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Free radical biology & medicine  2009;46(12):1626-1632.
Cobalamins (Cbl) are important co-factors for methionine synthase and methylmalonyl-coA mutase. Certain corrins also bind nitric oxide (NO), quenching its bioactivity. To determine if corrins would inhibit NO synthase (NOS), we measured their effects on 14-C-L-arginine-to-14-C-L-citrulline conversion by NOS1, NOS2, and NOS3. Hydroxocobalamin (OH-Cbl), cobinamide (Cbi), and dicyanocobinamide (CN2-Cbi) potently inhibited all isoforms, whfile cyanocobalamin, methylcobalamin, and adenosylcobalamin had much less effect. OH-Cbl and CN2-Cbi prevented binding of the oxygen analog carbon monoxide (CO) to the reduced NOS1 and NOS2 heme active site. CN2-Cbi did not react directly with NO or CO. Spectral perturbation analysis showed that CN2-Cbi interacted directly with the purified NOS1 oxygenase domain. NOS inhibition by corrins was rapid and not reversed by dialysis with L-arginine, tetrahydrobiopterin. Molecular modeling indicated that corrins could access the unusually large heme and substrate-binding pocket of NOS. Best fits were obtained in the “base-off” conformation of the lower axial dimethylbenzimidazole ligand. CN2-Cbi inhibited interferon-γ-activated Raw264.7 mouse macrophage NO production. We show for the first time that certain corrins directly inhibit NOS, suggesting that these agents (or their derivatives) may have pharmacological utility. Endogenous cobalamins and cobinamides might play important roles regulating NOS activity in normal and pathological conditions.
PMCID: PMC2745708  PMID: 19328848
cobalamin; cobinamide; vitamin B12; nitric oxide; nitric oxide synthase; arginine; macrophage
2.  Arginine, nitric oxide, carbon monoxide, and endothelial function in severe malaria 
Purpose of review
Parasiticidal therapy of severe falciparum malaria improves outcome, but up to 30% of these patients die despite best therapy. Nitric oxide is protective against severe disease, and both nitric oxide and arginine (the substrate for nitric oxide synthase) are low in clinical malaria. Parasitized red blood cell interactions with endothelium are important in the pathophysiology of malaria. This review describes new information regarding nitric oxide, arginine, carbon monoxide, and endothelial function in malaria.
Recent findings
Low arginine, low nitric oxide production, and endothelial dysfunction are common in severe malaria. The degree of hypoargininemia and endothelial dysfunction (measured by reactive hyperemia peripheral artery tonometry) is proportional to parasite burden and severity of illness. Plasma arginase (an enzyme that catabolizes arginine) is elevated in severe malaria. Administering arginine intravenously reverses hypoargininemia and endothelial dysfunction. The cause(s) of hypoargininemia in malaria is unknown. Carbon monoxide (which shares certain functional properties with nitric oxide) protects against cerebral malaria in mice.
Replenishment of arginine and restoration of nitric oxide production in clinical malaria should diminish parasitized red blood cells adherence to endothelium and reduce the sequelae of these interactions (e.g., cerebral malaria). Arginine therapy given in addition to conventional antimalaria treatment may prove to be beneficial in severe malaria.
PMCID: PMC2732119  PMID: 18725795
arginine; carbon monoxide; endothelium; malaria; nitric oxide
3.  Interferon (IFN)-α Activation of Human Blood Mononuclear Cells In Vitro and In Vivo for Nitric Oxide Synthase (NOS) Type 2 mRNA and Protein Expression: Possible Relationship of Induced NOS2 to the Anti–Hepatitis C Effects of IFN-α In Vivo  
The Journal of Experimental Medicine  1997;186(9):1495-1502.
Although researchers have noted high level activation of rodent mononuclear phagocytes for nitric oxide (NO) synthase type 2 (S2) expression and NO production with a variety of agents such as interferon (IFN) γ and endotoxin, it has been difficult to demonstrate activation of human mononuclear phagocytes. The purpose of this study was to determine if IFN-α serves as an activator in vitro and in vivo in humans. Treatment of normal monocytes or mononuclear cells in vitro with IFN-α caused a dose-dependent increase in monocyte NOS2 activity and NO production, and increased expression of NOS2 protein and mRNA expression. To determine if in vivo administration of IFN-α also modulated NOS2, we studied blood cells from patients with hepatitis C before and after IFN-α therapy. Untreated patients with chronic hepatitis C virus infection had levels of NOS activity and NOS2 antigen in freshly isolated mononuclear cells similar to those of healthy subjects, and they expressed minimal or no NOS2 mRNA. However, IFN-α treatment of patients with hepatitis C infection was associated with a significant elevation in mononuclear cell NOS activity, NOS2 antigen content, and NOS2 mRNA content. IFN-α–treated patients had significant decreases in levels of serum alanine aminotransferase and plasma hepatitis C mRNA. The degree of IFN-α–enhanced mononuclear cell NOS2 antigen content correlated significantly with the degree of reduction in serum alanine aminotransferase levels. Thus, IFN-α treatment of cells in vitro or administration of IFN-α to hepatitis C patients in vivo increases expression of mononuclear cell NOS2 mRNA expression, NOS activity, NOS2 antigen expression, and NO production. Since NO has been reported to have antiviral activity for a variety of viruses, we speculate that induced NO production may be related to the antiviral action(s) of IFN-α in hepatitis C infection.
