Cold exposure induces vaso-occlusion and pain in sickle mice that depend on complement activation.

Vaso-occlusive pain episodes (VOE) cause severe pain in patients with sickle cell disease (SCD). Vaso-occlusive events promote ischemia/reperfusion pathobiology that activates complement. We hypothesized that complement activation is linked to VOE. We used cold to induce VOE in the Townes sickle homozygous for hemoglobin S (HbSS) mouse model and complement inhibitors to determine whether anaphylatoxin C5a mediates VOE. We used a dorsal skinfold chamber to measure microvascular stasis (vaso-occlusion) and von Frey filaments applied to the plantar surface of the hind paw to assess mechanical hyperalgesia in HbSS and control Townes mice homozygous for hemoglobin A (HbAA) mice after cold exposure at 10°C/50°F for 1 hour. Cold exposure induced more vaso-occlusion in nonhyperalgesic HbSS mice (33%) than in HbAA mice (11%) or HbSS mice left at room temperature (1%). Cold exposure also produced mechanical hyperalgesia as measured by paw withdrawal threshold in HbSS mice compared with that in HbAA mice or HbSS mice left at room temperature. Vaso-occlusion and hyperalgesia were associated with an increase in complement activation fragments Bb and C5a in plasma of HbSS mice after cold exposure. This was accompanied by an increase in proinflammatory NF-κB activation and VCAM-1 and ICAM-1 expression in the liver. Pretreatment of nonhyperalgesic HbSS mice before cold exposure with anti-C5 or anti-C5aR monoclonal antibodies (mAbs) decreased vaso-occlusion, mechanical hyperalgesia, complement activation, and liver inflammatory markers compared with pretreatment with control mAb. Anti-C5 or -C5aR mAb infusion also abrogated mechanical hyperalgesia in HbSS mice with ongoing hyperalgesia at baseline. These findings suggest that C5a promotes vaso-occlusion, pain, and inflammation during VOE and may play a role in chronic pain.

Sickle red blood cell-derived extracellular vesicles activate endothelial cells and enhance sickle red cell adhesion mediated by von Willebrand factor.

Endothelial activation and sickle red blood cell (RBC) adhesion are central to the pathogenesis of sickle cell disease (SCD). Quantitatively, RBC-derived extracellular vesicles (REVs) are more abundant from SS RBCs compared with healthy RBCs (AA RBCs). Sickle RBC-derived REVs (SS REVs) are known to promote endothelial cell (EC) activation through cell signalling and transcriptional regulation at longer terms. However, the SS REV-mediated short-term non-transcriptional response of EC is unclear. Here, we examined the impact of SS REVs on acute microvascular EC activation and RBC adhesion at 2 h. Compared with AA REVs, SS REVs promoted human pulmonary microvascular ECs (HPMEC) activation indicated by increased von Willebrand factor (VWF) expression. Under microfluidic conditions, we found abnormal SS RBC adhesion to HPMECs exposed to SS REVs. This enhanced SS RBC adhesion was reduced by haeme binding protein haemopexin or VWF cleaving protease ADAMTS13 to a level similar to HPMECs treated with AA REVs. Consistent with these observations, haemin- or SS REV-induced microvascular stasis in SS mice with implanted dorsal skin-fold chambers that was inhibited by ADAMTS13. The adhesion induced by SS REVs was variable and was higher with SS RBCs from patients with increased markers of haemolysis (lactate dehydrogenase and reticulocyte count) or a concomitant clinical diagnosis of deep vein thrombosis. Our results emphasise the critical contribution made by REVs to the pathophysiology of SCD by triggering acute microvascular EC activation and abnormal RBC adhesion. These findings may help to better understand acute pathophysiological mechanism of SCD and thereby the development of new treatment strategies using VWF as a potential target.

A novel, highly potent and selective phosphodiesterase-9 inhibitor for the treatment of sickle cell disease.

