a possible contributing factor to chronic pain in SCD. If confirmed in larger multi-center studies, these observations could provide a rationale for novel interventions for chronic pain in SCD, via inhibition of mast cell activation/c-kit (tyrosine kinase inhibitors) or via repurposing of existing anti-NGF monoclonal antibodies as an alternative to opioids, whose inefficacy in chronic pain is well documented. Disclosures Kutlar: Novartis: Consultancy, Honoraria, Other: Personal fees, Research Funding; Bluebird Bio: Other: DSMB Member; Sancilio: Other: DSMB Chair.

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Mast Cell Activation Contributes to Neurogenic Inflammation and Pain in Sickle Cell Disease

Blood ◽

10.1182/blood.v120.21.374.374 ◽

2012 ◽

Vol 120 (21) ◽

pp. 374-374

Author(s):

Lucile Vincent ◽

Julia Nguyen ◽

Derek Vang ◽

Oludare B Taiwo ◽

Kathryn Luk ◽

...

Keyword(s):

Mast Cell ◽

Cell Activation ◽

Neurogenic Inflammation ◽

Control Mouse ◽

Mast Cell Degranulation ◽

Mast Cell Activation ◽

Plasma Extravasation ◽

Cell Disease ◽

Gm Csf ◽

Skin Biopsies

Abstract Abstract 374 Sickle cell disease (SCD) is associated with inflammation, endothelial dysfunction and pain. We observed increased immunoreactivity (ir) of pro-inflammatory and vasoactive neuropeptides, substance P (SP) and calcitonin-gene related peptide (CGRP) accompanied by decreased mu opioid receptor (MOR)-ir in the skin of sickle as compared to control mice (Kohli et al., Blood 2010). SP activates mast cells (MC), which are tissue resident leukocytes, leading to the release of inflammatory cytokines, tryptase and neuropeptides. SP also stimulates vascular permeability resulting in plasma extravasation and neurogenic inflammation. We hypothesized that pain in SCD is associated with a persistent feed-forward cycle of mast cell degranulation and neurogenic inflammation characterized by increased release of SP and CGRP from activated nociceptors in the skin leading to neuroinflammation, plasma extravasation and pain. We examined this hypothesis using sickle (HbSS-BERK) and control (HbAA-BERK) mice expressing sickle and normal human hemoglobin, respectively; and MOR-knockout (MOR-KO) mice with their wild type 129S6 controls. We developed an ex-vivo system to analyze the release of inflammatory cytokines, mast cell degranulation markers (tryptase and beta-hexosaminidase) and neuropeptides in skin biopsies. Neurogenic inflammation was studied in vivo using the Miles' assay. Evans blue was injected into the tail vein and its extravasation in skin evoked by stimulation with SP and capsaicin was quantified. Skin biopsies from sickle mice exhibited constitutively enhanced release of several cytokines (IL6, MCP-1, TNFalpha, MIP-1alpha, GM-CSF, RANTES, etc), tryptase