Polymicrobial Oral Infection with Four Periodontal Bacteria Orchestrates a Distinct Inflammatory Response and Atherosclerosis in ApoEnull Mice

Periodontal disease (PD) develops from a synergy of complex subgingival oral microbiome, and is linked to systemic inflammatory atherosclerotic vascular disease (ASVD). To investigate how a polybacterial microbiome infection influences atherosclerotic plaque progression, we infected the oral cavity of ApoE^null mice with a polybacterial consortium of 4 well-characterized periodontal pathogens, Porphyromonas gingivalis, Treponema denticola, Tannerealla forsythia and Fusobacterium nucleatum, that have been identified in human atherosclerotic plaque by DNA screening. We assessed periodontal disease characteristics, hematogenous dissemination of bacteria, peripheral T cell response, serum inflammatory cytokines, atherosclerosis risk factors, atherosclerotic plaque development, and alteration of aortic gene expression. Polybacterial infections have established gingival colonization in ApoE^null hyperlipidemic mice and displayed invasive characteristics with hematogenous dissemination into cardiovascular tissues such as the heart and aorta. Polybacterial infection induced significantly higher levels of serum risk factors oxidized LDL (p < 0.05), nitric oxide (p < 0.01), altered lipid profiles (cholesterol, triglycerides, Chylomicrons, VLDL) (p < 0.05) as well as accelerated aortic plaque formation in ApoE^null mice (p < 0.05). Periodontal microbiome infection is associated with significant decreases in Apoa1, Apob, Birc3, Fga, FgB genes that are associated with atherosclerosis. Periodontal infection for 12 weeks had modified levels of inflammatory molecules, with decreased Fas ligand, IL-13, SDF-1 and increased chemokine RANTES. In contrast, 24 weeks of infection induced new changes in other inflammatory molecules with reduced KC, MCSF, enhancing GM-CSF, IFNγ, IL-1β, IL-13, IL-4, IL-13, lymphotactin, RANTES, and also an increase in select inflammatory molecules. This study demonstrates unique differences in the host immune response to a polybacterial periodontal infection with atherosclerotic lesion progression in a mouse model.     

Tetrahydrobiopterin attenuates homocysteine induced endothelial dysfunction

Homocysteine is an independent risk factor for atherosclerotic vascular disease. It impairs endothelial function via increasing superoxide production and quenching nitric oxide (NO) release. Tetrahydrobiopterin (BH₄) is a critical cofactor that couples nitric oxide synthase and facilitates the production of nitric oxide (vs. superoxide anions). In the first study, the effects of hyperhomocysteinemia (0.1 mM, 3 h) on endothelium-dependent vasorelaxation to ACh and A23187 were examined in isolated segments of rat aortae in the presence or absence of BH₄ (0.1 mM). In the second study, the effects of hyperhomocysteinemia (24 h) on nitric oxide production and superoxide release (using lucigenin chemiluminescence) were studied in human umbilical vein endothelial cells in the absence or presence of BH₄ (10 M). Homocysteine incubation impaired receptor-dependent and -independent endothelial function to ACh and A23187. This effect was attenuated by BH₄. Furthermore, homocysteine exposure increased superoxide production and impaired agonist-stimulated nitric oxide release. These effects were attenuated by BH₄ (p < 0.05). Hyperhomocysteinemia impairs endothelial function, in part due to a diminished bioavailability of BH₄ with resultant uncoupling of nitric oxide synthase. BH₄ may represent an important target for strategies aimed at improving endothelial dysfunction secondary to hyperhomocysteinemia.     

Evidence against tetrahydrobiopterin depletion of vascular tissue exposed to nitric oxide/superoxide or nitroglycerin