The most common treatment for patients with sickle cell disease (SCD) is the chemotherapeutic hydroxyurea, a therapy with pleiotropic effects, including increasing fetal hemoglobin (HbF) in red blood cells and reducing adhesion of white blood cells to the vascular endothelium. Hydroxyurea has been proposed to mediate these effects through a mechanism of increasing cellular cGMP levels. An alternative path to increasing cGMP levels in these cells is through the use of phosphodiesterase-9 inhibitors that selectively inhibit cGMP hydrolysis and increase cellular cGMP levels. We have developed a novel, potent and selective phosphodiesterase-9 inhibitor (IMR-687) specifically for the treatment of SCD. IMR-687 increased cGMP and HbF in erythroid K562 and UT-7 cells and increased the percentage of HbF positive erythroid cells generated in vitro using a two-phase liquid culture of CD34+ progenitors from sickle cell blood or bone marrow. Oral daily dosing of IMR-687 in the Townes transgenic mouse SCD model, increased HbF and reduced red blood cell sickling, immune cell activation and microvascular stasis. The IMR-687 reduction in red blood cell sickling and immune cell activation was greater than that seen with physiological doses of hydroxyurea. In contrast to other described phosphodiesterase-9 inhibitors, IMR-687 did not accumulate in the central nervous system, where it would inhibit phosphodiesterase-9 in neurons, or alter rodent behavior. IMR-687 was not genotoxic or myelotoxic and did not impact fertility or fetal development in rodents. These data suggest that IMR-687 may offer a safe and effective oral alternative for hydroxyurea in the treatment of SCD.

Critical role of C5a in sickle cell disease.

Innate immune complement activation may contribute to sickle cell disease (SCD) pathogenesis. Ischemia-reperfusion physiology is a key component of the inflammatory and vaso-occlusive milieu in SCD and is associated with complement activation. C5a is an anaphylatoxin, a potent pro-inflammatory mediator that can activate leukocytes, platelets, and endothelial cells, all of which play a role in vaso-occlusion. We hypothesize that hypoxia-reoxygenation (H/R) in SCD mice activates complement, promoting inflammation and vaso-occlusion. At baseline and after H/R, sickle Townes-SS mice had increased C3 activation fragments and C5b-9 deposition in kidneys, livers and lungs and alternative pathway Bb fragments in plasma compared to control AA-mice. Activated complement promoted vaso-occlusion (microvascular stasis) in SS-mice; infusion of zymosan-activated, but not heat-inactivated serum, induced substantial vaso-occlusion in the skin venules of SS-mice. Infusion of recombinant C5a induced stasis in SS, but not AA-mice that was blocked by anti-C5a receptor (C5aR) IgG. C5a-mediated stasis was accompanied by inflammatory responses in SS-mice including NF-κB activation and increased expression of TLR4 and adhesion molecules VCAM-1, ICAM-1, and E-selectin in the liver. Anti-C5aR IgG blocked these inflammatory responses. Also, C5a rapidly up-regulated Weibel-Palade body P-selectin and von Willebrand factor on the surface of human umbilical vein endothelial cells in vitro and on vascular endothelium in vivo. In SS-mice, a blocking antibody to P-selectin inhibited C5a-induced stasis. Similarly, an antibody to C5 that blocks murine C5 cleavage or an antibody that blocks C5aR inhibited H/R-induced stasis in SS-mice. These results suggest that inhibition of C5a may be beneficial in SCD.

Hepatic Overexpression of Hemopexin Inhibits Inflammation and Vascular Stasis in Murine Models of Sickle Cell Disease.