PMCID: PMC2199112  PMID: 9348307
4.  Clinical and Serologic Manifestations of Autoimmune Disease in MRL-lpr/lpr Mice Lacking Nitric Oxide Synthase Type 2  
Nitric oxide (NO) is an important mediator of the inflammatory response. MRL–lpr/lpr mice overexpress inducible nitric oxide synthase (NOS2) and overproduce NO in parallel with the development of an autoimmune syndrome with a variety of inflammatory manifestations. In previous studies, we showed that inhibiting NO production with the nonselective nitric oxide synthase (NOS) inhibitor NG-monomethyl–arginine reduced glomerulonephritis, arthritis, and vasculitis in MRL–lpr/lpr mice. To define further the role of NO and NOS2 in disease in MRL–lpr/lpr mice, mice with targeted disruption of NOS2 were produced by homologous recombination and bred to MRL–lpr/lpr mice to the N4 generation. MRL–lpr/lpr littermates homozygous for disrupted NOS2 (−/−), heterozygous for disrupted NOS2 (+/−), or wildtype (+/+) were derived for this study. Measures of NO production were markedly decreased in the MRL-lpr/lpr (−/−) mice compared with MRL-lpr/lpr (+/+) mice, with intermediate production by the MRL-lpr/lpr (+/−) mice. There was no detectable NOS2 protein by immunoblot analysis of the spleen, liver, kidney, and peritoneal macrophages of the (−/−) animals, whereas that of (+/+) was high and (+/−) intermediate. The (−/−) mice developed glomerular and synovial pathology similar to that of the (+/−) and (+/+) mice. However, (−/−) mice and (+/−) mice had significantly less vasculitis of medium-sized renal vessels than (+/+) mice. IgG rheumatoid factor levels were significantly lower in the (−/−) mice as compared with (+/+) mice, but levels of anti-DNA antibodies were comparable in all groups. Our findings show that NO derived from NOS2 has a variable impact on disease manifestations in MRL-lpr/lpr mice, suggesting heterogeneity in disease mechanisms.
PMCID: PMC2199001  PMID: 9236188
5.  Increased plasma arginase activity in human sepsis: association with increased circulating neutrophils 
The pathophysiology of sepsis is incompletely understood. Impaired bioavailability of L-arginine, the substrate for NO synthesis, is linked to sepsis severity, and plasma arginase has been linked to hypoargininemia in other disease states. Circulating neutrophils are increased in sepsis and constitutively express arginase. We investigated whether plasma arginase activity is increased in human sepsis and whether this is associated with neutrophil numbers and activation.
We used HPLC and a radiometric assay to evaluate plasma amino acid concentrations and plasma arginase activity. The relationships between plasma arginase activity, neutrophil count, neutrophil activity and plasma L-arginine and arginine metabolites were evaluated in 44 sepsis patients and 25 controls.
Plasma arginase activity was increased in sepsis patients, correlated with neutrophil count (r=0.44; p=0.003), but was independent of sepsis severity (SOFA or APACHE II score). Plasma HNP1-3 correlated with neutrophil count (r=0.31; p=0.04), was elevated in shock (median 180 ng/mL versus 83 ng/mL sepsis without shock, p=0.0006) and correlated with SOFA score. Sepsis patients with high neutrophil counts had significantly higher plasma HNP1-3 and arginase activity and lower plasma L-arginine concentrations than those with lower neutrophil counts and controls.
Plasma arginase activity, potentially derived in part from neutrophil activation, is elevated in sepsis, and may contribute to impaired bioavailability of L-arginine in sepsis.
PMCID: PMC4431981  PMID: 24166672
plasma arginase activity; L-arginine; sepsis; hypoargininemia
6.  Impaired Systemic Tetrahydrobiopterin Bioavailability and Increased Oxidized Biopterins in Pediatric Falciparum Malaria: Association with Disease Severity 
PLoS Pathogens  2015;11(3):e1004655.
Decreased bioavailability of nitric oxide (NO) is a major contributor to the pathophysiology of severe falciparum malaria. Tetrahydrobiopterin (BH4) is an enzyme cofactor required for NO synthesis from L-arginine. We hypothesized that systemic levels of BH4 would be decreased in children with cerebral malaria, contributing to low NO bioavailability. In an observational study in Tanzania, we measured urine levels of biopterin in its various redox states (fully reduced [BH4] and the oxidized metabolites, dihydrobiopterin [BH2] and biopterin [B0]) in children with uncomplicated malaria (UM, n = 55), cerebral malaria (CM, n = 45), non-malaria central nervous system conditions (NMC, n = 48), and in 111 healthy controls (HC). Median urine BH4 concentration in CM (1.10 [IQR:0.55–2.18] μmol/mmol creatinine) was significantly lower compared to each of the other three groups — UM (2.10 [IQR:1.32–3.14];p<0.001), NMC (1.52 [IQR:1.01–2.71];p = 0.002), and HC (1.60 [IQR:1.15–2.23];p = 0.005). Oxidized biopterins were increased, and the BH4:BH2 ratio markedly decreased in CM. In a multivariate logistic regression model, each Log10-unit decrease in urine BH4 was independently associated with a 3.85-fold (95% CI:1.89–7.61) increase in odds of CM (p<0.001). Low systemic BH4 levels and increased oxidized biopterins contribute to the low NO bioavailability observed in CM. Adjunctive therapy to regenerate BH4 may have a role in improving NO bioavailability and microvascular perfusion in severe falciparum malaria.
