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1.  Effect of intra-arterial injection of lidocaine and methyl-prednisolone into middle meningeal artery on intractable headaches 
The present report describes the effect of intra-arterial injection of a dose of 40 mg lidocaine and 20 mg methylprednisolone into the middle meningeal artery of two patients suffering from severe headaches. The effect of injection of lidocaine and methylprednisolone was short lasting with effect manifesting within 5 min and lasting 5–8 h after injection. Both patients reported improvement in headache intensity after 24 h post-procedure. Intra-arterial injection of lidocaine and methylprednisolone may represent another treatment strategy for headaches not responsive to standard treatment.
PMCID: PMC4280869  PMID: 25566345
2.  Percutaneous Inferior Cervical Sympathetic Ganglion Blockade for the Treatment of Ventricular Tachycardia Storm: Case Report and Review of the Literature 
Running Title: Sympathetic Block for Ventricular Tachycardia Storm
We present the case of a patient with ventricular tachycardia storm refractory to medical therapy and multiple catheter ablations, successfully managed by percutaneous left inferior cervical sympathetic ganglion block.
A 70-year-old man with a history of ischemic cardiomyopathy and previous placement of implantable defibrillator developed intractable ventricular tachycardia recalcitrant to intravenous amiodarone, lidocaine, and multiple catheter ablations with radiofrequency energy and direct current. The patient received numerous defibrillator shocks that did not result in sustained restoration of sinus rhythm. A percutaneous inferior cervical sympathetic ganglion block was performed under fluoroscopic guidance, with the administration of bupivacaine by infiltration of the tissue between the left internal carotid artery and the cervical vertebral bodies.
Two and a half hours after the procedure, ventricular tachycardia converted to sinus rhythm. One month after discharge from the hospital, the patient remained free from sustained ventricular tachycardia and did not report discharges from his implantable defibrillator.
Percutaneous cervical sympathetic ganglion blockade appears to be an effective intervention in the treatment of ventricular tachycardia storm. Additional data are required before incorporating this technique into the management algorithm of incessant ventricular tachycardia.
PMCID: PMC4280879  PMID: 25566341
Sympathetic ganglion; ventricular storm; tachycardia; ganglion block
4.  Sentinel Hospital-Based Surveillance for Assessment of Burden of Rotavirus Gastroenteritis in Children in Pakistan 
PLoS ONE  2014;9(10):e108221.
To determine the burden and molecular epidemiology of rotavirus gastroenteritis in children hospitalized with severe acute watery diarrhea in Pakistan prior to introduction of rotavirus vaccine.
A cross-sectional study was carried out over a period of two years from 2006 – 2008 at five sentinel hospitals in the cities of Karachi, Lahore, Rawalpindi, and Peshawar. Stool samples collected from children under five years of age hospitalized with severe acute watery diarrhea were tested for rotavirus antigen via enzyme immunoassay (EIA) (IDEA REF K6020 Oxoid Ltd (Ely), Cambridge, United Kingdom). A subset of EIA positive stool samples were further processed for genotyping.
6679 children were enrolled and stool specimens of 2039 (30.5%) were positive for rotavirus. Rotavirus positivity ranged from 16.3% to 39.4% in the 5 hospitals with highest positivity in Lahore. 1241 (61%) of all rotavirus cases were in infants under one year of age. Among the strains examined for G-serotypes, the occurrence of G1, G2, G9 and G4 strains was found to be 28%, 24%, 14% and 13%, respectively. Among P-types, the most commonly occurring strains were P6 (31.5%) followed by P8 (20%) and P4 (12%). Prevalent rotavirus genotype in hospitalized children of severe diarrhea were G1P[8] 11.6% (69/593), followed by G2P[4] 10.4% (62/593), and G4P[6] 10.1% (60/593).
Approximately one third of children hospitalized with severe gastroenteritis in urban centers in Pakistan have rotavirus. Introduction of rotavirus vaccine in Pakistan's national immunization program could prevent many severe episodes and diarrheal deaths.
PMCID: PMC4189951  PMID: 25295613
5.  Outcome of Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Low Left Ventricular Ejection Fraction 
A high risk of regimen-related toxicity with allogeneic hematopoietic stem cell transplantation (allo-HSCT) limits this potentially curative treatment for patients with a left ventricular ejection fraction (LVEF) of ≥50%. We evaluated the frequency of cardiac complications and 100-day nonrelapse mortality (NRM) in 56 patients with a LVEF of ≤45%, who received allo HCTat our institution. The results were retrospectively compared with a matched control group with LVEF of ≥50%, which received an allogeneic stem cell transplantation (allo-SCT). After a median follow-up of 29 months in the study group, grade ≥2 cardiac complications were seen in 7 of 56 (12.5%) patients and cumulative incidence of 100-day NRM was 12.5% with no deaths from cardiac causes. In contrast, after a median follow-up of 49 months in the control group, grade >2 cardiac complications were seen in 19 of 161 patients (11.8%; P = 1.00) and cumulative incidence of 100-day NRM was 14.9% (P =.82). The presence of at least 1 of the 7 pretransplant cardiac risk factors (past history of smoking, hypertension, hyperlipidemia, coronary artery disease, arrhythmia, prior myocardial infarction, and congestive heart failure) was associated with a higher cardiac complication rate in the study group (P = .03). In conclusion, selected patients with a LVEF of ≤45% can safely receive allo-HCT without a significant increase in cardiac toxicity or NRM.
PMCID: PMC4112360  PMID: 19747634
Allogeneic hematopoietic stem cell transplantation; Low ejection fraction
6.  Dural venous sinuses distortion and compression with supratentorial mass lesions: a mechanism for refractory intracranial hypertension? 
To determine the effect of supratentorial intraparenchymal mass lesions of various volumes on dural venous sinuses structure and transluminal pressures.
