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1.  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
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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  A Genomic Approach to Improve Prognosis and Predict Therapeutic Response in Chronic Lymphocytic Leukemia 
Chronic lymphocytic leukemia (CLL) is a B-cell malignancy characterized by a variable clinical course. Several parameters have prognostic capabilities but are associated with altered response to therapy in only a small subset of patients.
Experimental Design
We used gene expression profiling methods to generate predictors of therapy response and prognosis. Genomic signatures that reflect progressive disease and responses to chemotherapy or chemo-immunotherapy were created using cancer cell lines and patient leukemia cell samples. We validated and applied these three signatures to independent clinical data from four cohorts representing a total of 301 CLL patients.
A genomic signature of prognosis created from patient leukemic cell gene expression data coupled with clinical parameters significantly differentiated patients with stable disease from those with progressive disease in the training dataset. The progression signature was validated in two independent datasets, demonstrating a capacity to accurately identify patients at risk for progressive disease. In addition, genomic signatures that predict response to chlorambucil or pentostatin, cyclophosphamide, and rituximab were generated and could accurately distinguish responding and non-responding CLL patients.
Thus, microarray analysis of CLL lymphocytes can be used to refine prognosis and predict response to different therapies. These results have implications for standard and investigational therapeutics in CLL patients.
PMCID: PMC2783430  PMID: 19861443
12.  NOS2A, TLR4, and IFNGR1 interactions influence pulmonary tuberculosis susceptibility in African-Americans 
Human genetics  2009;126(5):643-653.
Tuberculosis (TB) has substantial mortality worldwide with 5-10% of those exposed progressing to active TB disease. Studies in mice and humans indicate that the inducible nitric oxide synthase (iNOS) molecule plays an important role in immune response to TB. A mixed case-control association study of individuals with TB, relatives, or close contact controls was performed in 726 individuals (279 case and 166 control African-Americans; 198 case and 123 control Caucasians). Thirty-nine single nucleotide polymorphisms (SNPs) were selected from the NOS2A gene for single SNP, haplotype, and multilocus interaction analyses with other typed candidate genes using generalized estimating equations. In African-Americans, ten NOS2A SNPs were associated with TB. The strongest associations were observed at rs2274894 (odds ratio (OR) = 1.84, 95% confidence interval (CI) [1.23-2.77], p = 0.003) and rs7215373 (OR 1.67, 95% CI [1.17-2.37], p = 0.004), both of which passed a false discovery rate (FDR) correction for multiple comparisons (q*=0.20). The strongest gene-gene interactions were observed between NOS2A rs2248814 and IFNGR1 rs1327474 (p = 0.0004) and NOS2A rs944722 and IFNGR1 rs1327474 (p = 0.0006). Three other SNPs in NOS2A interacted with TLR4 rs5030729 and five other NOS2A SNPs interacted with IFNGR1 rs1327474. No significant associations were observed in Caucasians. These results suggest that NOS2A variants may contribute to TB susceptibility, particularly in individuals of African descent, and may act synergistically with SNPs in TLR4 and IFNGR1.
PMCID: PMC2881538  PMID: 19575238
tuberculosis; epistasis; complex disease; infectious disease; genetic epidemiology
13.  Single cell analysis reveals oligoclonality among “low count” monoclonal B cell lymphocytosis 
Monoclonal B cell lymphocytosis (MBL) is a pre-clinical hematologic syndrome characterized by small accumulations of CD5+ B lymphocytes. Most MBL share phenotypic characteristics with chronic lymphocytic leukemia (CLL). While some MBL progress to CLL, most MBL have apparently limited potential for progression to CLL, particularly those MBL with normal absolute B cell counts (“low count” MBL). Most CLL are monoclonal and it is not known whether MBL are monoclonal or oligoclonal; this is important because it is unclear whether MBL represent indolent CLL or represent a distinct pre-malignant precursor prior to the development of CLL. We used flow cytometry analysis and sorting to determine immunophenotypic characteristics, clonality, and molecular features of MBL from familial CLL kindreds. Single cell analysis indicated 4 of 6 low count MBL consisted of two or more unrelated clones; the other 2 MBL were monoclonal. 87% of low count MBL clones had mutated immunoglobulin genes, and no immunoglobulin heavy chain rearrangements of VH family 1 were observed. Some MBL were diversified, clonally related populations with evidence of antigen-drive. We conclude that while low count MBL share many phenotypic characteristics with CLL, many MBL are oligoclonal. This supports a model for step-wise development of MBL into CLL.