and the neuropeptides SP, and CGRP as compared to control mouse skin (p<0.05 for each). Increased RANTES and GM-CSF are suggestive of mast cell recruitment. Mast cell tryptase-ir was increased 2-fold while MOR-ir (but not delta- or kappa-OR-ir), was reduced by ∼50% in the skin of sickle as compared to control mice, suggestive of enhanced MC degranulation in sickle. In MC cultures prepared from sickle skin increased c-kit/CD117-, FCeR- and tryptase-ir were observed as compared to control mouse MCs. The plasma of sickle exhibited a ∼60–80% increase in MC degranulation markers, tryptase and beta-hexosaminidase, acute phase protein, serum amyloid protein, and neuropeptides, SP and CGRP, as compared to control mice (p<0.01 for each). These correlative molecular changes in the plasma and skin were accompanied by increased SP- and capsaicin-induced Evans blue dye leakage in the skin of sickle mice suggestive of neurogenic inflammation as compared to control (p<0.001 for each). MOR-KO mice also exhibited increased SP- and CGRP-ir in the skin and neurogenic inflammation, indicative of a contribution by MOR to the neuroinflamamtory process. In sickle mice treated with the mast cell stabilizer cromolyn sodium (CS), or the c-kit inhibitor, Imatinib, for 5 days, the inflammatory cytokine and neuropeptide release from the skin and the neurogenic inflammation were ameliorated as compared to vehicle (p<0.01). Additionally, morphine at a dose of 10 mg/Kg was ineffective in treating tonic cutaneous and thermal hyperalgesia, but effectively reduced hyperalgesia in CS and Imatinib treated sickle mice. Thus, MC degranulation contributes to neurogenic inflammation and pain in sickle mice. Imatinib treatment by itself reduced tonic hyperalgesia and significantly decreased GM-CSF release from the skin (p<0.05) correlative to the reduced white blood cell (WBC) count in sickle mice vs vehicle. In addition to inhibiting MC activity, Imatinib may be inhibiting protein tyrosine kinases involved in cytokine processing, vascular function and nociception. Together, our observations demonstrate that MCs contribute to a vicious cycle of pain and neurogenic inflammation mediated by increased neuropeptides in SCD. It is likely that mast cell inhibitors such as Imatinib may have a therapeutic effect on pain, inflammation and vascular dysfunction in SCD by reducing mast cell activation and neurogenic inflammation. Since, Imatinib decreased GM-CSF levels and WBC, it may even increase HbF levels, which are negatively regulated by GM-CSF in SCD. We therefore speculate that therapies targeting mast cells may potentiate therapeutic outcomes of analgesics, anti-inflammatory agents and Hydroxyurea® in SCD. Disclosures: No relevant conflicts of interest to declare.