Author Summary
Vascular nitric oxide (NO) bioavailability is decreased in severe falciparum malaria and associated with microvascular dysfunction, increased activation of the cells lining blood vessels (endothelial cells) and increased parasite biomass. Tetrahydrobiopterin (BH4) is an essential cofactor for nitric oxide synthase (NOS) enzymatic conversion of L-arginine to NO and L-citrulline. But when BH4 is low, NOS is “uncoupled” and produces superoxide instead of NO. In oxidative conditions, BH4 is oxidized to dihydrobiopterin (BH2) and biopterin (B0). BH2 competes with remaining BH4 at its NOS binding site, further decreasing NOS-catalyzed NO production. We measured BH4, BH2 and B0 in the urine of children with coma due to falciparum malaria (cerebral malaria), uncomplicated falciparum malaria, children with non-malaria central nervous system conditions and healthy controls. Urine BH4 was significantly decreased and BH2 significantly increased in cerebral malaria compared to uncomplicated malaria, non-malaria central nervous conditions and healthy controls, suggesting increased oxidative stress and insufficient recycling of BH2 back to BH4. Urine BH4 concentration was independently associated with increased risk of cerebral malaria. Given that safe therapies for regenerating BH4 have been studied in chronic vascular disease, this finding of low BH4 in pediatric cerebral malaria offers a new area of investigation for adjunctive therapies aimed at improving NO bioavailability and, consequently, clinical outcomes in severe falciparum malaria.
PMCID: PMC4357384  PMID: 25764173
7.  Impaired Systemic Tetrahydrobiopterin Bioavailability and Increased Dihydrobiopterin in Adult Falciparum Malaria: Association with Disease Severity, Impaired Microvascular Function and Increased Endothelial Activation 
PLoS Pathogens  2015;11(3):e1004667.
Tetrahydrobiopterin (BH4) is a co-factor required for catalytic activity of nitric oxide synthase (NOS) and amino acid-monooxygenases, including phenylalanine hydroxylase. BH4 is unstable: during oxidative stress it is non-enzymatically oxidized to dihydrobiopterin (BH2), which inhibits NOS. Depending on BH4 availability, NOS oscillates between NO synthase and NADPH oxidase: as the BH4/BH2 ratio decreases, NO production falls and is replaced by superoxide. In African children and Asian adults with severe malaria, NO bioavailability decreases and plasma phenylalanine increases, together suggesting possible BH4 deficiency. The primary three biopterin metabolites (BH4, BH2 and B0 [biopterin]) and their association with disease severity have not been assessed in falciparum malaria. We measured pterin metabolites in urine of adults with severe falciparum malaria (SM; n=12), moderately-severe malaria (MSM, n=17), severe sepsis (SS; n=5) and healthy subjects (HC; n=20) as controls. In SM, urinary BH4 was decreased (median 0.16 ¼mol/mmol creatinine) compared to MSM (median 0.27), SS (median 0.54), and HC (median 0.34)]; p<0.001. Conversely, BH2 was increased in SM (median 0.91 ¼mol/mmol creatinine), compared to MSM (median 0.67), SS (median 0.39), and HC (median 0.52); p<0.001, suggesting increased oxidative stress and insufficient recycling of BH2 back to BH4 in severe malaria. Overall, the median BH4/BH2 ratio was lowest in SM [0.18 (IQR: 0.04-0.32)] compared to MSM (0.45, IQR 0.27-61), SS (1.03; IQR 0.54-2.38) and controls (0.66; IQR 0.43-1.07); p<0.001. In malaria, a lower BH4/BH2 ratio correlated with decreased microvascular reactivity (r=0.41; p=0.03) and increased ICAM-1 (r=-0.52; p=0.005). Decreased BH4 and increased BH2 in severe malaria (but not in severe sepsis) uncouples NOS, leading to impaired NO bioavailability and potentially increased oxidative stress. Adjunctive therapy to regenerate BH4 may have a role in improving NO bioavailability and microvascular perfusion in severe falciparum malaria.
Author Summary
Vascular nitric oxide (NO) bioavailability is decreased in severe falciparum malaria and associated with microvascular dysfunction and increased endothelial activation. Nitric oxide synthase (NOS) requires tetrahydrobiopterin (BH4) as a co-factor to convert L-arginine to NO, but when BH4 is low, NOS is “uncoupled” and produces superoxide instead of NO. In conditions of increased oxidative stress, BH4 is converted to dihydrobiopterin (BH2) and biopterin (B0): the resulting BH2 competes with remaining BH4 as a competitive inhibitor of NOS, further decreasing NO production. We measured BH4 and BH2 in the urine of adults with severe and uncomplicated falciparum malaria and compared results to those of controls or those with sepsis. There was a significant decrease in urinary BH4 and increase in BH2 in severe malaria compared to uncomplicated malaria, sepsis, and controls, suggesting increased oxidative stress and insufficient recycling of BH2 back to BH4. The BH4/BH2 ratio was associated with increased risk of severe disease, endothelial activation and microvascular dysfunction, likely through impaired NOS function. This additional mechanism of decreased NO in severe malaria suggests that trials evaluating use of adjunctive L-arginine to increase NO in severe malaria may require concurrent therapy to regenerate BH4.
PMCID: PMC4357386  PMID: 25764397
8.  Decreased Endothelial Nitric Oxide Bioavailability, Impaired Microvascular Function, and Increased Tissue Oxygen Consumption in Children with Falciparum Malaria 
The Journal of infectious diseases  2014;210(10):1627-1632.
Endothelial nitric oxide (NO) bioavailability, microvascular function, and host oxygen consumption have not been assessed in pediatric malaria. We measured NO-dependent endothelial function by using peripheral artery tonometry to determine the reactive hyperemia index (RHI), and microvascular function and oxygen consumption (VO2) using near infrared resonance spectroscopy in 13 Indonesian children with severe falciparum malaria and 15 with moderately severe falciparum malaria. Compared with 19 controls, children with severe malaria and those with moderately severe malaria had lower RHIs (P = .03); 12% and 8% lower microvascular function, respectively (P = .03); and 29% and 25% higher VO2, respectively. RHIs correlated with microvascular function in all children with malaria (P < .001) and all with severe malaria (P < .001). Children with malaria have decreased endothelial and microvascular function and increased oxygen consumption, likely contributing to the pathogenesis of the disease.