Sickle cell disease (SCD) patients have low serum hemopexin (Hpx) levels due to chronic hemolysis. We hypothesize that in SCD mice, hepatic overexpression of hemopexin will scavenge the proximal mediator of vascular activation, heme, and will inhibit inflammation and microvascular stasis. To examine the protective role of Hpx in SCD, we transplanted bone marrow from NY1DD SCD mice into Hpx™/™ or Hpx+/+ C57BL/6 mice. Dorsal skin fold chambers were implanted in week 13 post-transplant and microvascular stasis (% non-flowing venules) evaluated in response to heme infusion. Hpx™/™ sickle mice had significantly greater microvascular stasis in response to heme infusion than Hpx+/+ sickle mice (p<0.05), demonstrating the protective effect of Hpx in SCD. We utilized Sleeping Beauty (SB) transposon-mediated gene transfer to overexpress wild-type rat Hpx (wt-Hpx) in NY1DD and Townes-SS SCD mice. Control SCD mice were treated with lactated Ringer's solution (LRS) or a luciferase (Luc) plasmid. Plasma and hepatic Hpx were significantly increased compared to LRS and Luc controls. Microvascular stasis in response to heme infusion in NY1DD and Townes-SS mice overexpressing wt-Hpx had significantly less stasis than controls (p<0.05). Wt-Hpx overexpression markedly increased hepatic nuclear Nrf2 expression, HO-1 activity and protein, the heme-Hpx binding protein and scavenger receptor, CD91/LRP1 and decreased NF-κB activation. Two missense (ms)-Hpx SB-constructs that bound neither heme nor the Hpx receptor, CD91/LRP1, did not prevent heme-induced stasis. In conclusion, increasing Hpx levels in transgenic sickle mice via gene transfer activates the Nrf2/HO-1 anti-oxidant axis and ameliorates inflammation and vaso-occlusion.

Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease.

Treatment of sickle cell disease (SCD) is hampered by incomplete understanding of pathways linking hemolysis to vaso-occlusion. We investigated these pathways in transgenic sickle mice. Infusion of hemoglobin or heme triggered vaso-occlusion in sickle, but not normal, mice. Methemoglobin, but not heme-stabilized cyanomethemoglobin, induced vaso-occlusion, indicating heme liberation is necessary. In corroboration, hemoglobin-induced vaso-occlusion was blocked by the methemoglobin reducing agent methylene blue, haptoglobin, or the heme-binding protein hemopexin. Untreated HbSS mice, but not HbAA mice, exhibited ∼10% vaso-occlusion in steady state that was inhibited by haptoglobin or hemopexin infusion. Antibody blockade of adhesion molecules P-selectin, von Willebrand factor (VWF), E-selectin, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, platelet endothelial cell (EC) adhesion molecule 1, α4ß1, or αVß3 integrin prevented vaso-occlusion. Heme rapidly (5 minutes) mobilized Weibel-Palade body (WPB) P-selectin and VWF onto EC and vessel wall surfaces and activated EC nuclear factor κB (NF-κB). This was mediated by TLR4 as TAK-242 blocked WPB degranulation, NF-κB activation, vaso-occlusion, leukocyte rolling/adhesion, and heme lethality. TLR4(-/-) mice transplanted with TLR4(+/+) sickle bone marrow exhibited no heme-induced vaso-occlusion. The TLR4 agonist lipopolysaccharide (LPS) activated ECs and triggered vaso-occlusion that was inhibited by TAK-242, linking hemolysis- and infection-induced vaso-occlusive crises to TLR4 signaling. Heme and LPS failed to activate VWF and NF-κB in TLR4(-/-) ECs. Anti-LPS immunoglobulin G blocked LPS-induced, but not heme-induced, vaso-occlusion, illustrating LPS-independent TLR4 signaling by heme. Inhibition of protein kinase C, NADPH oxidase, or antioxidant treatment blocked heme-mediated stasis, WPB degranulation, and oxidant production. We conclude that intravascular hemolysis in SCD releases heme that activates endothelial TLR4 signaling leading to WPB degranulation, NF-κB activation, and vaso-occlusion.

Mast cell extracellular trap formation underlies vascular and neural injury and hyperalgesia in sickle cell disease.

Sickle cell disease (SCD) is the most common inherited monogenetic disorder. Chronic and acute pain are hallmark features of SCD involving neural and vascular injury and inflammation. Mast cells reside in the vicinity of nerve fibers and vasculature, but how they influence these structures remains unknown. We therefore examined the mechanism of mast cell activation in a sickle microenvironment replete with cell-free heme and inflammation. Mast cells exposed to this environment showed an explosion of nuclear contents with the release of citrullinated histones, suggestive of mast cell extracellular trap (MCET) release. MCETs interacted directly with the vasculature and nerve fibers, a cause of vascular and neural injury in sickle cell mice. MCET formation was dependent upon peptidylarginine deiminase 4 (PAD4). Inhibition of PAD4 ameliorated vasoocclusion, chronic and acute hyperalgesia, and inflammation in sickle mice. PAD4 activation may also underlie neutrophil trap formation in SCD, thus providing a novel target to treat the sequelae of vascular and neural injury in SCD.