Several cardiovascular disorders, including atherosclerosis and tolerance to the antianginal drug nitroglycerin (GTN), may be associated with the generation of superoxide anions, which react with nitric oxide (NO) to yield peroxynitrite. According to a widely held view, oxidation of tetrahydrobiopterin (BH₄) by peroxynitrite causes uncoupling of endothelial NO synthase (eNOS), resulting in reduced NO bioavailability and endothelial dysfunction under conditions of oxidative stress. In this study we determined the levels of reduced biopterins and endothelial function in cultured cells exposed to peroxynitrite and GTN as well as in blood vessels isolated from GTN-tolerant guinea pigs and rats. BH₄ was rapidly oxidized by peroxynitrite and 3-morpholino sydnonimine (SIN-1) in buffer, but this was prevented by glutathione and not observed in endothelial cells exposed to SIN-1 or GTN. Prolonged treatment of the cells with 0.1 mM GTN caused slow N^G-nitro-l-arginine-sensitive formation of reactive oxygen species without affecting eNOS activity. Endothelial function and BH₄/BH₂ levels were identical in blood vessels of control and GTN-tolerant animals. Our results suggest that peroxynitrite-triggered BH₄ oxidation does not occur in endothelial cells or GTN-exposed blood vessels. GTN seems to trigger minor eNOS uncoupling that is unrelated to BH₄ depletion and without observable consequence on eNOS function.     

Inducible nitric oxide synthase-deficient mice show exacerbated inflammatory process and high production of both Th1 and Th2 cytokines during paracoccidioidomycosis

Paracoccidioidomycosis, the major systemic mycosis in Latin America, is caused by fungus Paracoccidioides brasiliensis. To analyze the influence of inducible nitric oxide synthase (iNOS) in this disease, iNOS-deficient (iNOS^−/−) and wild-type (WT) mice were infected intravenously with P. brasiliensis 18 isolate. We found that, unlike WT mice, iNOS^−/− mice did not control fungal proliferation, and began to succumb to infection by day 50 after inoculation of yeast cells. Typical inflammatory granulomas were found in WT mice, while, iNOS^−/− mice presented incipient granulomas with intense inflammatory process and necrosis. Additionally, splenocytes from iNOS^−/− mice did not produce nitric oxide, however, their proliferative response to Con-A was impaired, just like infected WT mice. Moreover, infected iNOS^−/− mice presented a mixed pattern of immune response, releasing high levels of both Th1 (IL-12, IFN-γ and TNF-α) and Th2 (IL-4 and IL-10) cytokines. These data suggest that the enzyme iNOS is a resistance factor during paracoccidioidomycosis by controlling fungal proliferation, by influencing cytokines production, and by appeasing the development of a high inflammatory response and consequently formation of necrosis. However, iNOS-derived nitric oxide seems not being the unique factor responsible for immunosuppression observed in infections caused by P. brasiliensis.     

Fusobacterium nucleatum Alters Atherosclerosis Risk Factors and Enhances Inflammatory Markers with an Atheroprotective Immune Response in ApoEnull Mice

The American Heart Association supports an association between periodontal disease (PD) and atherosclerotic vascular disease (ASVD) but does not as of yet support a causal relationship. Recently, we have shown that major periodontal pathogens Porphyromonas gingivalis and Treponema denticola are causally associated with acceleration of aortic atherosclerosis in ApoE^null hyperlipidemic mice. The aim of this study was to determine if oral infection with another significant periodontal pathogen Fusobacterium nucleatum can accelerate aortic inflammation and atherosclerosis in the aortic artery of ApoE^null mice. ApoE^null mice (n = 23) were orally infected with F. nucleatum ATCC 49256 and euthanized at 12 and 24 weeks. Periodontal disease assessments including F. nucleatum oral colonization, gingival inflammation, immune response, intrabony defects, and alveolar bone resorption were evaluated. Systemic organs were evaluated for infection, aortic sections were examined for atherosclerosis, and inflammatory markers were measured. Chronic oral infection established F. nucleatum colonization in the oral cavity, induced significant humoral IgG (P=0.0001) and IgM (P=0.001) antibody response (12 and 24 weeks), and resulted in significant (P=0.0001) alveolar bone resorption and intrabony defects. F. nucleatum genomic DNA was detected in systemic organs (heart, aorta, liver, kidney, lung) indicating bacteremia. Aortic atherosclerotic plaque area was measured and showed a local inflammatory infiltrate revealed the presence of F4/80⁺ macrophages and CD3⁺ T cells. Vascular inflammation was detected by enhanced systemic cytokines (CD30L, IL-4, IL-12), oxidized LDL and serum amyloid A, as well as altered serum lipid profile (cholesterol, triglycerides, chylomicrons, VLDL, LDL, HDL), in infected mice and altered aortic gene expression in infected mice. Despite evidence for systemic infection in several organs and modulation of known atherosclerosis risk factors, aortic atherosclerotic lesions were significantly reduced after F. nucleatum infection suggesting a potential protective function for this member of the oral microbiota.     