Three set of preparations were made using adult isolated head derived from fresh human cadaver. A supratentorial intraparenchymal balloon was introduced and inflated at various volumes and effect on dural venous sinuses was assessed by serial intravascular ultrasound, computed tomographic (CT), and magnetic resonance (MR) venograms. Contrast was injected through a catheter placed in sigmoid sinus for both CT and MR venograms. Serial trasluminal pressures were measured from middle part of superior sagittal sinus in another set of experiments.
At intraparenchymal balloon inflation of 90 cm3, there was attenuation of contrast enhancement of superior sagittal sinus with compression visualized in posterior part of the sinus without any evidence of compression in the remaining sinus. At intraparenchymal balloon inflation of 180 and 210 cm3, there was compression and obliteration of superior sagittal sinus throughout the length of the sinus. In the coronal sections, at intraparenchymal balloon inflations of 90 and 120 cm3, compression and obliteration of the posterior part of superior sagittal sinus were visualized. In the axial images, basal veins were not visualized with intraparenchymal balloon inflation of 90 cm3 or greater although straight sinus was visualized at all levels of inflation. Trasluminal pressure in the middle part of superior sagittal sinus demonstrated a mild increase from 0 cm H2O to 0.4 cm H2O and 0.5 cm H2O with inflation of balloon to volume of 150 and 180 cm3, respectively. There was a rapid increase in transluminal pressure from 6.8 cm H2O to 25.6 cm H2O as the supratentorial mass lesion increased from 180 to 200 cm3.
Our experiments identified distortion and segmental and global obliteration of dural venous sinuses secondary to supratentorial mass lesion and increase in transluminal pressure with large volume lesions. The secondary involvement of dural venous sinuses may represent a mechanism for refractory intracranial hypertension.
PMCID: PMC4051903  PMID: 24920987
dural venous sinus; mass lesion, intracranial hypertension; superior sagittal sinus; transverse sinus
Innate immunity  2011;18(2):268-278.
Francisella tularensis is a coccobacillus that causes tularemia. F. tularensis lipopolysaccharide (LPS) has nominal biological activity. Currently there is controversy regarding the structure of the lipid A obtained from F. tularensis LVS. Therefore, to resolve this controversy, the purification and structural identification of this LPS was crucial. To achieve this, LPS from F. tularensis was acid hydrolyzed to obtain crude lipid A, which was methylated and purified by HPLC. HPLC peak fractions were analyzed by mass spectrometry. The structure of the major lipid A of F. tularensis LVS comprised the glucosamine disaccharide backbone substituted with four fatty acyl groups, and a phosphate (1-position) with molecular masses of 1505. The major lipid A component had 18:0[3-O (16)] in the distal subunit and two 18:0 (3-OH) fatty acyl chains at the 2- or the 3- positions of the reducing subunit. Other lipid A variants in fatty acyl groups, include a phosphate or a phosphoryl galactosamine at the 1-position, and a hexose attached to the lipid A at the 4’ or 6’ position and these have not been described before for the F. tularensis LVS. This analysis revealed that the lipid A from F. tularensis LVS is far more complex than originally believed.
PMCID: PMC3990266  PMID: 21709054
8.  Nutritional Supplement-5 with a Combination of Proteasome Inhibitors (Resveratrol, Quercetin, δ-Tocotrienol) Modulate Age-Associated Biomarkers and Cardiovascular Lipid Parameters in Human Subjects 
Age-associated altered redox imbalances and dysregulated immune function, contribute to the development of a variety of age associated diseases. Inflammatory markers and lipid profiles are useful prognostic indicators of a variety of age-associated and cardiovascular diseases. We have previously studied the impact of several proteasome inhibitors on several markers of inflammation and lipid profiles in vitro, in vivo, in cell lines, animal models, and in human subjects. The current study represents an extension of this work. Our main hypothesis is that a combination of various naturally-occurring proteasome inhibitors, which inhibits nitric oxide (NO), and C-reactive protein (CRP) mediated inflammation, will have better efficacy in the prevention and treatment of age-associated disorders including cardiovascular disease.
Two double blind, randomized, placebo-controlled cross-over trials were conducted to determine the impact of a mixture of NS-5 (resveratrol, pterostilbene, quercetin, δ-tocotrienol, nicotinic acid) on serum NO, CRP, γ-glutamyl-transferase (γ-GT) activity, total antioxidant status (TAS), total cholesterol, LDL-cholesterol, HDL-cholesterol and triglycerides levels. Healthy seniors (Group-1; n = 32) free-living (A, B; 16/group), and hypercholesterolemic (Group-2; n = 64) subjects on AHA-Step-1-diet were divided into two groups (C, D; 32/group). Baseline levels were established for parameters as mentioned above. Groups A, C were administered 4-capsules/d of NS-5 and groups B, D, placebo (starch) for 6-weeks. Groups were crossed-over, followed by a 2-week wash-out period. Groups A, C were given 4-capsules/d of placebo and groups B, D, 4-capsules/d of NS-5 for 6-weeks. Groups C, D were continued on AHA-Step-1-diet.
All the subjects completed each phase in both studies without any complaints. There were significant (P < 0.01 – 0.05) decreases in the serum levels of NO (30%, 26%), CRP (29%, 21%), γ-GT activity (14%, 17%), and blood pressure (systolic/diastolic, 3/6%, 3/3%) of Groups A and B, respectively, of free-living healthy seniors without affecting the total, HDL-, LDL-cholesterol or triglycerides compared to their respective baseline values. However, serum levels of NO (36%, 43%), CRP (31%, 48%), γ-GT (17%, 20%), total cholesterol (19%, 15%), LDL-cholesterol (28%, 20%), triglycerides (11%, 18%) of Groups C and D were significantly (P < 0.01-0.05) decreased with NS-5 treatment of hypercholesterolemic subjects compared to baseline values, without affecting the serum HDL-cholesterol levels. The serum levels of total antioxidant status (TAS) were increased (10%, 14%; P < 0.05) in Groups A and B, increased (19%, 24%; P < 0.02), and blood pressure (systolic/diastolic, 5/6%, 3/5%) in Groups C and D with NS-5 treatment, compared to respective baseline values.