PMCID: PMC4334794  PMID: 24879801
severe malaria; cerebral malaria; Plasmodium falciparum; endothelial function; microvascular function; oxygen consumption; tissue hypoxia
9.  Neutrophils with myeloid derived suppressor function deplete arginine and constrain T cell function in septic shock patients 
Critical Care  2014;18(4):R163.
Impaired T cell function in sepsis is associated with poor outcome, but the mechanisms are unclear. In cancer, arginase-expressing myeloid derived suppressor cells (MDSCs) deplete arginine, impair T cell receptor CD3 zeta-chain expression and T cell function and are linked to poor clinical outcome, but their role during acute human infectious disease and in particular sepsis remains unknown. Hypoarginemia is prevalent in sepsis. This study aimed to determine whether neutrophils that co-purify with PBMC express arginase, and if arginine depletion constrains T cell CD3 zeta-chain expression and function in human sepsis.
Using flow cytometry, cell culture, HPLC, arginase activity and mRNA detection, our study examined whether neutrophils, with reduced buoyant density isolated in the Ficoll interface, metabolise L-arginine and suppress T cell proliferation in sepsis. A total of 35 sepsis patients (23 with septic shock) and 12 hospital controls in a tertiary referral hospital in tropical Australia were evaluated.
Only sepsis patients had interphase neutrophils, neutrophils co-purifying with mononuclear cells (≤1.077 specific gravity). The percentage of interphase neutrophils in sepsis was proportional to sepsis severity and correlated with plasma IL-6 concentrations. Ex vivo, sepsis-derived interphase neutrophils expressed arginase, metabolised culture L-arginine and suppressed T cell proliferation and CD3 zeta-chain expression. In vivo, in septic shock there was a longitudinal inverse association between interphase neutrophil number and CD3 zeta-chain expression. Depletion or inhibition of interphase neutrophils in vitro restored zeta-chain expression and T cell function.
For the first time during an acute human infection, interphase neutrophils that express arginase were found to circulate in sepsis, in proportion to disease severity. These neutrophil-MDSCs impair T cell CD3 zeta-chain expression and T cell function via L-arginine metabolism, and likely contribute to the T cell dysfunction seen in sepsis. Modulation of neutrophil-MDSC or their downstream effects warrant consideration as targets for novel adjunctive therapies in sepsis.
Electronic supplementary material
The online version of this article (doi:10.1186/cc14003) contains supplementary material, which is available to authorized users.
PMCID: PMC4261583  PMID: 25084831
10.  Immunologic aspects of monoclonal B cell lymphocytosis 
Immunologic research  2011;49(0):269-280.
Monoclonal B cell lymphocytosis (MBL) is a preclinical hematologic condition wherein small numbers of clonal B cells can be detected in the blood of otherwise healthy individuals. Most MBL have a surface immunophenotype nearly identical to that of chronic lymphocytic leukemia (CLL), though other phenotypes can also be identified. MBL has been shown to be a precursor state for CLL, but most MBL clones are quite small and apparently have minimal potential to progress of CLL or other B cell lymphoproliferative disorder (B-LPD). The investigation of MBL as a precursor state for CLL will likely lead to important insights into mechanisms of disease pathogenesis. The review will cover clinical and translational aspects of MBL, with a particular emphasis on the prevalence of MBL; the relationship between MBL, CLL, and other B-LPDs; and the capacity of MBL to modulate the normal B and T cell compartments.
PMCID: PMC4102133  PMID: 21161696
Monoclonal B Lymphocytosis; Chronic Lymphocytic Leukemia; Non-Hodgkin’s Lymphoma; B Cell Receptor; B Cell Development
11.  Mapping of the IRF8 gene identifies a 3’ UTR variant associated with risk of chronic lymphocytic leukemia but not other common non-Hodgkin lymphoma subtypes 
Our genome-wide association study (GWAS) of chronic lymphocytic leukemia (CLL) identified 4 highly-correlated intronic variants within the IRF8 gene that were associated with CLL. These results were further supported by a recent meta-analysis of our GWAS with two other GWAS of CLL, supporting the IRF8 gene as a strong candidate for CLL risk.
To refine the genetic association of CLL risk, we performed Sanger sequencing of IRF8 in 94 CLL cases and 96 controls. We then performed fine-mapping by genotyping 39 variants (of which 10 were identified from sequencing) in 745 CLL cases and 1521 controls. We also assessed these associations with risk of other non-Hodgkin lymphoma (NHL) subtypes.
The strongest association with CLL risk was observed with a common SNP located within the 3’ UTR of IRF8 (rs1044873, log additive odds ratio = 0.7, P=1.81×10−6). This SNP was not associated with the other NHL subtypes (all P>0.05).
We provide evidence that rs1044873 in the IRF8 gene accounts for the initial GWAS signal for CLL risk. This association appears to be unique to CLL with little support for association with other common NHL subtypes. Future work is needed to assess functional role of IRF8 in CLL etiology.
These data provide support that a functional variant within the 3’ UTR of IRF8 may be driving the GWAS signal seen on 16q24.1 for CLL risk.