Nitric Oxide Synthase Promotes Distension-Induced Tracheal Venular Leukocyte Adherence

The process of leukocyte recruitment to the airways in real time has not been extensively studied, yet airway inflammation persists as a major contributor to lung pathology. We showed previously in vivo, that neutrophils are recruited acutely to the large airways after periods of airway distension imposed by the application of positive end-expiratory pressure (PEEP). Given extensive literature implicating products of nitric oxide synthase (NOS) in lung injury after ventilatory over-distension, we questioned whether similar mechanisms exist in airway post-capillary venules. Yet, endothelial nitric oxide has been shown to be largely anti-inflammatory in other systemic venules. Using intravital microscopy to visualize post-capillary tracheal venules in anesthetized, ventilated mice, the number of adherent leukocytes was significantly decreased in eNOS^-/- mice under baseline conditions (2±1 cell/60 min observation) vs wild type (WT) C57BL/6 mice (7±2 cells). After exposure to PEEP (8 cmH₂O for 1 min; 5 times), adherent cells increased significantly (29±5 cells) in WT mice while eNOS^-/- mice demonstrated a significantly decreased number of adherent cells (11±4 cells) after PEEP. A similar response was seen when thrombin was used as the pro-inflammatory stimulus. In addition, mouse tracheal venular endothelial cells studied in vitro after exposure to cyclic stretch (18% elongation) or thrombin both demonstrated increased p-selectin expression that was significantly attenuated by N^G-nitro-L-arginine methyl ester, N-acetylcysteine amide (NACA) and excess BH₄. In vivo treatment with the ROS inhibitor NACA or co-factor BH₄ abolished completely the PEEP-induced leukocyte adherence. These results suggest that pro-inflammatory stimuli cause leukocyte recruitment to tracheal endothelium in part due to eNOS uncoupling.     

L-Arginine and tetrahydrobiopterin supported nitric oxide production is crucial for the microbicidal activity of neutrophils

Recent report from this lab has shown role of Rac2 in the translocation of inducible nitric oxide synthase (iNOS) to the phagosomal compartment of polymorphonuclear leukocytes (PMNs) following phagocytosis of beads. This study was undertaken to further assess the status and role of tetrahydrobiopterin (BH₄), a redox-sensitive cofactor, L-arginine, and the substrate of nitric oxide synthase (NOS) in sustained nitric oxide (˙NO) production in killing of phagocytosed microbes (Escherichia coli) by human PMNs. Time-dependent study revealed consistent NO and reactive oxygen species (ROS) production in the PMNs following phagocytosis of beads. In addition, levels of L-arginine and BH₄ were maintained or increased simultaneously to support the enzymatic activity of NOS in the bead activated PMNs. Moreover, translocation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) subunits along with iNOS was reconfirmed in the isolated phagosomes. We demonstrate that increase in the level of NO was supported by L-arginine and BH₄ to kill E. coli, by using PMNs from NOS2^?/? mice, human PMNs treated with biopterin inhibitor, N-acetyl serotonin (NAS), or by suspending human PMNs in L-arginine deficient medium. Altogether, this study demonstrates that following phagocytosis, sustained. NO production in the PMNs was well-maintained by redox sensitive cofactor, BH₄ and substrate, and L-arginine to enable microbial killing. Further results suggest NO production in the human PMNs, along with ROS and myeloperoxidase (MPO) is important to execute antimicrobial activity.     

Analysis of Plasma Biopterin Levels in Psychiatric Disorders Suggests a Common BH<Subscript>4</Subscript> Deficit in Schizophrenia and Schizoaffective Disorder

Tetrahydrobiopterin (BH₄) is an essential cofactor for amine neurotransmitter synthesis. BH₄ also stimulates and modulates the glutamatergic system, and regulates the synthesis of nitric oxide by nitric oxide synthases. A connection between BH₄ deficiencies and psychiatric disorders has been previously reported; major depression and obsessive-compulsive disorder have been found in subjects with a BH₄ deficiency disorder and more recently we have observed a robust plasma deficit of biopterin (a measure of BH₄), in a large group of schizophrenic patients compared to control subjects. To extend our previous finding in schizophrenia, we analyzed plasma biopterin levels from patients with schizoaffective and bipolar disorders. A significant difference in biopterin was seen among the diagnostic groups (P < 0.0001). Post hoc analyses indicated significant biopterin deficits relative to the normal control group for the schizoaffective group, who had biopterin levels comparable to the schizophrenic group. Bipolar disorder subjects had plasma biopterin levels that were higher that the schizoaffective disorder group and significantly higher than the schizophrenic group. The demonstrated significant biopterin deficit in both schizophrenia and schizoaffective disorder, may suggest an etiological role of a BH₄ deficit in these two disorders, via dysregulation of neurotransmitter systems.     