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Mast Cell Activation Syndrome as a Significant Comorbidity in Sickle Cell Disease

The American Journal of the Medical Sciences ◽

10.1097/maj.0000000000000325 ◽

2014 ◽

Vol 348 (6) ◽

pp. 460-464 ◽

Cited By ~ 10

Author(s):

Lawrence B. Afrin

Keyword(s):

Mast Cell ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Cell Activation ◽

Mast Cell Activation ◽

Cell Disease ◽

Mast Cell Activation Syndrome ◽

Activation Syndrome

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Acupuncture Led Attenuation of Inflammatory and Nociceptive Peripheral and Central Microenvironment in Sickle Mice

Blood ◽

10.1182/blood.v126.23.2169.2169 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2169-2169

Author(s):

Ying Wang ◽

Jianxun Lei ◽

Yann Y Lamarre ◽

Ritu Jha ◽

Fei Peng ◽

...

Keyword(s):

Spinal Cord ◽

Mast Cell ◽

Pain Relief ◽

Substance P ◽

Inflammatory Response ◽

Cell Activation ◽

Culture Medium ◽

Neurogenic Inflammation ◽

Mast Cell Activation ◽

Skin Biopsies

Abstract 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 (<20% pain relief) to EA. In positive responders, EA significantly reduced white blood cells (p<.001 Vs moderate- and non-responders), serum amyloid protein (p<.01 Vs untreated), IL-1beta (p<.05 Vs untreated, p<.01 Vs non-responders), and substance P (p<.05 Vs untreated and p<.001 Vs non-responders and p<.05 Vs moderate-responders). Concurrently, spinal cord analysis of EA treated positive-responders showed reduced substance P (p<.05 Vs untreated and non-responders), IL-1 beta (p<.01 Vs untreated), TNF alpha (p<.05 and p<.01 Vs moderate- and non-responders, respectively). Consistent with this central and peripheral anti-inflammatory response, culture medium from skin biopsies of positive responders demonstrated reduced substance P (p<.01 Vs moderate- and non-responders) and tryptase (p<.01 Vs untreated, moderate- and non-responders), and significantly less toluidine blue stained degranulating mast cells in the skin (p<.05 Vs untreated and non-responders) suggestive of attenuation of mast cell and peripheral nervous system activation. Functionally, capsaicin and substance P-induced neurogenic inflammation were significantly attenuated in positive-responders vs non-responders (p<.05) or untreated (p<.05). Peripheral and central attenuation of inflammatory and neurogenic response to EA was accompanied by inhibition of nociceptive signaling in the spinal cord. Spinal phosphorylation of p38 MAPK decreased in EA treated mice (p<.05 Vs sham-EA and untreated control; and positive-responders Vs non-responders). Conclusions: EA treatment on conscious free-moving mice simulates clinical conditions in patients and excludes the potential influence due to restraint or anesthetics. EA leads to peripheral and central neuromodulation and anti-inflammatory response by attenuating mast cell activation, substance P, and cytokine release in the periphery and by abrogating spinal nociceptive signaling of p38MAPK and inflammation. Together, these molecular and cellular effects lead to EA-induced attenuation of neurogenic inflammation and hyperalgesia in sickle mice. Importantly, these data explain the cause of variable effectiveness of EA in SCD. Disclosures No relevant conflicts of interest to declare.

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Hemin-Induced Mast Cell-Extracellular Traps Impart Resistance to Therapy in a Sickle Microenvironment

Blood ◽

10.1182/blood.v126.23.3385.3385 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3385-3385 ◽

Cited By ~ 4

Author(s):

Huy Tran ◽

Ritu Jha ◽

Julia Nguyen ◽

Sarita Jarrett ◽

Jonathan Rodriguez ◽

...

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Sickle Cell Disease ◽

Cell Activation ◽

Mast Cell Activation ◽

Cell Disease ◽

Tnf Α ◽

Free Heme ◽

Cp 55,940 ◽

Extracellular Trap

Abstract 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.

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XANTATINA INHIBE LA ACTIVACIÓN DE MASTOCITOS INDUCIDA POR NEUROPÉPTIDOS PRO-INFLAMATORIOS

Revista Argentina de Anatomía Clínica ◽

10.31051/1852.8023.v2.n1.13859 ◽

2016 ◽

Vol 2 (1) ◽

pp. 16-24

Author(s):