PMCID: PMC3596428  PMID: 23307532
CLL; NHL; SNPs; IRF8; risk locus
12.  Genome-wide Association Study Identifies Multiple Risk Loci for Chronic Lymphocytic Leukemia 
Berndt, Sonja I. | Skibola, Christine F. | Joseph, Vijai | Camp, Nicola J. | Nieters, Alexandra | Wang, Zhaoming | Cozen, Wendy | Monnereau, Alain | Wang, Sophia S. | Kelly, Rachel S. | Lan, Qing | Teras, Lauren R. | Chatterjee, Nilanjan | Chung, Charles C. | Yeager, Meredith | Brooks-Wilson, Angela R. | Hartge, Patricia | Purdue, Mark P. | Birmann, Brenda M. | Armstrong, Bruce K. | Cocco, Pierluigi | Zhang, Yawei | Severi, Gianluca | Zeleniuch-Jacquotte, Anne | Lawrence, Charles | Burdette, Laurie | Yuenger, Jeffrey | Hutchinson, Amy | Jacobs, Kevin B. | Call, Timothy G. | Shanafelt, Tait D. | Novak, Anne J. | Kay, Neil E. | Liebow, Mark | Wang, Alice H. | Smedby, Karin E | Adami, Hans-Olov | Melbye, Mads | Glimelius, Bengt | Chang, Ellen T. | Glenn, Martha | Curtin, Karen | Cannon-Albright, Lisa A. | Jones, Brandt | Diver, W. Ryan | Link, Brian K. | Weiner, George J. | Conde, Lucia | Bracci, Paige M. | Riby, Jacques | Holly, Elizabeth A. | Smith, Martyn T. | Jackson, Rebecca D. | Tinker, Lesley F. | Benavente, Yolanda | Becker, Nikolaus | Boffetta, Paolo | Brennan, Paul | Foretova, Lenka | Maynadie, Marc | McKay, James | Staines, Anthony | Rabe, Kari G. | Achenbach, Sara J. | Vachon, Celine M. | Goldin, Lynn R | Strom, Sara S. | Lanasa, Mark C. | Spector, Logan G. | Leis, Jose F. | Cunningham, Julie M. | Weinberg, J. Brice | Morrison, Vicki A. | Caporaso, Neil E. | Norman, Aaron D. | Linet, Martha S. | De Roos, Anneclaire J. | Morton, Lindsay M. | Severson, Richard K. | Riboli, Elio | Vineis, Paolo | Kaaks, Rudolph | Trichopoulos, Dimitrios | Masala, Giovanna | Weiderpass, Elisabete | Chirlaque, María-Dolores | Vermeulen, Roel C H | Travis, Ruth C. | Giles, Graham G. | Albanes, Demetrius | Virtamo, Jarmo | Weinstein, Stephanie | Clavel, Jacqueline | Zheng, Tongzhang | Holford, Theodore R | Offit, Kenneth | Zelenetz, Andrew | Klein, Robert J. | Spinelli, John J. | Bertrand, Kimberly A. | Laden, Francine | Giovannucci, Edward | Kraft, Peter | Kricker, Anne | Turner, Jenny | Vajdic, Claire M. | Ennas, Maria Grazia | Ferri, Giovanni M. | Miligi, Lucia | Liang, Liming | Sampson, Joshua | Crouch, Simon | Park, Ju-hyun | North, Kari E. | Cox, Angela | Snowden, John A. | Wright, Josh | Carracedo, Angel | Lopez-Otin, Carlos | Bea, Silvia | Salaverria, Itziar | Martin, David | Campo, Elias | Fraumeni, Joseph F. | de Sanjose, Silvia | Hjalgrim, Henrik | Cerhan, James R. | Chanock, Stephen J. | Rothman, Nathaniel | Slager, Susan L.
Nature genetics  2013;45(8):868-876.
PMCID: PMC3729927  PMID: 23770605
13.  Chronic Lymphocytic Leukemia and Regulatory B Cells Share IL-10-Competence and Immunosuppressive Function 
Leukemia  2012;27(1):170-182.
Chronic lymphocytic leukemia (CLL) can be immunosuppressive in humans and mice, and CLL cells share multiple phenotypic markers with regulatory B cells that are competent to produce IL-10 (B10 cells). To identify functional links between CLL cells and regulatory B10 cells, the phenotypes and abilities of leukemia cells from 93 patients with overt CLL to express IL-10 were assessed. CD5+ CLL cells purified from 90% of the patients were IL-10-competent and secreted IL-10 following appropriate ex vivo stimulation. Serum IL-10 levels were also significantly elevated in CLL patients. IL-10-competent cell frequencies were higher among CLLs with IgVH mutations, and correlated positively with TCL1 expression. In the TCL1-transgenic (TCL1-Tg) mouse model of CLL, IL-10-competent B cells with the cell-surface phenotype of B10 cells expanded significantly with age, preceding the development of overt, CLL-like leukemia. Malignant CLL cells in TCL1-Tg mice also shared immunoregulatory functions with mouse and human B10 cells. Serum IL-10 levels varied in TCL1-Tg mice, but in vivo low-dose lipopolysaccharide treatment induced IL-10 expression in CLL cells and high levels of serum IL-10. Thus, malignant IL-10-competent CLL cells exhibit regulatory functions comparable to normal B10 cells that may contribute to the immunosuppression observed in patients and TCL1-Tg mice.
PMCID: PMC3742013  PMID: 22713648
B10 cells; regulatory B cell; CLL; leukemia; IL-10; immunosuppression
14.  Common variants within 6p21.31 locus are associated with chronic lymphocytic leukaemia and potentially other non-Hodgkin lymphoma subtypes 
British journal of haematology  2012;159(5):572-576.
A recent meta-analysis of three genome-wide association studies of chronic lymphocytic leukaemia (CLL) identified two common variants at the 6p21.31 locus that are associated with CLL risk. To verify and further explore the association of these variants with other non-Hodgkin lymphoma (NHL) subtypes, we genotyped 1196 CLL cases, 1699 NHL cases, and 2410 controls. We found significant associations between the 6p21.31 variants and CLL risk (rs210134: P=0.01; rs210142: P=6.8×10−3). These variants also showed a trend towards association with some of the other NHL subtypes. Our results validate the prior work and support specific genetic pathways for risk among NHL subtypes.