Altered renal immune complexes deposition in female BWF1 lupus mice following Plasmodium chabaudi infection

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease that has a mysterious relationship with malaria infection. The current study was designated to compare between the effect of the live and the gamma irradiated Plasmodium chabaudi infection on BWF1 lupus murine model. A total of 30 female BWF1 mice were randomly divided into three groups (10 mice/group) as follows: group (I) lupus group (lupus non infected); group (II) live malaria infected group (lupus + live malaria infection); and group (III) irradiated malaria-infected group (lupus + gamma irradiated malaria infection). Live P. chabaudi infection was accompanied with a decrease in survival rate and food consumption in comparison to the control group of mice while gamma irradiated P. chabaudi -infection was unable to do this effect. Additionally, live P. chabaudi infection was accompanied with an increased level of proteinuria and increased rate of immune complexes deposition in kidney. Moreover, infection with live, but not gamma-irradiated P. chabaudi was accompanied with an increase in nitric oxide (NO), hydrogen peroxide (H₂O₂), and malondialdehyde (MDA) levels in plasma of lupus mice. The levels of both total cholesterol and triglycerides in plasma of lupus mice after live P. chabaudi infection were obviously decreased in comparison to the control group. On the other hand, gamma-irradiated P. chabaudi infection resembled the control group. Our data revealed that infection of lupus mice with live but not gamma-irradiated P. chabaudi has several histological and biochemical effects.     

Impairment of Endothelial-Myocardial Interaction Increases the Susceptibility of Cardiomyocytes to Ischemia/Reperfusion Injury

Endothelial-myocardial interactions may be critically important for ischemia/reperfusion injury. Tetrahydrobiopterin (BH₄) is a required cofactor for nitric oxide (NO) production by endothelial NO synthase (eNOS). Hyperglycemia (HG) leads to significant increases in oxidative stress, oxidizing BH₄ to enzymatically incompetent dihydrobiopterin. How alterations in endothelial BH₄ content impact myocardial ischemia/reperfusion injury remains elusive. The aim of this study was to examine the effect of endothelial-myocardial interaction on ischemia/reperfusion injury, with an emphasis on the role of endothelial BH₄ content. Langendorff-perfused mouse hearts were treated by triton X-100 to produce endothelial dysfunction and subsequently subjected to 30 min of ischemia followed by 2 h of reperfusion. The recovery of left ventricular systolic and diastolic function during reperfusion was impaired in triton X-100 treated hearts compared with vehicle-treated hearts. Cardiomyocytes (CMs) were co-cultured with endothelial cells (ECs) and subsequently subjected to 2 h of hypoxia followed by 2 h of reoxygenation. Addition of ECs to CMs at a ratio of 1∶3 significantly increased NO production and decreased lactate dehydrogenase activity compared with CMs alone. This EC-derived protection was abolished by HG. The addition of 100 µM sepiapterin (a BH₄ precursor) or overexpression of GTP cyclohydrolase 1 (the rate-limiting enzyme for BH₄ biosynthesis) in ECs by gene trasfer enhanced endothelial BH₄ levels, the ratio of eNOS dimer/monomer, eNOS phosphorylation, and NO production and decreased lactate dehydrogenase activity in the presence of HG. These results demonstrate that increased BH₄ content in ECs by either pharmacological or genetic approaches reduces myocardial damage during hypoxia/reoxygenation in the presence of HG. Maintaining sufficient endothelial BH₄ is crucial for cardioprotection against hypoxia/reoxygenation injury.     