Patricia M. Vargas ◽

Elia Martino ◽

Teresa H. Fogal ◽

Carlos E. Tonn ◽

Alicia B. Penissi

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Substance P ◽

Cell Activation ◽

Neurogenic Inflammation ◽

Light And Electron Microscopy ◽

Serotonin Release ◽

Mast Cell Activation ◽

Morphological Studies ◽

Peritoneal Mast Cells

Los mastocitos son células del tejido conectivo que participan en la génesis y modulación de las respuestas inflamatorias. Previamente hemos demos-trado que xanthatina (xanthanólido sesquiterpeno aislado de Xanthium cavanillesii Schouw) inhibe la activación de mastocitos inducida por secretagogos experimentales. Sin embargo, se desconoce su efecto sobre la activación de mastocitos inducida por estímulos fisiopatológicos. Estos estímulos incluyen, entre otros, los neuropéptidos pro-inflamatorios sustancia P y neurotensina, responsables de una de las principales vías de inflamación neurogénica. El objetivo del presente trabajo fue estudiar el efecto de xanthatina sobre la activación de mastocitos inducida por sustancia P y neurotensina. Mastocitos peritoneales de rata se incubaron con: 1) PBS (basal); 2) sustancia P (100 µm); 3) neurotensina (50 µm); 4) xanthatina (8-320 µm)+sustancia P; 5) xanthatina (8-320 µm)+neurotensina. Se llevaron a cabo los siguientes estudios: análisis dosis-respuesta de la liberación de serotonina inducida por neuropéptidos proinflamatorios, vitalidad celular, morfología mastocitaria por microscopía óptica y electrónica, análisis de estabilidad de xanthatina por cromatografía en capa fina. Los ensayos de liberación de serotonina y los estudios morfológicos mostraron la efectividad de xanthatina para estabilizar mastocitos. El presente estudio provee la primer evidencia a favor de la hipótesis de que xanthatina inhibe la liberación de serotonina inducida por sustancia P y neurotensina a partir de mastocitos peritoneales. Este sesquiterpeno podría representar una nueva alternativa fármacológica en la regulación de la activación mastocitaria para el tratamiento de las inflamaciones neurogénicas. Mast cells are connective tissue cells involved in the genesis and modulation of inflammatory responses. We have previously shown that xanthatin (xanthanolide sesquiterpene isolated from Xanthium cavanillesii Schouw) inhibits mast cell activation induced by experimental secretagogues. However, the effect of xanthatin on mast cell activation induced by pathophysiological stimuli remains unknown. These stimuli include, among others, the pro-inflammatory neuropeptide substance P and neurotensin, responsible for one of the main pathways of neurogenic inflammation. The present study was designed to examine the effects of xanthatin on mast cell activation induced by pro-inflammatory peptides, such as substance P and neurotensin. Rat peritoneal mast cells were incubated with: 1) PBS (basal); 2) substance P (100 µm); 3) neurotensin (50 µm); 4) xanthatin (8-320 µm)+substance P; 5) xanthatin (8-320 µm)+neurotensin. Concentration-response studies of mast cell serotonin release evoked by pro-inflammatory neuropeptides, evaluation of mast cell viability and morphology by light and electron microscopy, and drug stability analysis by thin layer chromatography were performed. Serotonin release studies, carried out together with morphological studies, showed the effectiveness of xanthatin to stabilize mast cells. The present study provides the first strong evidence in favour of the hypothesis that xanthatin inhibits substance P - and neurotensin-induced serotonin release from peritoneal mast cells. Our findings may provide an insight into the design of novel pharmacological agents which may be used to regulate the mast cell response in neurogenic inflammation.

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Evolution of Chronic Pain in Sickle Cell Disease

Blood ◽

10.1182/blood.v128.22.1297.1297 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1297-1297 ◽

Cited By ~ 6

Author(s):

Michael Pope ◽

Camila Albo ◽

Kyle Michael Kidwell ◽

Hongyan Xu ◽

Latanya Bowman ◽

...

Keyword(s):