PMCID: PMC3614403  PMID: 23025533
CLL; NHL; SNPs; BAK1; risk locus
15.  The inhibition by interleukin 1 of MSC chondrogenesis and the development of biomechanical properties in biomimetic 3D woven PCL scaffolds 
Biomaterials  2012;33(35):8967-8974.
Tissue-engineered constructs designed to treat large cartilage defects or osteoarthritic lesions may be exposed to significant mechanical loading as well as an inflammatory environment upon implantation in an injured or diseased joint. We hypothesized that a three-dimensionally (3D) woven poly(ε-caprolactone) (PCL) scaffold seeded with bone marrow-derived mesenchymal stem cells (MSCs) would provide biomimetic mechanical properties in early stages of in vitro culture as the MSCs assembled a functional, cartilaginous extracellular matrix (ECM). We also hypothesized that these properties would be maintained even in the presence of the pro-inflammatory cytokine interleukin-1 (IL-1), which is found at high levels in injured or diseased joints. MSC-seeded 3D woven scaffolds cultured in chondrogenic conditions synthesized a functional ECM rich in collagen and proteoglycan content, reaching an aggregate modulus of ~0.75 MPa within 14 days of culture. However, the presence of pathophysiologically relevant levels of IL-1 limited matrix accumulation and inhibited any increase in mechanical properties over baseline values. On the other hand, the mechanical properties of constructs cultured in chondrogenic conditions for 4 weeks prior to IL-1 exposure were protected from deleterious effects of the cytokine. These findings demonstrate that IL-1 significantly inhibits the chondrogenic development and maturation of MSC-seeded constructs; however, the overall mechanical functionality of the engineered tissue can be preserved through the use of a 3D woven scaffold designed to recreate the mechanical properties of native articular cartilage.
PMCID: PMC3466362  PMID: 22999467
cytokine; articular cartilage; tissue engineering; textile; stem cell; mesenchymal stem cell; differentiation; fiber; inflammation; fabric
16.  Relationship of Cell-Free Hemoglobin to Impaired Endothelial Nitric Oxide Bioavailability and Perfusion in Severe Falciparum Malaria 
The Journal of infectious diseases  2009;200(10):1522-1529.
Hemolysis causes anemia in falciparum malaria, but its contribution to microvascular pathology in severe malaria (SM) is not well characterized. In other hemolytic diseases, release of cell-free hemoglobin causes nitric oxide (NO) quenching, endothelial activation, and vascular complications. We examined the relationship of plasma hemoglobin and myoglobin to endothelial dysfunction and disease severity in malaria.
Cell-free hemoglobin (a potent NO quencher), reactive hyperemia peripheral arterial tonometry (RH-PAT) (a measure of endothelial NO bioavailability), and measures of perfusion and endothelial activation were quantified in adults with moderately severe (n = 78) or severe (n = 49) malaria and control subjects (n = 16) from Papua, Indonesia.
Cell-free hemoglobin concentrations in patients with SM (median, 5.4 μmol/L; interquartile range [IQR], 3.2–7.4 μmol/L) were significantly higher than in those with moderately severe malaria (2.6 μmol/L; IQR, 1.3–4.5 μmol/L) or controls (1.2 μmol/L; IQR, 0.9–2.4 μmol/L; P < .001). Multivariable regression analysis revealed that cell-free hemoglobin remained inversely associated with RH-PAT, and in patients with SM, there was a significant longitudinal association between improvement in RH-PAT index and decreasing levels of cell-free hemoglobin (P = .047). Cell-free hemoglobin levels were also independently associated with lactate, endothelial activation, and proinflammatory cytokinemia.
Hemolysis in falciparum malaria results in NO quenching by cell-free hemoglobin, and may exacerbate endothelial dysfunction, adhesion receptor expression and impaired tissue perfusion. Treatments that increase NO bioavailability may have potential as adjunctive therapies in SM.
PMCID: PMC3740798  PMID: 19803726
17.  Clinical and Biological Relevance of Genomic Heterogeneity in Chronic Lymphocytic Leukemia 
PLoS ONE  2013;8(2):e57356.
Chronic lymphocytic leukemia (CLL) is typically regarded as an indolent B-cell malignancy. However, there is wide variability with regards to need for therapy, time to progressive disease, and treatment response. This clinical variability is due, in part, to biological heterogeneity between individual patients’ leukemias. While much has been learned about this biological variation using genomic approaches, it is unclear whether such efforts have sufficiently evaluated biological and clinical heterogeneity in CLL.
To study the extent of genomic variability in CLL and the biological and clinical attributes of genomic classification in CLL, we evaluated 893 unique CLL samples from fifteen publicly available gene expression profiling datasets. We used unsupervised approaches to divide the data into subgroups, evaluated the biological pathways and genetic aberrations that were associated with the subgroups, and compared prognostic and clinical outcome data between the subgroups.
Using an unsupervised approach, we determined that approximately 600 CLL samples are needed to define the spectrum of diversity in CLL genomic expression. We identified seven genomically-defined CLL subgroups that have distinct biological properties, are associated with specific chromosomal deletions and amplifications, and have marked differences in molecular prognostic markers and clinical outcomes.
Our results indicate that investigations focusing on small numbers of patient samples likely provide a biased outlook on CLL biology. These findings may have important implications in identifying patients who should be treated with specific targeted therapies, which could have efficacy against CLL cells that rely on specific biological pathways.
PMCID: PMC3585365  PMID: 23468975
18.  Plasma Plasmodium falciparum Histidine-Rich Protein-2 Concentrations Are Associated with Malaria Severity and Mortality in Tanzanian Children 
PLoS ONE  2012;7(5):e35985.