The consumption of NS-5 mixture decreased significantly serum NO, CRP and γ-GT levels, improved TAS and lipid profiles at risk cardiovascular and hold promise for delaying onset of age-associated diseases.
PMCID: PMC3851026  PMID: 24319627
Anti-inflammatory and anti-ageing agents; Resveratrol; Quercetin; δ-tocotrienol; Nitric oxide (NO); C-reactive protein (CRP); γ-glutamyl-transferase (γ-GT); Total antioxidant status (TAS)
9.  Suppression of Nitric Oxide Production and Cardiovascular Risk Factors in Healthy Seniors and Hypercholesterolemic Subjects by a Combination of Polyphenols and Vitamins 
Dysregulated immune function associated with ageing has been implicated in a variety of human diseases. We have demonstrated the anti-inflammatory properties of resveratrol, pterostilbene, morin hydrate, quercetin, δ-tocotrienol, riboflavinin a variety of experimental animal models, and determined that these compounds act by inhibiting proteasome activity.
To determine whether serum nitric oxide (NO) levels increase with age in humans, and whether the combined cholesterol-lowering and inflammation-reducing properties of resveratrol, pterostilbene, Morin hydrate, quercetin, δ-tocotrienol, riboflavin, and nicotinic acid would reduce cardiovascular risk factors in humans when used as nutritional supplements with, or without, other dietary changes.
Elderly human subjects were stratified into two groups based on total serum cholesterol levels. Initial total serum cholesterol levels were normal and elevated in Group 1 and 2 subjects, respectively. Baseline serum NO, C-reactive protein (CRP), γ-glutamyltransferase (γ-GT) activity, uric acid, total antioxidant status (TAS), total cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides levels were established over a four week period. Group 1 subjects subsequently received nutritional supplementation with one of two different combinations (NS-7 = 25 mg of each, resveratrol, pterostilbene, quercetin, δ-tocotrienol, nicotinic acid, morin hydrate or NS-6 = morin hydrate replaced with quercetin, 50 mg/capsule). Group 2 subjects also received these nutritional supplements (two capsules/d), but an AHA Step-1 diet was also implemented. After these interventions were administered for four weeks, the above parameters were re-measured and changes from baseline levels determined. Nitric acid (NO) levels in children, young adults, and seniors were also compared.
The key results of the current study were: 1) that serum NO levels were significantly increased in seniors compared to both children (~80%) and young adults (~65%); 2) that the intake of two capsules/d of NS-7 or NS-6 for four weeks significantly (P < 0.05) decreased serum NO (39%, 24%), CRP (19%, 21%), uric acid (6%, 12%) levels, and γ-GT activity (8%, 6%), respectively in free-living healthy seniors; 3) that serum NO (36%, 29%), CRP (29%, 20%), uric acid (6%, 9%) γ-GT activity (9%, 18%), total cholesterol (8%, 11%), LDL-cholesterol (10%, 13%), and triglycerides (16%, 23%) levels were significantly (P < 0.02) decreased in hypercholesterolemic subjects restricted to AHA Step-1 diet plus intake of SN-7 or SN-6 (two capsules/d), respectively; 4) that TAS was increased (3%, 9%; P < 0.05) in free-living healthy seniors receiving NS-7 or NS-6 alone, and in hypercholesterolemic subjects plus AHA Step-1 diet (20%, 12%; P < 0.02) with either of the combinations tested.
Serum NO levels are elevated in elderly humans compared to children or young adults. Diet supplementation with combinations of resveratrol, pterostilbene, morin hydrate, quercetin, δ-tocotrienol, riboflavin, and nicotinic acid reduce cardiovascular risk factors in humans when used as nutritional supplements with, or without, other dietary changes.
PMCID: PMC3486425  PMID: 23125945
Resveratrol; Quercetin; δ-Tocotrienol; C-Reactive Protein; Nitric Oxide (NO); Tumor Necrosis Factor-alpha (TNF-α); Lipid Parameters
10.  Erythromelalgia: An Uncommon Presentation Precipitated by Aspirin Withdrawal 
Case Reports in Medicine  2012;2012:616125.
Erythromelalgia is a rare disorder frequently associated with myeloproliferative disorders. We describe a case of elderly patient diagnosed with myeloproliferative disorder in remission. The patient was on aspirin for secondary prevention of stroke and was taken off aspirin and developed erythromelalgia within two weeks of withdrawal of aspirin. After restarting aspirin, patient's symptoms improved within 2 weeks.
PMCID: PMC3403327  PMID: 22844295
11.  Inhibition of nitric oxide and inflammatory cytokines in LPS-stimulated murine macrophages by resveratrol, a potent proteasome inhibitor 
Altered immune function during ageing results in increased production of nitric oxide (NO) and other inflammatory mediators. Recently, we have reported that NO production was inhibited by naturally-occurring proteasome inhibitors (quercetin, δ-tocotrienol, and riboflavin) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, and thioglycolate-elicited peritoneal macrophages from C57BL/6 mice. In a continuous effort to find more potent, non-toxic, commercially available, naturally-occurring proteasome inhibitors that suppress inflammation, the present study was carried out to describe the inhibition of NF-κB activation and NO, TNF-α, IL-6, IL-1β, and iNOS expression by trans-resveratrol, trans-pterostilbene, morin hydrate, and nicotinic acid in LPS-induced RAW 264.7 cells and thioglycolate-elicited peritoneal macrophages from C57BL/6 and BALB/c mice.