Electronic health records perception among three healthcare providers specialties in Saudi Arabia: A cross-sectional study.

Worldwide, more health care facilities are adapting the use of electronic health record (EHR). Healthcare providers (HCP) have different perceptions toward the use of EHR. To investigate the perception of three classes of HCP in Saudi Arabia toward using EHR, a questionnaire (targeting satisfaction, easiness, and benefits of use as major perception indicators) was prepared. The questionnaire was assessed by an expert panel for content validity. The questionnaire internal consistency was examined using Cronbach's alpha. 108 physicians, physical therapists (PT) and respiratory care therapists (RT) from different hospitals in Saudi Arabia answered the questionnaire. Most of respondents perceived EHR systems as beneficial and made work easier. Most HCP were satisfied with the use of EHR, however, with the use of EHR more time was needed to finish the work. Age, experience, job, and job rank of HCP are of different importance in determining responses, perception, and obstacles of using EHR. Moreover, the perception of using EHR seems to be field specific. There is a positive perception among Saudi Arabia HCP about EHR use.

Psychometric properties of an Arabic translation of the chronic pain acceptance questionnaire (CPAQ) in a sample of patients with chronic pain.

PURPOSE: Chronic pain (CP) acceptance is a major factor in determining the well-being of patients with chronic pain. The chronic pain acceptance questionnaire (CPAQ) was translated and validated into Arabic (CPAQ-Ar). METHODS: 244 patients with CP completed the CPAQ-Ar, the Beck Depression Inventory-II (BDI-II), the short form health survey (SF-36), the Pain Catastrophizing Scale (PCS), the Pittsburgh Sleep Quality Index (PSQI), the Modified Fatigue Impact Scale (MFIS), and the Depression Anxiety Stress Scale 21 (DASS-21). 110 patients completed the CPAQ-Ar twice separated by two weeks to investigate test-retest reliability. RESULTS: Cronbach's α was 0.902 while the intraclass correlation coefficient (ICC) was 0.917. The standard error measurement (SEM) was seven points while the minimal detectable change with 95% confidence interval (MDC95) was seventeen points. The CPAQ-Ar showed moderate to high correlations with the PCS, the BDI-II, the SF-36, the MFIS, the PSQI, and the DASS-21 indicating a good concurrent validity. Exploratory factor analysis confirmed that the CPAQ-Ar consists of two subscales. Better pain acceptance associated with male gender, older people, employed participants, low pain intensity, and single pain site. CONCLUSIONS: The CPAQ-Ar is a valid and reliable tool for the measurement of pain acceptance in Arabic speaking patients with CP.

The BACH1 inhibitor ASP8731 inhibits inflammation and vaso-occlusion and induces fetal hemoglobin in sickle cell disease.

In sickle cell disease (SCD), heme released during intravascular hemolysis promotes oxidative stress, inflammation, and vaso-occlusion. Conversely, free heme can also activate expression of antioxidant and globin genes. Heme binds to the transcription factor BACH1, which represses NRF2-mediated gene transcription. ASP8731, is a selective small molecule inhibitor of BACH1. We investigated the ability of ASP8731 to modulate pathways involved in SCD pathophysiology. In HepG2 liver cells, ASP8731 increased HMOX1 and FTH1 mRNA. In pulmonary endothelial cells, ASP8731 decreased VCAM1 mRNA in response to TNF-α and blocked a decrease in glutathione in response to hemin. Townes-SS mice were gavaged once per day for 4 weeks with ASP8731, hydroxyurea (HU) or vehicle. Both ASP8731 and HU inhibited heme-mediated microvascular stasis and in combination, ASP8731 significantly reduced microvascular stasis compared to HU alone. In Townes-SS mice, ASP8731 and HU markedly increased heme oxygenase-1 and decreased hepatic ICAM-1, NF-kB phospho-p65 protein expression in the liver, and white blood cell counts. In addition, ASP8731 increased gamma-globin expression and HbF+ cells (F-cells) as compared to vehicle-treated mice. In human erythroid differentiated CD34+ cells, ASP8731 increased HGB mRNA and increased the percentage of F-cells 2-fold in manner similar to HU. ASP8731 and HU when given together induced more HbF+ cells compared to either drug alone. In CD34+ cells from one donor that was non-responsive to HU, ASP8731 induced HbF+ cells ~2-fold. ASP8731 and HU also increased HBG and HBA, but not HBB mRNA in erythroid differentiated CD34+ cells derived from SCD patients. These data indicate that BACH1 may offer a new therapeutic target to treat SCD.