Plasma Plasmodium falciparum histidine-rich protein-2 (PfHRP-2) concentrations, a measure of parasite biomass, have been correlated with malaria severity in adults, but not yet in children. We measured plasma PfHRP-2 in Tanzanian children with uncomplicated (n = 61) and cerebral malaria (n = 45; 7 deaths). Median plasma PfHRP-2 concentrations were higher in cerebral malaria (1008 [IQR 342–2572] ng/mL) than in uncomplicated malaria (465 [IQR 36–1426] ng/mL; p = 0.017). In cerebral malaria, natural log plasma PfHRP-2 was associated with coma depth (r = −0.42; p = 0.006) and mortality (OR: 3.0 [95% CI 1.03–8.76]; p = 0.04). In this relatively small cohort study in a mesoendemic transmission area of Africa, plasma PfHRP-2 was associated with pediatric malaria severity and mortality. Further studies among children in areas of Africa with higher malaria transmission and among children with different clinical manifestations of severe malaria will help determine the wider utility of quantitative PfHRP-2 as a measure of parasite biomass and prognosis in sub-Saharan Africa.
PMCID: PMC3346811  PMID: 22586457
19.  Immnuophenotypic and Gene Expression Analysis of Monoclonal B Cell Lymphocytosis Shows Biologic Characteristics Associated With Good Prognosis CLL 
Monoclonal B cell lymphocytosis (MBL) is a hematologic condition wherein small B cell clones can be detected in the blood of asymptomatic individuals. Most MBL have an immunophenotype similar to chronic lymphocytic leukemia (CLL), and “CLL-like” MBL is a precursor to CLL. We used flow cytometry to identify MBL from unaffected members of CLL kindreds. We identified 101 MBL cases from 622 study subjects; of these, 82 individuals with MBL were further characterized. Ninety-one unique MBL clones were detected: 73 CLL-like MBL (CD5+CD20dimsIgdim), 11 atypical MBL (CD5+CD20+sIg+), and 7 CD5neg MBL (CD5negCD20+sIgneg). Extended immunophenotypic characterization of these MBL subtypes was performed, and significant differences in cell surface expression of CD23, CD49d, CD79b, and FMC-7 were observed among the groups. Markers of risk in CLL such as CD38, ZAP70, and CD49d were infrequently expressed in CLL-like MBL, but were expressed in the majority of atypical MBL. Interphase cytogenetics was performed in 35 MBL cases, and del 13q14 was most common (22/30 CLL-like MBL cases). Gene expression analysis using oligonucleotide arrays was performed on 7 CLL-like MBL, and showed activation of B cell receptor associated pathways. Our findings underscore the diversity of MBL subtypes and further clarify the relationship between MBL and other lymphoproliferative disorders.
PMCID: PMC3164475  PMID: 21617698
20.  Interleukin-1 and tumor necrosis factor alpha inhibit repair of the porcine meniscus in vitro 
Injury or removal of the knee meniscus leads to progressive joint degeneration, and current surgical therapies for meniscal tears seek to maximally preserve meniscal structure and function. However, the factors that influence intrinsic repair of the meniscus are not well understood. The goal of this study was to investigate the capacity of meniscus tissue to repair a simulated defect in vitro and to examine the effect of pro-inflammatory cytokines on this process.
Cylindrical explants were harvested from the outer one-third of medial porcine menisci. To simulate a full-thickness defect, a central core was removed and reinserted immediately into the defect. Explants were cultured for 2, 4, or 6 weeks in serum-containing media in the presence or absence of interleukin-1 (IL-1) or tumor necrosis factor alpha (TNF-alpha), and meniscal repair was investigated using mechanical testing and fluorescence confocal microscopy.
Meniscal lesions in untreated samples showed a significant capacity for intrinsic repair in vitro, with increasing cell accumulation and repair strength over time in culture. In the presence of IL-1 or TNF-alpha, no repair was observed despite the presence of abundant viable cells.
This study demonstrates that the meniscus exhibits an intrinsic repair response in vitro. However, the presence of pro-inflammatory cytokines completely inhibited repair. These findings suggest that increased levels of pro-inflammatory cytokines post-injury or under arthritic conditions may inhibit meniscal repair. Therefore, inhibition of these cytokines may provide a means of accelerating repair of damaged or injured menisci in vivo.
PMCID: PMC3217205  PMID: 17448702
Meniscus; Injury; Cartilage; Integrative repair; IL-1; TNF-α; Osteoarthritis
21.  Common Occurrence of Monoclonal B-cell Lymphocytosis Among Members of High-Risk CLL Families 
British journal of haematology  2010;151(2):152-158.
Monoclonal B-cell lymphocytosis (MBL) is an asymptomatic haematological condition characterized by low absolute levels of B-cell clones with a surface immunophenotype similar to that of chronic lymphocytic leukaemia (CLL). In the general population, MBL increases with age with a prevalence of 5–9% in individuals over age 60 years. It has been reported to be higher among first-degree relatives from CLL families. We report results of multi-parameter flow cytometry among 505 first-degree relatives with no personal history of lymphoproliferative disease from 140 families having at least two cases of CLL. Seventeen percent of relatives had MBL. Age was the most important determinant where the probability for developing MBL by age 90 years was 61%. MBL clustered in certain families but clustering was independent of the number of known CLL cases in a family. As is the case with CLL, males had a significantly higher risk for MBL than did females (p=0.04). MBL patients had significantly higher mean absolute lymphocyte counts (2.4 × 109/l) and B-cell counts (0.53 × 109/l) than those with a normal B-cell immunophenotype. Our findings show that MBL occurs at a very high rate in high risk CLL families. Both the age and gender distribution of MBL are parallel to CLL, implying a shared inherited risk.