PMCID: PMC2806490  PMID: 19946263
Monoclonal B Lymphocytosis; Chronic Lymphocytic Leukemia; B Cell Repertoire; Human B Cells
14.  Inhibition of Matrix Metalloproteinases Enhances In Vitro Repair of the Meniscus 
Damage or injury of the meniscus is associated with onset and progression of knee osteoarthritis (OA). The intrinsic repair capacity of the meniscus is inhibited by inflammatory cytokines, such as interleukin-1 (IL-1). Using an in vitro meniscal repair model system, we examined the hypothesis that inhibition of matrix metalloproteinases (MMPs) in the presence of IL-1 will enhance repair of meniscal lesions. Integrative repair of the meniscus was examined between two concentric explants cultured with IL-1 and various MMP inhibitors for 14 days. Throughout the culture period, we assessed total specific MMP activity in the media. At harvest, biomechanical testing to assess the strength of repair and histologic staining were performed. IL-1 decreased the shear strength of repair, as compared with control explants. In the presence of IL-1, the broad-spectrum MMP inhibitor GM 6001 decreased the MMP activity in the media, increased the shear strength of repair, and enhanced tissue repair in the interface. However, individual MMP inhibitors did not alter the shear strength of repair in either the presence or absence of IL-1. These findings suggest IL-1 may inhibit meniscal repair through upregulation of MMPs, but inhibition of multiple MMPs may be necessary to promote integrative meniscal repair.
PMCID: PMC2674160  PMID: 18975039
16.  Oligoclonal TCRVβ gene usage among CD8+ T cells in monoclonal B lymphocytosis and CLL 
British journal of haematology  2009;145(4):535-537.
PMCID: PMC2754701  PMID: 19298246
Chronic lymphocytic leukemia; monoclonal B lymphocytosis; T cell repertoire; spectratyping
17.  Increased Asymmetric Dimethylarginine in Severe Falciparum Malaria: Association with Impaired Nitric Oxide Bioavailability and Fatal Outcome 
PLoS Pathogens  2010;6(4):e1000868.
Asymmetrical dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), is a predictor of mortality in critical illness. Severe malaria (SM) is associated with decreased NO bioavailability, but the contribution of ADMA to the pathogenesis of impaired NO bioavailability and adverse outcomes in malaria is unknown. In adults with and without falciparum malaria, we tested the hypotheses that plasma ADMA would be: 1) increased in proportion to disease severity, 2) associated with impaired vascular and pulmonary NO bioavailability and 3) independently associated with increased mortality. We assessed plasma dimethylarginines, exhaled NO concentrations and endothelial function in 49 patients with SM, 78 with moderately severe malaria (MSM) and 19 healthy controls (HC). Repeat ADMA and endothelial function measurements were performed in patients with SM. Multivariable regression was used to assess the effect of ADMA on mortality and NO bioavailability. Plasma ADMA was increased in SM patients (0.85 µM; 95% CI 0.74–0.96) compared to those with MSM (0.54 µM; 95%CI 0.5–0.56) and HCs (0.64 µM; 95%CI 0.58–0.70; p<0.001). ADMA was an independent predictor of mortality in SM patients with each micromolar elevation increasing the odds of death 18 fold (95% CI 2.0–181; p = 0.01). ADMA was independently associated with decreased exhaled NO (rs = −0.31) and endothelial function (rs = −0.32) in all malaria patients, and with reduced exhaled NO (rs = −0.72) in those with SM. ADMA is increased in SM and associated with decreased vascular and pulmonary NO bioavailability. Inhibition of NOS by ADMA may contribute to increased mortality in severe malaria.