The HDAC inhibitors trichostatin A and suberoylanilide hydroxamic acid exhibit multiple modalities of benefit for the vascular pathobiology of sickle transgenic mice.

The vascular pathobiology of sickle cell anemia involves inflammation, coagulation, vascular stasis, reperfusion injury, iron-based oxidative biochemistry, deficient nitric oxide (NO) bioavailability, and red cell sickling. These disparate pathobiologies intersect and overlap, so it is probable that multimodality therapy will be necessary for this disease. We have, therefore, tested a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), for efficacy in reducing endothelial activation. We found that pulmonary vascular endothelial VCAM-1 and tissue factor (TF) expression (both are indicators of endothelial activation) are powerfully and significantly inhibited by TSA. This is seen both with pretreatment before the inducing stress of hypoxia/reoxygenation (NY1DD sickle transgenic mouse), and upon longer-term therapy after endothelial activation has already occurred (hBERK1 sickle mouse at ambient air). In addition, TSA prevented vascular stasis in sickle mice, it exhibited activity as an iron chelator, and it induced expression of the antisickling hemoglobin, hemoglobin F. Notably, the TSA analog SAHA (suberoylanilide hydroxaminc acid) that is already approved for human clinical use exhibits the same spectrum of biologic effects as TSA. We suggest that SAHA possibly could provide true, multimodality, salubrious effects for prevention and treatment of the chronic vasculopathy of sickle cell anemia.

Factors that influence quality of life in patients with multiple sclerosis in Saudi Arabia.

PURPOSE: To study the factors which may contribute to quality of life (QOL) in patients with multiple sclerosis (pwMS) in Saudi Arabia. METHODS: 175 pwMS and 71 age-, gender-, and BMI-matched healthy subjects participated in this cross-sectional study. QOL was studied by the multiple sclerosis quality of life-54 (MSQOL-54) while depression, disability, and fatigue were measured by the beck depression inventory-II (BDI-II), the expanded disability status scale (EDSS), and the modified fatigue impact scale (MFIS), respectively. The effects of demographic and clinical characteristics on MSQOL-54 were studied. RESULTS: QOL was worse in pwMS. A better QOL in pwMS was linked to being male, having relapsing-remitting MS, having lower BMI, being employed, having a low disability, having no or minimal depression, and not fatigued. Age, disease duration, marital status, living status, and level of education did not affect the QOL. QOL showed a moderate to strong correlation with depression and fatigue and a weak correlation with EDSS. Depression and fatigue were the strongest predictors of QOL. Other predictors included gender and BMI but not EDSS. CONCLUSIONS: Many of the factors which seem to influence QOL in pwMS are modifiable. Evaluation and management of such factors may improve QOL in pwMS.Implications for rehabilitationAssessment of QOL (using a proper tool) should be part of every pwMS evaluation.Depression and fatigue are the main predictors of QOL in pwMs, therefore, attention should be paid for their evaluation and management.Sexual dysfunction and pain should be assessed and managed early in the course of the disease.

Varied Composition and Underlying Mechanisms of Gut Microbiome in Neuroinflammation.