PMCID: PMC2966536  PMID: 20738309
chronic lymphocytic leukaemia; high risk families; monoclonal B-cell lymphocytosis; flow cytometry
22.  Age-related susceptibility to severe malaria associated with galectin-2 in highland Papuans 
The Journal of infectious diseases  2010;202(1):117-124.
Age and host genetics are important determinants of malaria severity. Lymphotoxin-alpha (LTα) has been linked to the development of cerebral malaria (CM) and other severe malaria (SM) syndromes. Mutations in genes regulating LTα production contribute to other acute vascular diseases and may contribute to malaria pathogenesis.
We tested the association between rs7291467, a single nucleotide polymorphism (SNP) in the LTα-related gene encoding galectin-2 (LGALS2), disease severity and function in a case-control study of ethnic Highland Papuan adults and children with SM (n=380) and asymptomatic malaria-exposed controls (n=356), originating from a non-malaria-endemic region but residing in a lowland malaria-endemic area of Papua, Indonesia.
The LGALS2 SNP showed significant association with susceptibility to SM (including CM), in children (OR 2.02 [95% CI:1.14-3.57]) but not adults. In SM, the C-allele at rs7291467 was associated with enhanced galectin-2 transcript levels. In a separate group of Tanzanian children originating from a malaria-endemic region, we found preservation of the major ancestral LGALS2 allele and no association with susceptibility to CM.
Results suggest differences in the inflammatory contribution to the development of SM between children and adults in the same population, and potential differences between individuals originating from malaria-endemic and non-endemic areas.
PMCID: PMC2880661  PMID: 20500087
Malaria; Galectin-2; Inflammation; Gene regulation; Age; Severe Malaria; Cerebral Malaria
23.  Dynamic Loading Enhances Integrative Meniscal Repair in the Presence of Interleukin-1 
Meniscal tears are a common knee injury and increased levels of interleukin-1 (IL-1) have been measured in injured and degenerated joints. Studies have shown that IL-1 decreases the shear strength, cell accumulation, and tissue formation in meniscal repair interfaces. While mechanical stress and IL-1 modulate meniscal biosynthesis and degradation, the effects of dynamic loading on meniscal repair are unknown. The purpose of this study was to determine the effects of mechanical compression on meniscal repair under normal and inflammatory conditions.
Experimental Design
Explants were harvested from porcine medial menisci. To simulate a full-thickness defect, a central core was removed and reinserted. Explants were loaded for 4 hours/day at 1Hz and 0 – 26% strain for 14 days in the presence of 0 or 100pg/mL of IL-1. Media were assessed for matrix metalloproteinase (MMP) activity, aggrecanase activity, sulfated glycosaminoglycan (S-GAG) release, and nitric oxide (NO) production. After 14 days, biomechanical testing and histological analyses were performed.
IL-1 increased MMP activity, S-GAG release, and NO production, while decreasing the shear strength and tissue repair in the interface. Dynamic loading antagonized IL-1-mediated inhibition of repair at all strain amplitudes. Neither IL-1 treatment nor strain altered aggrecanase activity. Additionally, strain alone did not alter meniscal healing, except at the highest strain magnitude (26%), a level that enhanced the strength of repair.
Dynamic loading blocked the catabolic effects of IL-1 on meniscal repair, suggesting that joint loading through physical therapy may be beneficial in promoting healing of meniscal lesions under inflammatory conditions.
PMCID: PMC2872683  PMID: 20202487
meniscus; tissue repair; mechanical loading; interleukin-1; matrix metalloproteinase; aggrecanase; nitric oxide
24.  Genetic susceptibility variants for chronic lymphocytic leukemia 
There is strong and consistent evidence that a genetic component contributes to the etiology of chronic lymphocytic leukemia (CLL). A recent genome-wide association study (GWAS) of CLL identified 7 genetic variants that increased the risk of CLL within a European population.
We evaluated the association of these variants, or variants in linkage disequilibrium (LD) with these variants, with CLL risk in an independent sample of 438 CLL cases and 328 controls.
Of these 7 SNPs, 6 had p-trend < 0.05 and had estimated odds ratios (ORs) that were strikingly comparable to those of the previous study. Associations were seen for rs9378805 (OR = 1.47, 95% CI: 1.19, 1.80, p-trend = 0.0003) near IRF4 and rs735665 near GRAMD1B (OR= 1.47; 95% CI: 1.14, 1.89; p-trend = 0.003). However, no associations (P> 0.05) were found for rs11083846, nor were any found for any SNPs in LD with rs11083846.
Our results confirm the previous findings and further support the role of a genetic basis in the etiology of CLL; however, more research is needed to elucidate the causal SNP(s) and the potential manner in which these SNPs or linked SNPs function in CLL pathogenesis.
PMCID: PMC2852480  PMID: 20332261
IRF4; CLL; genetic association
25.  Genome-wide association study of follicular lymphoma identifies a risk locus at 6p21.32 
Nature genetics  2010;42(8):661-664.
To identify susceptibility loci for non-Hodgkin lymphoma (NHL) subtypes, we conducted a three-stage genome-wide association study. We identified two variants associated with follicular lymphoma (FL) in 1,465 FL cases/6,958 controls at 6p21.32 (rs10484561, rs7755224, r2=1.0; combined p-values=1.12×10-29, 2.00×10-19), providing further support that MHC genetic variation influences FL susceptibility. Confirmatory evidence of a previously reported association was also found between chronic lymphocytic leukemia/small lymphocytic lymphoma and rs735665 (combined p-value=4.24×10-9).
PMCID: PMC2913472  PMID: 20639881

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