Evidence of Infectious Asthma Phenotype: Chlamydia-Induced Allergy and Pathogen-Specific IgE in a Neonatal Mouse Model

Asthma is a chronic respiratory disease whose etiology is poorly understood. Recent studies suggest that early-life respiratory infections with atypical bacteria may play an important role in the induction or exacerbation of chronic respiratory disease. The current study utilized a neonatal mouse ovalbumin (OVA) sensitization model of asthma to determine the course of early-life respiratory tract infection by Chlamydia. Neonatal (day 1) and adult (6 wks) BALB/c mice were infected intranasally with Chlamydia (MoPn) and 7 weeks later were sensitized and challenged with ovalbumin. Allergic airway disease was characterized by examination of serum and bronchoalveolar lavage fluid (BAL) cellularity, cytokine production and antibody response. The presence of Chlamydia was determined by PCR and culture. Ova-specific IgE was quantified by ELISA and Chlamydia-specific IgE was determined via Western blot analysis. Chlamydial infection in neonatal mice induced increased production of Th₂ cytokines (IL-4, 5, 10, and 13) in both BAL and serum, while infected adult mice produced increased Th₁ cytokines (IL-2, IFN-γ). The BAL from infected neonates contained significantly elevated levels of eosinophils compared to infected adult mice. Although adult mice cleared the infection ∼30 days post infection (pi), neonates were still infected 66 days after initial infection. Chlamydia-specific IgE was detected in both the BAL and serum of neonatal mice beginning 28 days post infection, however, infected adult mice did not produce Chlamydia-specific IgE antibodies over the course of the study. When allergic airway was induced using Ova, infected neonatal mice increased their production of IL-4, IL-5 and IL-13 by >2 fold compared to uninfected controls and infected adult groups. Our findings demonstrate that early-life Chlamydia infection induces a Th₂-dominant cytokine response in the airways of neonatal mice, leading to chronic infection. More significantly, early life respiratory colonization with Chlamydia elicits pathogen-specific IgE production, which further supports an infectious asthma phenotype.     

Tetrahydrobiopterin Biosynthesis as a Potential Target of the Kynurenine Pathway Metabolite Xanthurenic Acid

Tryptophan metabolites in the kynurenine pathway are up-regulated by pro-inflammatory cytokines or glucocorticoids, and are linked to anti-inflammatory and immunosuppressive activities. In addition, they are up-regulated in pathologies such as cancer, autoimmune diseases, and psychiatric disorders. The molecular mechanisms of how kynurenine pathway metabolites cause these effects are incompletely understood. On the other hand, pro-inflammatory cytokines also up-regulate the amounts of tetrahydrobiopterin (BH₄), an enzyme cofactor essential for the synthesis of several neurotransmitter and nitric oxide species. Here we show that xanthurenic acid is a potent inhibitor of sepiapterin reductase (SPR), the final enzyme in de novo BH₄ synthesis. The crystal structure of xanthurenic acid bound to the active site of SPR reveals why among all kynurenine pathway metabolites xanthurenic acid is the most potent SPR inhibitor. Our findings suggest that increased xanthurenic acid levels resulting from up-regulation of the kynurenine pathway could attenuate BH₄ biosynthesis and BH₄-dependent enzymatic reactions, linking two major metabolic pathways known to be highly up-regulated in inflammation.     

GTP Cyclohydrolase I: Purification, Characterization, and Effects of Inhibition on Nitric Oxide Synthase in Nocardia Species

GTP cyclohydrolase I (GTPCH) catalyzes the first step in pteridine biosynthesis in Nocardia sp. strain NRRL 5646. This enzyme is important in the biosynthesis of tetrahydrobiopterin (BH₄), a reducing cofactor required for nitric oxide synthase (NOS) and other enzyme systems in this organism. GTPCH was purified more than 5,000-fold to apparent homogeneity by a combination of ammonium sulfate fractionation, GTP-agarose, DEAE Sepharose, and Ultragel AcA 34 chromatography. The purified enzyme gave a single band for a protein estimated to be 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme was estimated to be 253 kDa by gel filtration, indicating that the active enzyme is a homo-octamer. The enzyme follows Michaelis-Menten kinetics, with a K_m for GTP of 6.5 μM. Nocardia GTPCH possessed a unique N-terminal amino acid sequence. The pH and temperature optima for the enzyme were 7.8 and 56°C, respectively. The enzyme was heat stable and slightly activated by potassium ion but was inhibited by calcium, copper, zinc, and mercury, but not magnesium. BH₄ inhibited enzyme activity by 25% at a concentration of 100 μM. 2,4-Diamino-6-hydroxypyrimidine (DAHP) appeared to competitively inhibit the enzyme, with a K_i of 0.23 mM. With Nocardia cultures, DAHP decreased medium levels of NO₂⁻ plus NO₃⁻. Results suggest that in Nocardia cells, NOS synthesis of nitric oxide is indirectly decreased by reducing the biosynthesis of an essential reducing cofactor, BH₄.     