The present results indicate that resveratrol, pterostilbene, and morin hydrate caused significant inhibition (>70% to 90%; P < 0.02) in the activities of chymotrypsin-like, trypsin-like, and post-acidic (post-glutamase) proteasome sites in RAW 264.7 cells at a dose of only 20 μM. These compounds also inhibited the production of NO by RAW-264.7 cells stimulated with LPS alone (>40%; P < 0.05), or LPS + interferon-γ (IFN-γ; >60%; P < 0.02). Furthermore, resveratrol, pterostilbene, morin hydrate, and quercetin suppressed secretion of TNF-α (>40%; P < 0.05) in LPS-stimulated RAW 264.7 cells, and suppressed NF-κB activation (22% to 45%; P < 0.05) in LPS-stimulated HEK293T cells. These compounds also significantly suppressed LPS-induced expression of TNF-α, IL-1β, IL-6, and iNOS genes in RAW 264.7 cells, and also in thioglycolate-elicited peritoneal macrophages from C57BL/6 and BALB/c mice.
The present results clearly demonstrate that resveratrol and pterostilbene are particularly potent proteasome inhibitors that suppress expression of genes, and production of inflammatory products in LPS-stimulated RAW 264.7 cells, and macrophages from C57BL/6 and BALB/c mice. Resveratrol and pterostilbene which are present in grapes, blueberries, and red wine, have been implicated as contributing factors to the lower incidence of cardiovascular disease in the French population, despite their relatively high dietary fat intake. Consequently, it appears likely that the beneficial nutritional effects of resveratrol and pterostilbene are due at least in part, to their ability to inhibit NF-κB activation by the proteasome, thereby suppressing activation of pro-inflammatory cytokines and iNOS genes, resulting in decreased secretion of TNF-α, IL-1β, IL-6, and NO levels, in response to inflammatory stimuli. This is the first report demonstrating that resveratrol and pterostilbene act as proteasome inhibitors, thus providing a mechanism for their anti-inflammatory effects.
PMCID: PMC3393619  PMID: 22698256
Nitric oxide (NO); TNF-α; NF-κB; Cytokines; Resveratrol; Proteasome inhibitors
12.  LPS-Induced Formation of Immunoproteasomes: TNF-α and Nitric Oxide Production are Regulated by Altered Composition of Proteasome-Active Sites 
Cell biochemistry and biophysics  2011;60(1-2):77-88.
Stimulation of mouse macrophages with LPS leads to tumor necrosis factor (TNF-α) secretion and nitric oxide (NO) release at different times through independent signaling pathways. While the precise regulatory mechanisms responsible for these distinct phenotypic responses have not been fully delineated, results of our recent studies strongly implicate the cellular cytoplasmic ubiquitin–proteasome pathway as a key regulator of LPS-induced macrophage inflammatory responses. Our objective in this study was to define the relative contribution of specific proteasomal active-sites in induction of TNF-α and NO after LPS treatment of RAW 264.7 macrophages using selective inhibitors of these active sites. Our data provide evidence that LPS stimulation of mouse macrophages triggers a selective increase in the levels of gene and protein expression of the immunoproteasomes, resulting in a modulation of specific functional activities of the proteasome and a corresponding increase in NO production as compared to untreated controls. These findings suggest the LPS-dependent induction of immunoproteasome. In contrast, we also demonstrate that TNF-α expression is primarily dependent on both the chymotrypsin- and the trypsin-like activities of X, Y, Z subunits of the proteasome. Proteasome-associated post-acidic activity alone also contributes to LPS-induced expression of TNF-α. Taken together; our results indicate that LPS-induced TNF-α in macrophages is differentially regulated by each of the three proteasome activities. Since addition of proteasome inhibitors to mouse macrophages profoundly affects the degradation of proteins involved in signal transduction, we conclude that proteasome-specific degradation of several signaling proteins is likely involved in differential regulation of LPS-dependent secretion of proinflammatory mediators.
PMCID: PMC3119080  PMID: 21455682
Inflammation; Endotoxic shock; Cytokines; Nitric oxide
13.  The Immunoproteasomes Regulate Lps-Induced Trif/Tram Signaling Pathway in Murine Macrophages 
Cell biochemistry and biophysics  2011;60(1-2):119-126.
We have proposed the novel concept that the macrophage ubiquitin-proteasome (UP) pathway functions as a key regulator of LPS-induced inflammation signaling. Our findings suggest that proteasome-associated protease subunits X, Y, and Z, are replaced by LMP subunits after LPS treatment of RAW 264.7 cells. Our objective here was to determine the contribution of selective LMP proteasomal subunits to LPS-induced nitric oxide (NO) and TNF-α production in primary murine macrophages. Accordingly, thioglycollate-elicited macrophages from LMP7, LMP2, LMP10 (MECL-1), and LMP7/MECL-1 double knockout mice were stimulated in vitro with LPS, and were found to generate markedly reduced NO levels compared to wild-type (WT) mice, whereas TNF-α levels responses were essentially unaltered relative to wild-type responses. Our recent studies suggest that the TRIF/TRAM pathway is defective in LMP knockouts which may explain why iNOS/NO are not robustly induced in LPS-treated macrophages from knockouts. Treating these macrophages with IFN-γ and LPS, however, reverses this defect, leading to robust NO induction. TNF-α is induced by LPS in the LMP knockout macrophages because IκB and IRAK are degraded normally via the MyD88 pathway. Collectively, these findings strongly support the concept that LMP7/MECL-1 proteasomes subunits actively function to regulate LPS-induced NO production by affecting the TRIF/TRAM pathway.