Author Summary
Severe falciparum malaria is associated with impaired microvascular perfusion, lung injury and decreased bioavailability of nitric oxide (NO), but the causes of these processes are not fully understood. Asymmetrical dimethylarginine (ADMA), a competitive endogenous inhibitor of nitric oxide synthase (NOS), is an independent predictor of mortality in other critical illnesses, and can impair vascular function in chronic disease. ADMA can be produced by both the host and malaria parasites. The major novel findings of this study in malaria are that ADMA is an independent predictor of death in falciparum malaria, and is associated with decreased availability of nitric oxide in at least two organ systems affected by malaria parasites, the lining of blood vessels and the lungs. This study contributes to knowledge of regulation and availability of pulmonary and endothelial NO in critical illness and identifies pathogenic processes which may contribute to death in severe malaria. Therapies which increase the availability of NO or which reduce ADMA levels may have potential for adjunctive therapy of severe malaria.
PMCID: PMC2858698  PMID: 20421938
18.  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
19.  Inhibition of Integrative Repair of the Meniscus Following Acute Exposure to Interleukin-1 in vitro 
Damage or loss of the meniscus is associated with progressive osteoarthritic degeneration of the knee joint. Injured and degenerative joints are characterized by elevated levels of the pro-inflammatory cytokine interleukin-1 (IL-1), which with prolonged exposure can induce catabolic and anti-anabolic activities that inhibit tissue repair. We used an in vitro model system to examine the hypotheses that acute exposure to IL-1 inhibits meniscal repair, and that an IL-1-mediated increase in matrix metalloproteinase (MMP) activity is associated with the inhibition of repair. Integrative tissue repair was studied between concentric explants of porcine medial menisci that were treated with IL-1α acutely (100 pg/mL for 1 or 3 days) or chronically (100 pg/mL for entire culture duration). After 14 and 28 days in culture, biomechanical testing, cell viability, and histology were performed to assess meniscal repair. Total specific MMP activity in the culture media was measured using a quenched fluorescent substrate. As little as 1 day of IL-1 exposure significantly reduced shear strength, cell accumulation, and tissue repair compared to controls. IL-1 exposure for 1 or 3 days significantly increased MMP activity that subsided by day 9. With chronic IL-1 exposure, MMP activity remained elevated for the duration of culture and was negatively correlated with repair strength. Our study shows that short-term exposure to physiologically-relevant concentrations of IL-1 significantly reduces meniscal repair in vitro, and thus may potentially inhibit the intrinsic repair response in vivo. The suppression of IL-1 or MMP expression and/or activity warrant investigation as potential strategies for promoting meniscal repair.
PMCID: PMC2729761  PMID: 18050309
collagen; knee; articular cartilage; tissue engineering; collagenase
20.  Recovery of Endothelial Function in Severe Falciparum Malaria: Relationship with Improvement in Plasma L-Arginine and Blood Lactate Concentrations 
The Journal of infectious diseases  2008;198(4):602-608.
Severe malaria is characterized by microvascular obstruction, endothelial dysfunction, and reduced levels of L-arginine and nitric oxide (NO). L-Arginine infusion improves endothelial function in moderately severe malaria. Neither the longitudinal course of endothelial dysfunction nor factors associated with recovery have been characterized in severe malaria.
Endothelial function was measured longitudinally in adults with severe malaria (n = 49) or moderately severe malaria (n = 48) in Indonesia, using reactive hyperemia peripheral arterial tonometry (RH-PAT). In a mixed-effects model, changes in RH-PAT index values in patients with severe malaria were related to changes in parasitemia, lactate, acidosis, and plasma L-arginine concentrations.
Among patients with severe malaria, the proportion with endothelial dysfunction fell from 94% (46/49 patients) to 14% (6/42 patients) before discharge or death (P <.001). In severe malaria, the median time to normal endothelial function was 49 h (interquartile range, 20–70 h) after the start of antimalarial therapy. The mean increase in L-arginine concentrations in patients with severe malaria was 11 μmol/L/24 h (95% confidence interval [CI], 9–13 μmol/L/24 h), from a baseline of 49 μmol/L (95% CI, 37–45 μmol/L). Improvement of endothelial function in patients with severe malaria correlated with increasing levels of L-arginine (r = 0.56; P =.008) and decreasing levels of lactate (r = −0.44; P =.001).
Recovery of endothelial function in severe malaria is associated with recovery from hypoargininemia and lactic acidosis. Agents that can improve endothelial NO production and endothelial function, such as L-arginine, may have potential as adjunctive therapy early during the course of severe malaria.