Polymicrobial Infection with Major Periodontal Pathogens Induced Periodontal Disease and Aortic Atherosclerosis in Hyperlipidemic ApoEnull Mice

Periodontal disease (PD) and atherosclerosis are both polymicrobial and multifactorial and although observational studies supported the association, the causative relationship between these two diseases is not yet established. Polymicrobial infection-induced periodontal disease is postulated to accelerate atherosclerotic plaque growth by enhancing atherosclerotic risk factors of orally infected Apolipoprotein E deficient (ApoE^null) mice. At 16 weeks of infection, samples of blood, mandible, maxilla, aorta, heart, spleen, and liver were collected, analyzed for bacterial genomic DNA, immune response, inflammation, alveolar bone loss, serum inflammatory marker, atherosclerosis risk factors, and aortic atherosclerosis. PCR analysis of polymicrobial-infected (Porphyromonas gingivalis [P. gingivalis], Treponema denticola [T. denticola], and Tannerella forsythia [T. forsythia]) mice resulted in detection of bacterial genomic DNA in oral plaque samples indicating colonization of the oral cavity by all three species. Fluorescent in situ hybridization detected P. gingivalis and T. denticola within gingival tissues of infected mice and morphometric analysis showed an increase in palatal alveolar bone loss (p<0.0001) and intrabony defects suggesting development of periodontal disease in this model. Polymicrobial-infected mice also showed an increase in aortic plaque area (p<0.05) with macrophage accumulation, enhanced serum amyloid A, and increased serum cholesterol and triglycerides. A systemic infection was indicated by the detection of bacterial genomic DNA in the aorta and liver of infected mice and elevated levels of bacterial specific IgG antibodies (p<0.0001). This study was a unique effort to understand the effects of a polymicrobial infection with P. gingivalis, T. denticola and T. forsythia on periodontal disease and associated atherosclerosis in ApoE^null mice.     

Nitric Oxide Production by Necrotrophic Pathogen Macrophomina phaseolina and the Host Plant in Charcoal Rot Disease of Jute: Complexity of the Interplay between Necrotroph–Host Plant Interactions

M. phaseolina, a global devastating necrotrophic fungal pathogen causes charcoal rot disease in more than 500 host plants. With the aim of understanding the plant-necrotrophic pathogen interaction associated with charcoal rot disease of jute, biochemical approach was attempted to study cellular nitric oxide production under diseased condition. This is the first report on M. phaseolina infection in Corchorus capsularis (jute) plants which resulted in elevated nitric oxide, reactive nitrogen species and S nitrosothiols production in infected tissues. Time dependent nitric oxide production was also assessed with 4-Amino-5-Methylamino-2′,7′-Difluorofluorescein Diacetate using single leaf experiment both in presence of M. phaseolina and xylanases obtained from fungal secretome. Cellular redox status and redox active enzymes were also assessed during plant fungal interaction. Interestingly, M. phaseolina was found to produce nitric oxide which was detected in vitro inside the mycelium and in the surrounding medium. Addition of mammalian nitric oxide synthase inhibitor could block the nitric oxide production in M. phaseolina. Bioinformatics analysis revealed nitric oxide synthase like sequence with conserved amino acid sequences in M. phaseolina genome sequence. In conclusion, the production of nitric oxide and reactive nitrogen species may have important physiological significance in necrotrophic host pathogen interaction.     