PMCID: PMC3212439  PMID: 21455681
inflammation; endotoxic shock; macrophages; cytokines; nitric oxide
14.  Inhibition of nitric oxide in LPS-stimulated macrophages of young and senescent mice by δ-tocotrienol and quercetin 
Changes in immune function believed to contribute to a variety of age-related diseases have been associated with increased production of nitric oxide (NO). We have recently reported that proteasome inhibitors (dexamethasone, mevinolin, quercetin, δ-tocotrienol, and riboflavin) can inhibit lipopolysaccharide (LPS)-induced NO production in vitro by RAW 264.7 cells and by thioglycolate-elicited peritoneal macrophages derived from four strains of mice (C57BL/6, BALB/c, LMP7/MECL-1-/- and PPAR-α-/- knockout mice). The present study was carried out in order to further explore the potential effects of diet supplementation with naturally-occurring inhibitors (δ-tocotrienol and quercetin) on LPS-stimulated production of NO, TNF-α, and other pro-inflammatory cytokines involved in the ageing process. Young (4-week-old) and senescent mice (42-week old) were fed control diet with or without quercetin (100 ppm), δ-tocotrienol (100 ppm), or dexamethasone (10 ppm; included as positive control for suppression of inflammation) for 4 weeks. At the end of feeding period, thioglycolate-elicited peritoneal macrophages were collected, stimulated with LPS, LPS plus interferon-β (IFN-β), or LPS plus interferon-γ (IFN-γ), and inflammatory responses assessed as measured by production of NO and TNF-α, mRNA reduction for TNF-α, and iNOS genes, and microarray analysis.
Thioglycolate-elicited peritoneal macrophages prepared after four weeks of feeding, and then challenged with LPS (10 ng or 100 ng) resulted in increases of 55% and 73%, respectively in the production of NO of 46-week-old compared to 8-week-old mice fed control diet alone (respective control groups), without affecting the secretion of TNF-α among these two groups. However, macrophages obtained after feeding with quercetin, δ-tocotrienol, and dexamethasone significantly inhibited (30% to 60%; P < 0.02) the LPS-stimulated NO production, compared to respective control groups. There was a 2-fold increase in the production of NO, when LPS-stimulated macrophages of quercetin, δ-tocotrienol, or dexamethasone were also treated with IFN-β or IFN-γ compared to respective control groups. We also demonstrated that NO levels and iNOS mRNA expression levels were significantly higher in LPS-stimulated macrophages from senescent (0.69 vs 0.41; P < 0.05), compared to young mice. In contrast, age did not appear to impact levels of TNF-α protein or mRNA expression levels (0.38 vs 0.35) in LPS-stimulated macrophages. The histological analyses of livers of control groups showed lesions of peliosis and microvesicular steatosis, and treated groups showed Councilman body, and small or large lymphoplasmacytic clusters.
The present results demonstrated that quercetin and δ-tocotrienols inhibit the LPS-induced NO production in vivo. The microarray DNA analyses, followed by pathway analyses indicated that quercetin or δ-tocotrienol inhibit several LPS-induced expression of several ageing and pro-inflammatory genes (IL-1β, IL-1α, IL-6, TNF-α, IL-12, iNOS, VCAM1, ICAM1, COX2, IL-1RA, TRAF1 and CD40). The NF-κB pathway regulates the production of NO and inhibits the pro-inflammatory cytokines involved in normal and ageing process. These ex vivo results confirmed the earlier in vitro findings. The present findings of inhibition of NO production by quercetin and δ-tocotrienol may be of clinical significance treating several inflammatory diseases, including ageing process.
PMCID: PMC3267680  PMID: 22185406
15.  Suppression of nitric oxide induction and pro-inflammatory cytokines by novel proteasome inhibitors in various experimental models 
Inflammation has been implicated in a variety of diseases associated with ageing, including cancer, cardiovascular, and neurologic diseases. We have recently established that the proteasome is a pivotal regulator of inflammation, which modulates the induction of inflammatory mediators such as TNF-α, IL-1, IL-6, and nitric oxide (NO) in response to a variety of stimuli. The present study was undertaken to identify non-toxic proteasome inhibitors with the expectation that these compounds could potentially suppress the production of inflammatory mediators in ageing humans, thereby decreasing the risk of developing ageing related diseases. We evaluated the capacity of various proteasome inhibitors to suppress TNF-α, NO and gene suppression of TNF-α, and iNOS mRNA, by LPS-stimulated macrophages from several sources. Further, we evaluated the mechanisms by which these agents suppress secretion of TNF-α, and NO production. Over the course of these studies, we measured the effects of various proteasome inhibitors on the RAW 264.7 cells, and peritoneal macrophages from four different strains of mice (C57BL/6, BALB/c, proteasome double subunits knockout LMP7/MECL-1-/-, and peroxisome proliferator-activated receptor-α,-/- (PPAR-α,-/-) knockout mice. We also directly measured the effect of these proteasome inhibitors on proteolytic activity of 20S rabbit muscle proteasomes.
There was significant reduction of chymotrypsin-like activity of the 20S rabbit muscle proteasomes with dexamethasone (31%), mevinolin (19%), δ-tocotrienol (28%), riboflavin (34%), and quercetin (45%; P < 0.05). Moreover, quercetin, riboflavin, and δ-tocotrienol also inhibited chymotrypsin-like, trypsin-like and post-glutamase activities in RAW 264.7 whole cells. These compounds also inhibited LPS-stimulated NO production and TNF-α, secretion, blocked the degradation of P-IκB protein, and decreased activation of NF-κB, in RAW 264.7 cells. All proteasome inhibitors tested also significantly inhibited NO production (30% to 60% reduction) by LPS-induced thioglycolate-elicited peritoneal macrophages derived from all four strains of mice. All five compounds also suppressed LPS-induced TNF-α, secretion by macrophages from C57BL/6 and BALB/c mice. TNF-α, secretion, however, was not suppressed by any of the three proteasome inhibitors tested (δ-tocotrienol, riboflavin, and quercetin) with LPS-induced macrophages from LMP7/MECL-1-/- and PPAR-α,-/- knockout mice. Results of gene expression studies for TNF-α, and iNOS were generally consistent with results obtained for TNF-α, protein and NO production observed with four strains of mice.