PMCID: PMC2709993  PMID: 18605903
21.  Intraprotein Electron Transfer in Inducible Nitric Oxide Synthase Holoenzyme 
Intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in nitric oxide (NO) synthesis by NO synthase (NOS). Our previous laser flash photolysis studies provided a direct determination of the kinetics of the FMN–heme IET in a truncated oxyFMN construct of murine inducible NOS (iNOS), in which only the oxygenase and FMN domains along with the calmodulin (CaM) binding site are present [Feng et al. (2006) J. Am. Chem. Soc. 128, 3808-3811]. Here we report the kinetics of the IET in a human iNOS oxyFMN construct, a human iNOS holoenzyme and a murine iNOS holoenzyme, using CO photolysis in comparative studies on partially reduced NOS and a NOS oxygenase construct that lacks the FMN domain. The IET rate constants for the human and murine iNOS holoenzymes are 34 ± 5 s-1 and 35 ± 3 s-1, respectively, thereby providing a direct measurement of this IET between the catalytically significant redox couples of FMN and heme in the iNOS holoenzyme. These values are approximately an order of magnitude smaller than that in the corresponding iNOS oxyFMN construct, suggesting that in the holoenzyme the rate-limiting step in the IET is the conversion of the shielded electron-accepting (input) state to a new electron-donating (output) state. The fact that there is no rapid IET component in the kinetic traces obtained with the iNOS holoenzyme implies that the enzyme remains mainly in the input state. The IET rate constant value for the iNOS holoenzyme is similar to that obtained for a CaM-bound neuronal NOS (nNOS) holoenzyme, suggesting that CaM activation effectively removes the inhibitory effect of the unique autoregulatory insert in nNOS.
PMCID: PMC2596912  PMID: 18830722
Electron transfer; nitric oxide synthase; laser flash photolysis; heme; flavin
22.  Reactive nitrogen and oxygen species in interleukin-1-mediated DNA damage associated with osteoarthritis 
Osteoarthritis (OA) is associated with increased levels of reactive nitrogen and oxygen species and pro-inflammatory cytokines, such as interleukin-1 (IL-1). Nitric oxide (NO) can mediate a number of the catabolic effects of IL-1 in articular cartilage. The aims of this study were to determine if OA cartilage shows evidence of DNA damage, and if IL-1 could induce DNA damage in non-OA cartilage by increasing NO or superoxide.
Articular chondrocytes were isolated from porcine femoral condyles and embedded in 1.2% alginate. The effects of 24 hrs incubation with IL-1, the nitric oxide synthase 2 (NOS2) selective inhibitor, the free radical scavenger SOD, the NO donor NOC18, or the combined NO and peroxynitrite donor SIN-1 on DNA damage were tested, using the “comet” assay. NO production was measured using the Griess assay. The type of oxidative damage present was assessed using a modified comet assay.
OA cartilage had significantly more DNA damage than non-OA cartilage (p < 0.001). IL-1 caused an increase in DNA damage (p < 0.01), which was associated with increased NO production (p < 0.01). Both oxidative DNA strand breaks and base modifications of purines and pyrimidines were observed. IL-1-induced DNA damage was inhibited by a NOS2 inhibitor or by superoxide dismutase (p < 0.01). Furthermore, NOC-18 or SIN-1 caused DNA damage (p < 0.001).
Our work shows chondrocytes in osteoarthritic cartilage exhibit DNA damage, and that IL-1 induces DNA damage and reactive oxygen and nitrogen species in non-OA chondrocytes in alginate.
PMCID: PMC2430155  PMID: 17945515
23.  Regional differences in prostaglandin E2 and nitric oxide production in the knee meniscus in response to dynamic compression 
Injury or loss of the knee meniscus is associated with altered joint stresses that lead to progressive joint degeneration. The goal of this study was to determine if dynamic mechanical compression influences the production of inflammatory mediators by meniscal cells. Dynamic compression increased prostaglandin E2 (PGE2) and nitric oxide (NO) production over a range of stress magnitudes (0.0125-0.5 MPa) in a manner that depended on stress magnitude and zone of tissue origin. Inner zone explants showed greater increases in PGE2 and NO production as compared to outer zone explants. Meniscal tissue expressed NOS2 and NOS3 protein, but not NOS1. Mechanically-induced NO production was blocked by NOS inhibitors, and the non-selective NOS inhibitor L-NMMA augmented PGE2 production in the outer zone only. These findings suggest that the meniscus may serve as an intra-articular source of pro-inflammatory mediators, and that alterations in the magnitude or distribution of joint loading could significantly influence the production of these mediators in vivo.