The human gut microbiome has been implicated in a host of bodily functions and their regulation, including brain development and cognition. Neuroinflammation is a relatively newer piece of the puzzle and is implicated in the pathogenesis of many neurological disorders. The microbiome of the gut may alter the inflammatory signaling inside the brain through the secretion of short-chain fatty acids, controlling the availability of amino acid tryptophan and altering vagal activation. Studies in Korea and elsewhere highlight a strong link between microbiome dynamics and neurocognitive states, including personality. For these reasons, re-establishing microbial flora of the gut looks critical for keeping neuroinflammation from putting the whole system aflame through probiotics and allotransplantation of the fecal microbiome. However, the numerosity of the microbiome remains a challenge. For this purpose, it is suggested that wherever possible, a fecal microbial auto-transplant may prove more effective. This review summarizes the current knowledge about the role of the microbiome in neuroinflammation and the various mechanism involved in this process. As an example, we have also discussed the autism spectrum disorder and the implication of neuroinflammation and microbiome in its pathogenesis.

MASP-2 and MASP-3 inhibitors block complement activation, inflammation, and microvascular stasis in a murine model of vaso-occlusion in sickle cell disease.

Patients with sickle cell disease (SCD) have ongoing hemolysis that promotes endothelial injury, complement activation, inflammation, vaso-occlusion, ischemia-reperfusion pathophysiology, and pain. Complement activation markers are increased in SCD in steady-state and further increased during vaso-occlusive crisis (VOC). However, the mechanisms driving complement activation in SCD have not been completely elucidated. Ischemia-reperfusion and heme released from hemoglobin during hemolysis, events that characterize SCD pathophysiology, can activate the lectin pathway (LP) and alternative pathway (AP), respectively. Here we evaluated the role of LP and AP in Townes sickle (SS) mice using inhibitory monoclonal antibodies (mAb) to mannose binding lectin (MBL)-associated serine protease (MASP)-2 or MASP-3, respectively. Townes SS mice were pretreated with MASP-2 mAb, MASP-3 mAb, isotype control mAb, or PBS before they were challenged with hypoxia-reoxygenation or hemoglobin. Pretreatment of SS mice with MASP-2 or MASP-3 mAb, markedly reduced Bb fragments, C4d and C5a in plasma and complement deposition in the liver, kidneys, and lungs collected 4 hours after challenge compared to control mAb-treated mice. Consistent with complement inhibition, hepatic inflammation markers NF-ĸB phospho-p65, VCAM-1, ICAM-1, and E-selectin were significantly reduced in SS mice pretreated with MASP-2 or MASP-3 mAb. Importantly, MASP-2 or MASP-3 mAb pretreatment significantly inhibited microvascular stasis (vaso-occlusion) induced by hypoxia-reoxygenation or hemoglobin. These studies suggest that the LP and the AP are both playing a role in promoting inflammation and vaso-occlusion in SCD. Inhibiting complement activation via the LP or the AP might inhibit inflammation and prevent VOC in SCD patients.

Sciatic nerve excursion during neural mobilization with ankle movement using dynamic ultrasound imaging: a cross-sectional study.

PURPOSE: Ankle movement is used as a sensitizing maneuver for sciatica during neurodynamic techniques. In vivo studies on the sciatic nerve biomechanics associated with ankle movement during different positions of neighboring joints are scarce. The aim of this study was to investigate sciatic nerve excursion during ankle dorsiflexion in different positions in a healthy population. METHODS: This is a cross-sectional study. High-resolution dynamic ultrasound imaging was used to measure longitudinal excursion of the sciatic nerve in the posterior thigh of 27 healthy participants during ankle dorsiflexion in six positions of the neck, hip, and knee. Both the long and short distance of the nerve excursion were measured. Wilcoxon signed-rank tests were used for data analysis, and Eta squared (r) was used to quantify the effect size. RESULTS: Ankle dorsiflexion resulted in distal sciatic nerve excursion that was significantly higher in positions in which the knee was extended (median 0.7-1.6 mm) than in positions in which the knee was flexed (median 0.5-1.4 mm) (P ≤ 0.049, r ≥ 0.379). There were no significant differences in nerve excursion between positions where the neck was neutral compared with positions where the neck was flexed (P ≥ 0.710, r ≤ 0.072) or between positions where the hip was neutral compared with positions where the hip was flexed (P ≥ 0.456, r ≤ 0.143). CONCLUSION: The positions of adjacent joints, particularly the knee, had an impact on the excursion of the sciatic nerve in the thigh during ankle movement.