Results of the current study demonstrate that δ-tocotrienol, riboflavin, and quercetin inhibit NO production by LPS-stimulated macrophages of all four strains of mice, and TNF-α, secretion only by LPS-stimulated macrophages of C57BL/6 and BALB/c mice. The mechanism for this inhibition appears to be decreased proteolytic degradation of P-IκB protein by the inhibited proteasome, resulting in decreased translocation of activated NF-κB to the nucleus, and depressed transcription of gene expression of TNF-α, and iNOS. Further, these naturally-occurring proteasome inhibitors tested appear to be relatively potent inhibitors of multiple proteasome subunits in inflammatory proteasomes. Consequently, these agents could potentially suppress the production of inflammatory mediators in ageing humans, thereby decreasing the risk of developing a variety of ageing related diseases.
PMCID: PMC3206449  PMID: 21992595
Shock (Augusta, Ga.)  2010;34(4):390-401.
Our previous work has provided strong evidence that the proteasome is central to the vast majority of genes induced in mouse macrophages in response to lipopolysaccharide (LPS) stimulation. In the studies presented here, we evaluated the role of the macrophage proteasome in response to a second microbial product CpG DNA (unmethylated bacterial DNA). For these studies, we applied Affymetrix microarray analysis of RNA derived from murine macrophages stimulated with CpG DNA in the presence or absence of proteasome inhibitor, lactacystin. The results of these studies revealed that similar to LPS, a vast majority of those macrophage genes regulated by CpG DNA are also under the control of the proteasome at 4 h. In contrast to LPS stimulation, however, many of these genes were induced much later than 4 h, at 18 h, in response to CpG DNA. Lactacystin treatment of macrophages completely blocked the CpG DNA-induced gene expression of TNF-α and other genes involved in production of inflammatory mediators. These data strongly support the conclusion that, similar to LPS, the macrophage proteasome is a key regulator of CpG DNA-induced signaling pathways.
PMCID: PMC2943147  PMID: 20160661
17.  Preventive effects of phytoestrogens against postmenopausal osteoporosis as compared to the available therapeutic choices: An overview 
Estrogen deficiency is a major risk factor for osteoporosis in postmenopausal women. Although hormone replacement therapy (HRT) has been rampantly used to recompense for the bone loss, but the procedure is coupled with severe adverse effects. Hence, there is a boost in the production of newer synthetic products to ward off the effects of menopause-related osteoporosis. As of today, there are several prescription products available for the treatment of postmenopause osteoporosis; most of these are estrogenic agents and combination products. Nevertheless, in view of the lack of effect and/or toxicity of these products, majority of the postmenopausal women are now fascinated by highly publicized natural products. This is an offshoot of the generalized consensus that these products are more effective and free from any adverse effects. Recently, certain plant-derived natural products, mostly phytoestrogens (isoflavones, lignans, coumestanes, stilbenes, flavonoids) and many more novel estrogen-like compounds in plants have been immensely used to prevent menopause-related depletion in bone mineral density (BMD). Although, a number of papers are published on menopause-related general symptoms, sexual dysfunction, cardiovascular diseases, Alzheimer's disease, diabetes, colon, and breast cancers, there is paucity of literature on the accompanying osteoporosis and its treatment. In view of the controversies on synthetic hormones and drugs and drift of a major population of patients toward natural drugs, it was found worthwhile to investigate if these drugs are suitable to be used in the treatment of postmenopausal osteoporosis. Preparation of this paper is an attempt to review the (a) epidemiology of postmenopausal osteoporosis, (b) treatment modalities of postmenopausal osteoporosis by hormones and synthetic drugs and the associated drawbacks and adverse effects, and (c) prevention and treatment of postmenopausal osteoporosis by phytoestrogens, their drawbacks and toxicity. It is apparent that both the categories of treatment are useful and both have adverse effects, but the plant products are nonscientific and hence are not advised to be used till more studies are undertaken to ensure that the benefits clearly outweigh the risk, in addition to recognition by Food and Drug Administration.
PMCID: PMC3276006  PMID: 22346228
Menopause; osteoporosis; phytoestrogens
18.  Tocotrienols-induced inhibition of platelet thrombus formation and platelet aggregation in stenosed canine coronary arteries 
Dietary supplementation with tocotrienols has been shown to decrease the risk of coronary artery disease. Tocotrienols are plant-derived forms of vitamin E, which have potent anti-inflammatory, antioxidant, anticancer, hypocholesterolemic, and neuroprotective properties. Our objective in this study was to determine the extent to which tocotrienols inhibit platelet aggregation and reduce coronary thrombosis, a major risk factor for stroke in humans. The present study was carried out to determine the comparative effects of α-tocopherol, α-tocotrienol, or tocotrienol rich fraction (TRF; a mixture of α- + γ- + δ-tocotrienols) on in vivo platelet thrombosis and ex vivo platelet aggregation (PA) after intravenous injection in anesthetized dogs, by using a mechanically stenosed circumflex coronary artery model (Folts' cyclic flow model).