Chronic Pain ◽

Substance P ◽

Sickle Cell ◽

Cell Activation ◽

Statistical Significance ◽

Age Groups ◽

Mast Cell Activation ◽

Age Group ◽

Opioid Use ◽

Age Dependent

Abstract Vaso-occlusive episodes (VOE), are the most common cause of health care encounters and are considered to be a hallmark of sickle cell disease (SCD). It has been increasingly recognized that many SCD patients, especially adults experience daily chronic pain (PiSCES study). While adolescents and young adults experience mostly acute episodic nociceptive pain, a significant number of adults develop chronic neuropathic and centralized pain. Although the precise mechanisms underlying this age-related transition in pain phenotype are not clearly understood, some factors that contribute to this phenomenon include chronic inflammation, organ damage and opioid induced hyperalgesia. Vincent et al (Blood, 2013) showed that mast cell activation is an important contributor to neurogenic inflammation and chronic pain in a mouse model of SCD. In an observational translational study, we recently reported that SCD patients with chronic pain (those who report pain >50% of time) were older and had higher levels of mast cell activation markers, plasma tryptase and substance P, compared to SCD patients who did not have chronic pain (Kuei et al, Blood, 2015). We report here the results of a prospective study of SCD patients (SS and Sβ0 thal) in different age groups. We tested the hypotheses that i) evolution to chronic pain is an age-dependent phenomenon, with higher frequency in older subjects, and ii) serum tryptase and substance P levels can be used as markers of chronic pain. A total of 36 subjects with SCD from the patient population of Pediatric and Adult sickle cell clinics of the MCG Sickle Cell Center were enrolled; these included 6 subjects aged 15-19, and 10 adults each in the age groups of 20-29, 30-39, and ≥40, respectively. Informed consent was obtained. Information on the frequency of VOEs (hospitalizations and ED visits) in the last 2 years, presence of chronic pain, hydroxyurea (HU) exposure, opioid usage as morphine equivalents within the past year, CBC, retics, CMP and % Hb F were collected. Quantitative sensory testing (QST) was performed using three different modalities: pressure pain sensitivity using a hand held computerized algometer AlgoMed, Medoc, Israel); heat and cold sensitivity (Q-Sense, Medoc, Israel); and Von Frey monofilament for neuropathic pain. Plasma tryptase and substance P levels were assayed by ELISA using kits from Biomatik inc, and Enzo Life Sciences, respectively. The results (lab tests, QST, VOEs, HU usage, plasma tryptase and substance P levels) are summarized in Table 1. Overall, there was an age dependent increase in the frequency of subjects with chronic pain; while 16.7% of patients in the 15-19 age group experienced chronic pain, this increased to 60.0% in the 20-29 year olds, and to 63.6% in those 30-39 years of age as shown in Figure 1. Among patients ≥40 years, 40.0% reported experiencing chronic pain. VOE frequency (average of last 2 years) was highest in the 20-29 age group (11.0/year) followed by 5.6 in the 30-39 age group. It was 2.57/year among 15-19 year olds, and 2.70/year in patients ≥40 years (p=0.004). Although Hb F and MCV was highest among ≥40 group (20.4%, and 112.1 fl, respectively), this did not reach statistical significance. As seen in the Table, BUN and creatinine showed a significant age dependent increase indicative of progressive organ damage. There was no significant difference between different age groups in other laboratory parameters, QST results, and plasma tryptase and substance P levels. The significant finding of this study is the higher frequency of VOEs in the 20-39 age group, compared to adolescents and patients 40 years and above. Although this finding is paralleled by a trend in opioid use, and frequency of chronic pain, these did not reach statistical significance in this relatively small group. The other interesting observation is the relatively mild course of disease in those ≥40 years of age (lower frequency of chronic pain, lower opioid use, lower VOE frequency). This seemingly paradoxical finding can be explained either by a survival advantage of the relatively mild patients, or a better adherence to HU, as suggested by higher MCV and Hb F in this group. The transition from adolescence to young adulthood is associated with a higher morbidity in SCD. Factors contributing to this phenomenon should be studied in a larger cohort. Disclosures Kutlar: Novartis Pharmaceuticals: Research Funding.

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Targeting pain at its source in sickle cell disease

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00021.2018 ◽

2018 ◽

Vol 315 (1) ◽

pp. R104-R112 ◽

Cited By ~ 8

Author(s):

Kanika Gupta ◽

Om Jahagirdar ◽

Kalpna Gupta

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Cell Activation ◽

Molecular Mechanisms ◽

Genetic Disorder ◽

Somatosensory System ◽

Organ Damage ◽

Mast Cell Activation ◽

Cell Disease ◽

Central Nervous Systems

Sickle cell disease (SCD) is a genetic disorder associated with hemolytic anemia, end-organ damage, reduced survival, and pain. One of the unique features of SCD is recurrent and unpredictable episodes of acute pain due to vasoocclusive crisis requiring hospitalization. Additionally, patients with SCD often develop chronic persistent pain. Currently, sickle cell pain is treated with opioids, an approach limited by adverse effects. Because pain can start at infancy and continue throughout life, preventing the genesis of pain may be relatively better than treating the pain once it has been evoked. Therefore, we provide insights into the cellular and molecular mechanisms of sickle cell pain that contribute to the activation of the somatosensory system in the peripheral and central nervous systems. These mechanisms include mast cell activation and neurogenic inflammation, peripheral nociceptor sensitization, maladaptation of spinal signals, central sensitization, and modulation of neural circuits in the brain. In this review, we describe potential preventive/therapeutic targets and their targeting with novel pharmacologic and/or integrative approaches to ameliorate sickle cell pain.

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