Plasma-Derived Hemopexin as a Candidate Therapeutic Agent for Acute Vaso-Occlusion in Sickle Cell Disease: Preclinical Evidence.

People living with sickle cell disease (SCD) face intermittent acute pain episodes due to vaso-occlusion primarily treated palliatively with opioids. Hemolysis of sickle erythrocytes promotes release of heme, which activates inflammatory cell adhesion proteins on endothelial cells and circulating cells, promoting vaso-occlusion. In this study, plasma-derived hemopexin inhibited heme-mediated cellular externalization of P-selectin and von Willebrand factor, and expression of IL-8, VCAM-1, and heme oxygenase-1 in cultured endothelial cells in a dose-responsive manner. In the Townes SCD mouse model, intravenous injection of free hemoglobin induced vascular stasis (vaso-occlusion) in nearly 40% of subcutaneous blood vessels visualized in a dorsal skin-fold chamber. Hemopexin administered intravenously prevented or relieved stasis in a dose-dependent manner. Hemopexin showed parallel activity in relieving vascular stasis induced by hypoxia-reoxygenation. Repeated IV administration of hemopexin was well tolerated in rats and non-human primates with no adverse findings that could be attributed to human hemopexin. Hemopexin had a half-life in wild-type mice, rats, and non-human primates of 80-102 h, whereas a reduced half-life of hemopexin in Townes SCD mice was observed due to ongoing hemolysis. These data have led to a Phase 1 clinical trial of hemopexin in adults with SCD, which is currently ongoing.

Psychometric properties of an Arabic translation of the modified fatigue impact scale in patients with multiple sclerosis.

PURPOSE: To translate and validate the modified fatigue impact scale into Arabic (MFIS-A) in patients with multiple sclerosis (MS). METHODS: A total of 116 patients with relapsing remitting MS and 59 healthy participants were recruited. Fifty patients filled the MFIS-A twice with one week difference. Reliability was assessed by measuring Cronbach's α and intraclass correlation coefficient (ICC). The MFIS-A was correlated with the fatigue severity scale (FSS), the vitality domain of the Short Form 36 (SF-36V), the fatigue visual analogue scale (VAS-F), and the Beck Depression Inventory II (BDI-II) to assess validity. Dimensionality of the MFIS-A was investigated. A receiver operating characteristic (ROC) curve analysis was done and specificity and sensitivity were calculated. RESULTS: Factor analysis (based on 116 patients) revealed that the MFIS-A consists of two subscales: the physical/social and the cognitive subscales. The MFIS-A showed excellent test-retest reliability (ICC = 0.920) and internal consistency (Cronbach's α = 0.968). The minimal detectable change with 95% confidence interval was 14.68 (32.0%). The MFIS-A showed strong positive correlation with FSS and BDI-II, moderate positive correlation with VAS-F, negative moderate correlation with SF-36V, and weak correlation with EDSS. The MFIS-A differentiated healthy participants from patients with 79.3% sensitivity and 89.8% specificity. CONCLUSIONS: The MFIS-A showed good validity and reliability indicating its usefulness as an assessment measure for patients with MS.IMPLICATIONS FOR REHABILITATIONMFIS-A is a valid and reliable tool for fatigue evaluation for patients with relapsing remitting MS.The optimal cutoff scores of the total MFIS-A, the physical/social, and cognitive subscales which indicate fatigue are 35.5, 18.5, and 15.5, respectively.Changes of 14.68 or more points may indicate a clinically important change (a true change) in fatigue in patients with MS.

Soluble MD-2 and Heme in Sickle Cell Disease Plasma Promote Pro-Inflammatory Signaling in Endothelial Cells.