Collagen-induced platelet aggregation (PA) in platelet rich plasma (PRP) was decreased markedly after treatment with α-tocotrienol (59%; P < 0.001) and TRF (92%; P < 0.001). α-Tocopherol treatment was less effective, producing only a 22% (P < 0.05) decrease in PA. Adenosine diphosphate-induced (ADP) PA was also decreased after treatment with α-tocotrienol (34%; P < 0.05) and TRF (42%; P < 0.025). These results also indicate that intravenously administered tocotrienols were significantly better than tocopherols in inhibiting cyclic flow reductions (CFRs), a measure of the acute platelet-mediated thrombus formation. Tocotrienols (TRF) given intravenously (10 mg/kg), abolished CFRs after a mean of 68 min (range 22 -130 min), and this abolition of CFRs was sustained throughout the monitoring period (50 - 160 min).
Next, pharmacokinetic studies were carried out and tocol levels in canine plasma and platelets were measured. As expected, α-Tocopherol treatment increased levels of total tocopherols in post- vs pre-treatment specimens (57 vs 18 μg/mL in plasma, and 42 vs 10 μg/mL in platelets). However, treatment with α-tocopherol resulted in slightly decreased levels of tocotrienols in post- vs pre-treatment samples (1.4 vs 2.9 μg/mL in plasma and 2.3 vs 2.8 μg/mL in platelets). α-Tocotrienol treatment increased levels of both tocopherols and tocotrienols in post- vs pre-treatment samples (tocopherols, 45 vs 10 μg/mL in plasma and 28 vs 5 μg/mL in platelets; tocotrienols, 2.8 vs 0.9 μg/mL in plasma and 1.28 vs 1.02 μg/mL in platelets). Treatment with tocotrienols (TRF) also increased levels of tocopherols and tocotrienols in post- vs pre-treatment samples (tocopherols, 68 vs 20 μg/mL in plasma and 31.4 vs 7.9 μg/mL in platelets; tocotrienols, 8.6 vs 1.7 μg/mL in plasma and 3.8 vs 3.9 μg/mL in platelets).
The present results indicate that intravenously administered tocotrienols inhibited acute platelet-mediated thrombus formation, and collagen and ADP-induced platelet aggregation. α-Tocotrienols treatment induced increases in α-tocopherol levels of 4-fold and 6-fold in plasma and platelets, respectively. Interestingly, tocotrienols (TRF) treatment induced a less pronounced increase in the levels of tocotrienols in plasma and platelets, suggesting that intravenously administered tocotrienols may be converted to tocopherols. Tocotrienols, given intravenously, could potentially prevent pathological platelet thrombus formation and thus provide a therapeutic benefit in conditions such as stroke and myocardial infarction.
PMCID: PMC3096575  PMID: 21489303
19.  δ-Tocotrienol and quercetin reduce serum levels of nitric oxide and lipid parameters in female chickens 
Chronic, low-grade inflammation provides a link between normal ageing and the pathogenesis of age-related diseases. A series of in vitro tests confirmed the strong anti-inflammatory activities of known inhibitors of NF-κB activation (δ-tocotrienol, quercetin, riboflavin, (-) Corey lactone, amiloride, and dexamethasone). δ-Tocotrienol also suppresses β-hydroxy-β-methylglutaryl coenzyme A (HMG-CoA) reductase activity (the rate-limiting step in de novo cholesterol synthesis), and concomitantly lowers serum total and LDL cholesterol levels. We evaluated these compounds in an avian model anticipating that a dietary additive combining δ-tocotrienol with quercetin, riboflavin, (-) Corey lactone, amiloride, or dexamethasone would yield greater reductions in serum levels of total cholesterol, LDL-cholesterol and inflammatory markers (tumor necrosis factor-α [TNF-α], and nitric oxide [NO]), than that attained with the individual compounds.
The present results showed that supplementation of control diets with all compounds tested except riboflavin, (-) Corey lactone, and dexamethasone produced small but significant reductions in body weight gains as compared to control. (-) Corey lactone and riboflavin did not significantly impact body weight gains. Dexamethasone significantly and markedly reduced weight gain (>75%) compared to control. The serum levels of TNF-α and NO were decreased 61% - 84% (P < 0.001), and 14% - 67%, respectively, in chickens fed diets supplemented with δ-tocotrienol, quercetin, riboflavin, (-) Corey lactone, amiloride, or dexamethasone as compared to controls. Significant decreases in the levels of serum total and LDL-cholesterol were attained with δ-tocotrienol, quercetin, riboflavin and (-) Corey lactone (13% - 57%; P < 0.05), whereas, these levels were 2-fold higher in dexamethasone treated chickens as compared to controls. Parallel responses on hepatic lipid infiltration were confirmed by histological analyses. Treatments combining δ-tocotrienol with the other compounds yielded values that were lower than individual values attained with either δ-tocotrienol or the second compound. Exceptions were the significantly lower total and LDL cholesterol and triglyceride values attained with the δ-tocotrienol/(-) Corey lactone treatment and the significantly lower triglyceride value attained with the δ-tocotrienol/riboflavin treatment. δ-Tocotrienol attenuated the lipid-elevating impact of dexamethasone and potentiated the triglyceride lowering impact of riboflavin. Microarray analyses of liver samples identified 62 genes whose expressions were either up-regulated or down-regulated by all compounds suggesting common impact on serum TNF-α and NO levels. The microarray analyses further identified 41 genes whose expression was differentially impacted by the compounds shown to lower serum lipid levels and dexamethasone, associated with markedly elevated serum lipids.
This is the first report describing the anti-inflammatory effects of δ-tocotrienol, quercetin, riboflavin, (-) Corey lactone, amiloride, and dexamethasone on serum TNF-δ and NO levels. Serum TNF-δ levels were decreased by >60% by each of the experimental compounds. Additionally, all the treatments except with dexamethasone, resulted in lower serum total cholesterol, LDL-cholesterol and triglyceride levels. The impact of above mentioned compounds on the factors evaluated herein was increased when combined with δ-tocotrienol.