PMCID: PMC2258009  PMID: 17517372
osteoarthritis; proteoglycan; collagen; prostaglandin; mechanobiology; knee
24.  Safety Profile of L-Arginine Infusion in Moderately Severe Falciparum Malaria 
PLoS ONE  2008;3(6):e2347.
L-arginine infusion improves endothelial function in malaria but its safety profile has not been described in detail. We assessed clinical symptoms, hemodynamic status and biochemical parameters before and after a single L-arginine infusion in adults with moderately severe malaria.
Methodology and Findings
In an ascending dose study, adjunctive intravenous L-arginine hydrochloride was infused over 30 minutes in doses of 3 g, 6 g and 12 g to three separate groups of 10 adults hospitalized with moderately severe Plasmodium falciparum malaria in addition to standard quinine therapy. Symptoms, vital signs and selected biochemical measurements were assessed before, during, and for 24 hours after infusion. No new or worsening symptoms developed apart from mild discomfort at the intravenous cannula site in two patients. There was a dose-response relationship between increasing mg/kg dose and the maximum decrease in systolic (ρ = 0.463; Spearman's, p = 0.02) and diastolic blood pressure (r = 0.42; Pearson's, p = 0.02), and with the maximum increment in blood potassium (r = 0.70, p<0.001) and maximum decrement in bicarbonate concentrations (r = 0.53, p = 0.003) and pH (r = 0.48, p = 0.007). At the highest dose (12 g), changes in blood pressure and electrolytes were not clinically significant, with a mean maximum decrease in mean arterial blood pressure of 6 mmHg (range: 0–11; p<0.001), mean maximal increase in potassium of 0.5 mmol/L (range 0.2–0.7 mmol/L; p<0.001), and mean maximal decrease in bicarbonate of 3 mEq/L (range 1–7; p<0.01) without a significant change in pH. There was no significant dose-response relationship with blood phosphate, lactate, anion gap and glucose concentrations. All patients had an uncomplicated clinical recovery.
Infusion of up to 12g of intravenous L-arginine hydrochloride over 30 minutes is well tolerated in adults with moderately severe malaria, with no clinically important changes in hemodynamic or biochemical status. Trials of adjunctive L-arginine can be extended to phase 2 studies in severe malaria.
Trial Registration NCT00147368
PMCID: PMC2405947  PMID: 18545693
25.  Impaired nitric oxide bioavailability and l-arginine–reversible endothelial dysfunction in adults with falciparum malaria 
The Journal of Experimental Medicine  2007;204(11):2693-2704.
Severe falciparum malaria (SM) is associated with tissue ischemia related to cytoadherence of parasitized erythrocytes to microvascular endothelium and reduced levels of NO and its precursor, l-arginine. Endothelial function has not been characterized in SM but can be improved by l-arginine in cardiovascular disease. In an observational study in Indonesia, we measured endothelial function using reactive hyperemia–peripheral arterial tonometry (RH-PAT) in 51 adults with SM, 48 patients with moderately severe falciparum malaria (MSM), and 48 controls. The mean RH-PAT index was lower in SM (1.41; 95% confidence interval [CI] = 1.33–1.47) than in MSM (1.82; 95% CI = 1.7–2.02) and controls (1.93; 95% CI = 1.8–2.06; P < 0.0001). Endothelial dysfunction was associated with elevated blood lactate and measures of hemolysis. Exhaled NO was also lower in SM relative to MSM and controls. In an ascending dose study of intravenous l-arginine in 30 more patients with MSM, l-arginine increased the RH-PAT index by 19% (95% CI = 6–34; P = 0.006) and exhaled NO by 55% (95% CI = 32–73; P < 0.0001) without important side effects. Hypoargininemia and hemolysis likely reduce NO bioavailability. Endothelial dysfunction in malaria is nearly universal in severe disease, is reversible with l-arginine, and likely contributes to its pathogenesis. Clinical trials in SM of adjunctive agents to improve endothelial NO bioavailability, including l-arginine, are warranted.
PMCID: PMC2118490  PMID: 17954570

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