Recent evidence indicates that hemolysis in sickle cell disease (SCD) promotes inflammation via innate immune signaling through toll-like receptor 4 (TLR4). Free heme released by hemolyzed red blood cells can bind to myeloid differentiation factor-2 (MD-2) and activate TLR4 pro-inflammatory signaling on endothelium to promote vaso-occlusion and acute chest syndrome in murine models of SCD. MD-2 is co-expressed with TLR4 on cell membranes, but in inflammatory conditions, soluble MD-2 (sMD-2) is elevated in plasma. sMD-2 levels were significantly increased in human and murine sickle (SS) plasma as compared to normal (AA) plasma. Human umbilical vein endothelial cells (HUVEC) and human lung microvascular endothelial cells incubated with human SS plasma had significant increases in pro-inflammatory IL-8, IL-6, and soluble VCAM-1 secretion compared to endothelial cells incubated with AA plasma. The increase in HUVEC IL-8 secretion was blocked by depletion of sMD-2 from SS plasma and enhanced by the addition of sMD-2 to AA plasma. The TLR4 signaling inhibitor, TAK-242, inhibited HUVEC IL-8 secretion in response to SS plasma by 85%. Heme-agarose pull-down assays and UV/Vis spectroscopy demonstrated that heme binds to sMD-2. Hemopexin, a high affinity heme-binding protein, inhibited HUVEC IL-8 secretion induced by SS plasma or SS and AA plasma supplemented with sMD-2. These data suggest that sMD-2 bound to heme might play an important role in pro-inflammatory signaling by endothelium in SCD.

Endothelial nitric oxide synthase and nitric oxide regulate endothelial tissue factor expression in vivo in the sickle transgenic mouse.

Activation of the coagulation system is a characteristic feature of sickle cell anemia, which also includes clinical thrombosis. The sickle transgenic mouse abnormally expresses tissue factor (TF) on the pulmonary vein endothelium. Knowing that this aberrancy is stimulated by inflammation, we sought to determine whether nitric oxide (NO) contributes to regulation of endothelial TF expression in the sickle mouse model. We used the NY1DD sickle mouse, which exhibits a low-TF to high-TF phenotype switch on exposure to hypoxia/reoxygenation. Manipulations of NO biology, such as breathing NO or addition of arginine or L-NAME (N-nitro-L-arginine-methyl-ester) to the diet, caused significant modulations of TF expression. This was also seen in hBERK1 sickle mice, which have a different genetic background and already have high-TF even at ambient air. Study of NY1DD animals bred to overexpress endothelial nitric oxide synthase (eNOS; eNOS-Tg) or to have an eNOS knockout state (one eNOS(-/-) animal and several eNOS(+/-) animals) demonstrated that eNOS modulates endothelial TF expression in vivo by down-regulating it. Thus, the biodeficiency of NO characteristic of patients with sickle cell anemia may heighten risk for activation of the coagulation system.

The antinociceptive and anti-inflammatory effects of Salvia officinalis leaf aqueous and butanol extracts.

CONTEXT: The leaf of sage Salvia officinalis L. (Lamiaceae) is reputed in the folk medicine of Arabia, and Jordan in particular, to relieve pain associated with gastrointestinal disturbance. OBJECTIVES: Evaluation of the antinociceptive and anti-inflammatory activities of aqueous and butanol extracts of S. officinalis leaf. MATERIALS AND METHODS: The analgesic effects of the aqueous extract (10, 31.6, 100, 316, 1000 mg/kg) and butanol extract (10, 31.6, 100, 316 mg/kg) were studied using the hot-plate test for mice and the formalin-induced paw licking in rats. The effects were compared to those of morphine and the influence of naloxone on these effects was also evaluated. The same concentrations of both extracts were used to evaluate their anti-inflammatory effects using the cotton pellet granuloma and carrageenan-induced paw edema in rats. RESULTS: The aqueous extract (10, 31.6, 100, 316, 1000 mg/kg) and butanol extract (10, 31.6, 100, 316 mg/kg) caused analgesic effect in the hot-plate latency assay as well as in early and late phases of formalin-induced paw licking in rats. These effects were reduced by the opioid receptor antagonist, naloxone (5 mg/kg). The same range of doses of both extracts caused dose-dependent inhibition of carrageenan-induced paw edema in rats as well as inhibition of cotton pellet granuloma. DISCUSSION AND CONCLUSION: These observations suggest that the sage leaf aqueous and butanol extracts have analgesic and anti-inflammatory effects, confirming the traditional use of this plant for pain alleviation.