PMCID: PMC3053241  PMID: 21356098
20.  Tocotrienols inhibit lipopolysaccharide-induced pro-inflammatory cytokines in macrophages of female mice 
Inflammation has been implicated in cardiovascular disease, and the important role of proteasomes in the development of inflammation and other macrophage functions has been demonstrated. Tocotrienols are potent hypocholesterolemic agents that inhibit β-hydroxy-β-methylglutaryl coenzyme A reductase activity, which is degraded via the ubiquitin-proteasome pathway. Our objective was to evaluate the effect of tocotrienols in reducing inflammation. Lipopolysaccharide (LPS) was used as a prototype for inflammation in murine RAW 264.7 cells and BALB/c female mice.
The present results clearly demonstrate that α-, γ-, or δ-tocotrienol treatments inhibit the chymotrypsin-like activity of 20 S rabbit muscle proteasomes (> 50%; P < 0.05). Chymotrypsin, trypsin, and post-glutamase activities were decreased > 40% (P < 0.05) with low concentrations (< 80 μM), and then increased gradually with concentrations of (80 - 640 μM) in RAW 264.7 whole cells. Tocotrienols showed 9 - 33% (P < 0.05) inhibitions in TNF-α secretion in LPS-stimulated RAW 264.7 cells. Results of experiments carried out in BALB/c mice demonstrated that serum levels of TNF-α after LPS treatment were also reduced (20 - 48%; P < 0.05) by tocotrienols with doses of 1 and 10 μg/kg, and a corresponding rise in serum levels of corticosterone (19 - 41%; P < 0.05) and adrenocorticotropic hormone (81 - 145%; P < 0.02) was observed at higher concentrations (40 μM). Maximal inhibition of LPS-induced TNF-α was obtained with δ-tocotrienol (10 μg/kg). Low concentrations of δ-Tocotrienols (< 20 μM) blocked LPS-induced gene expression of TNF-α, IL-1β, IL-6 and iNOS (> 40%), while higher concentrations (40 μM) increased gene expression of the latter in peritoneal macrophages (prepared from BALB/c mice) as compared to control group.
These results represent a novel approach by using natural products, such as tocotrienols as proteasome modulators, which may lead to the development of new dietary supplements of tocotrienols for cardiovascular diseases, as well as others that are based on inflammation.
PMCID: PMC3016328  PMID: 21162750
21.  Gallstone ileus an unusual reason for right iliac fossa pain in Crohn's disease: a case report 
Cases Journal  2009;2:9142.
The symptoms of Crohn's disease can vary significantly among afflicted individuals. It is a chronic inflammatory disease, which has many complications involving gastrointestinal and other symptoms. The diagnosis of gallstone ileus is difficult in Crohn's disease and an early diagnosis can reduce mortality. We present a case of 72 year old female with known Crohn's disease with acute right iliac fossa pain. She was investigated with an abdominal CT, giving a definite diagnosis of gallstone ileus.
PMCID: PMC2803939  PMID: 20062659
22.  Gallstone ileus an unusual reason for right iliac fossa pain in Crohn's disease: a case report 
Cases Journal  2009;2:9285.
The symptoms of Crohn's disease can vary significantly among afflicted individuals. It is a chronic inflammatory disease, which has many complications involving gastrointestinal and other symptoms. The diagnosis of gallstone ileus is difficult in Crohn's disease and an early diagnosis can reduce mortality. We present a case of 72 year old female with known Crohn's disease with acute right iliac fossa pain. She was investigated with an abdominal CT, giving a definite diagnosis of gallstone ileus.
PMCID: PMC2827046  PMID: 20184714
23.  A combination of proteasome inhibitors and antibiotics prevents lethality in a septic shock model 
Innate immunity  2008;14(5):319-329.
Our recent studies with lactacystin, a prototype proteasome inhibitor, have suggested that the proteasome is a key regulator of LPS-induced signaling pathways contributing to the inflammatory process. Moreover, lactacystin protects animals from LPS-induced shock. Therefore, we sought to identify other less toxic compounds that would block the chymotrypsin-like activity of the proteasome or LPS-induced nitric oxide (NO). After screening over 100 natural compounds (based on chemistry and inhibition of LPS-induced biological activities), we now report for the first time that quercetin, like lactacystin (the prototype proteasome inhibitor), and mevinolin are also inhibitors of the chymotrypsin-like activity of the cellular proteasome within living cells. In addition, this study also suggests that mevinolin and quercetin both have relatively potent anti-inflammatory effects on LPS-treated macrophages in vitro. Interestingly, both of these compounds behave like lactacystin in that they block LPS-induced NO to a greater extent than TNF-α. The results of our experiments clearly suggest that mevinolin, in combination with the antibiotic imipenem, can provide protection against polymicrobial septic lethality induced by cecal-ligation and puncture in mice. Collectively, these studies strongly support the conclusion that therapeutic targeting of cellular proteasomes, in conjunction with standard antimicrobial therapy, may be of considerable survival benefit in the treatment of septic shock.
PMCID: PMC2666041  PMID: 18809656
Inflammation; endotoxic shock; macrophages; statins; Primaxin; cytokines; nitric oxide
25.  Cryofibrinogenaemia: not just skin deep 
BMJ Case Reports  2013;2013:bcr2012008102.
A Caucasian woman in her 60s with a history of rheumatoid arthritis presented to our institution complaining of skin ulceration. Her initial course was complicated by superinfection and sepsis until a diagnosis of cryofibrinogenaemia was finally established. Cryofibrinogenaemia remains as an under-recognised entity in part, because it can mimic other causes of skin ulcerations. In addition, its diagnosis can be challenging because of the particular handling techniques required of lab specimens. This case exemplifies some of the diagnostic and treatment challenges encountered while managing the patient with cryofibrinogenaemia.
PMCID: PMC3604005  PMID: 23429017

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