Effect of dietary arginine supplementation on reproductive performance of mice with porcine circovirus type 2 infection

The objective of this study was to investigate whether supplemental dietary arginine increases reproductive performance in mice infected with porcine circovirus type2 (PCV2). A total of 50KM female mice were allotted randomly to the arginine group (0.6% arginine?+?gestation diet) and control group (1.22% alanine?+?gestation diet). All the mice began to mate after 14?days of treatment with our prepared feed and challenged with PCV2 at the dose of 100 TCID₅₀ (50% tissue culture infection dose, TCID₅₀) after 7?days of pregnancy. Abortion rate, litter number, litter birth weight, the daily weight gain in the first 7?days and survival rate in the first 2?weeks of the neonates were calculated. The serum progesterone, estrogen, nitric oxide and superoxide dismutase (SOD) activity and total antioxidant capacity (T-AOC) on the 14th day of pregnancy were measured. Arginine supplementation decreased the abortion rate of pregnant mice and mortality of neonates caused by PCV2 infection. Further, litter number, litter birth weight and the daily weight gain of neonates increased in the arginine group compared to the control group. Arginine supplementation increased significantly the serum progesterone (P?<?0.01) and nitric oxide levels (P?<?0.05), but had little effect on the serum estrogen level. SOD activity and T-AOC in the arginine group were significantly higher (P?<?0.01) than the control group. In conclusion, arginine supplementation partially reversed the reproductive failure in mice caused by PCV2 infection.     

Influence of Parasite Load on Renal Function in Mice Acutely Infected with Trypanosoma cruzi

Background

Chagas disease is a neglected tropical disease caused by Trypanosoma cruzi. Despite the vast number of studies evaluating the pathophysiological mechanisms of the disease, the influence of parasite burden on kidney lesions remains unclear. Thus, the main goal of this work was to evaluate the effect of T. cruzi infection on renal function and determine whether there was a correlation between parasite load and renal injury using an acute experimental model of the disease.

Methodology/Principal Findings

Low, medium and high parasite loads were generated by infecting C57BL/6 mice with 300 (low), 3,000 (medium) or 30,000 (high) numbers of “Y” strain trypomastigotes. We found that mice infected with T. cruzi trypomastigotes show increased renal injury. The infection resulted in reduced urinary excretion and creatinine clearance. We also observed a marked elevation in the ratio of urine volume to kidney and body weight, blood urea nitrogen, chloride ion, nitric oxide, pro- and anti-inflammatory cytokines and the number of leukocytes in the blood and/or renal tissues of infected mice. Additionally, we observed the presence of the parasite in the cortical/medullary and peri-renal region, an increase of inflammatory infiltrate and of vascular permeability of the kidney. Overall, most renal changes occurred mainly in animals infected with high parasitic loads.

Conclusions/Significance

These data demonstrate that T. cruzi impairs kidney function, and this impairment is more evident in mice infected with high parasitic loads. Moreover, these data suggest that, in addition to the extensively studied cardiovascular effects, renal injury should be regarded as an important indicator for better understanding the pan-infectivity of the parasite and consequently for understanding the disease in experimental models.     

Magnetic circular dichroism spectroscopic characterization of the NOS-like protein from Geobacillus stearothermophilus (gsNOS)

Nitric oxide synthase (NOS) catalyzes the NADPH- and O₂-dependent oxidation of l-arginine (l-Arg) to nitric oxide (NO) and citrulline via an N^G-hydroxy-l-arginine (NHA) intermediate. Mammalian NOSs have been studied quite extensively; other eukaryotes and some prokaryotes appear to express NOS-like proteins comparable to the oxygenase domain of mammalian NOSs. In this study, a recombinant NOS-like protein from the thermostable bacterium Geobacillus stearothermophilus (gsNOS) has been characterized using magnetic circular dichroism (MCD) and UV–Vis absorption spectroscopic techniques. Spectral comparisons of ligand complexes (with O₂, NO and CO) of substrate-bound (l-Arg or NHA) gsNOS, including the key oxyferrous complex studied at ?50 °C in cryogenic mixed solvents, with analogous mammalian NOS complexes indicate overall spectroscopic similarities between gsNOS and mammalian NOSs. However, more detailed spectral comparisons reflect subtle structural differences between gsNOS and mammalian NOSs. This may be due to an incomplete tetrahydrobiopterin (BH₄)-binding site and low BH₄-binding affinity, which may become even lower in the presence of cryosolvent in gsNOS. Although BH₄-binding may be altered, gsNOS appears to require the pterin for NO production since formation of the stable ferric-NO product complex was only observed when excess BH₄ (>150 μM) over gsNOS was present upon single turnover reaction in which O₂ was bubbled into dithionite-reduced NHA-bound protein solution at ?35 °C or ?50 °C.