Combined inhibition of indoleamine 2,3-dioxygenase and nitric oxide synthase modulates neurotoxin release by interferon-gamma-activated macrophages

We evaluated the synthesis of nitric oxide (NO) and of the neurotoxic kynurenine metabolites 3OH-kynurenine and quinolinic acid (QUIN) in interferon-gamma (IFN-gamma)-activated macrophages of the murine BACl.2F5 cell line with the aim of investigating the roles of mononuclear phagocytes in inflammatory neurological disorders. IFN-gamma induced indoleamine 2,3-dioxygenase (IDO) and NO synthase (NOS) and increased the synthesis of 3OH-kynurenine, QUIN, and NO that accumulated in the incubation medium where they reached neurotoxic levels. Macrophage exposure to norharmane, an IDO inhibitor, resulted in a decreased formation of not only the kynurenine metabolites but also NO. The inhibition of NO synthesis could not be ascribed to reduced NADPH availability or decreased NOS induction. Norharmane inhibited NOS activity also in coronary vascular endothelial cells and in isolated aortic rings. Our findings suggest that activated macrophages release large amounts of neurotoxic molecules and that norharmane may represent a prototype compound to study macrophage involvement in inflammatory brain damage.     

SOFA score and left ventricular systolic function as predictors of short-term outcome in patients with sepsis

In a group of septic patients, we assess the short-term prognostic value of LV systolic performance, evaluated through conventional left ventricular ejection fraction (LVEF) and left ventricular global longitudinal strain (LV GLS). One hundred forty-seven patients with sepsis were recruited; LVEF by planimetry and peak GLS by 2D speckle tracking could be assessed within 24 h. The study population was stratified according to SOFA tertiles assessed at the time of the echocardiogram (G1: SOFA score <5; G2: SOFA score 5–7; G3: SOFA score >7). Day-7 follow-up data were used as reference. Patients in G2 and G3 show a significant hemodynamic derangement, paralleling the more pronounced organ damage by definition; nevertheless, LVEF and GLS are comparable among the three groups (both p > 0.1). All-cause mortality at day-7 follow-up is slightly lower in G1 (9%) versus G2 and G3 (14 and 26%, respectively, p = NS). Analyses through ROC curves focusing on day-7 mortality show that the SOFA score fairly correlates with events (AUC 0.635, p = 0.037), while low LVEF (AUC 0.35, p = 0.022) and less negative GLS (AUC 0.73, p = 0.001) do so. In multivariate analyses, mortality by day-7 follow-up is more likely per higher GLS (i.e., indicative of worst systolic dysfunction, HR 1.22/%, p = 0.005) and per increasing SOFA score (HR 1.22/unit, p = 0.010), whereas LVEF, adjusted for age and SOFA score, does not enter the prognostic model. In the very short term in patients with severe sepsis, LV systolic function assessment by means of GLS predicts the short-term prognosis, independent of SOFA.     

alpha(1D)-adrenoceptors cause endothelium-dependent vasodilatation in the rat mesenteric vascular bed

The vasodilator activity of alpha(1)-adrenoceptor agonists was tested in the rat mesenteric vascular bed (MVB), and the mechanism involved was investigated in cultured endothelial cells isolated from the bovine coronary vascular bed. In preparations preconstricted by U46619, noradrenaline and phenylephrine induced a slight relaxant effect at nanomolar concentrations. This effect was abolished in endothelium-denuded preparations and in preparations pretreated with 100 microM N(omega)-nitro-L-arginine methyl ester plus 3 microM indomethacin. Both the phospholipase C inhibitor U73122 and the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin inhibited the vasorelaxant effect of phenylephrine. The cellular level of inositol monophosphate (IP(1)) in bovine endothelial cells doubled after a 15-min exposure to 0.03 to 0.1 nM phenylephrine. The activity of cNOS was significantly increased following exposure to the same concentrations of phenylephrine. Both chloroethylclonidine and the selective alpha(1D)-adrenoceptor antagonist BMY 7378 reduced, in a concentration-dependent manner, the relaxant effect induced by phenylephrine, whereas the selective alpha(1A)-adrenoceptor antagonist (+)-niguldipine was ineffective. BMY 7378 also blocked the cNOS activation induced by phenylephrine. Conversely, the increase in perfusion pressure induced by micromolar concentrations of phenylephrine was blocked by 1 nM (+)-niguldipine, but was unaffected by BMY 7378. These findings demonstrate that nanomolar concentrations of phenylephrine, which are devoid of any contractile effect, induced a slight endothelium-dependent vasorelaxation in the rat MVB through the stimulation of alpha(1D)-adrenoceptors, located on endothelial cells, which act through phospholipase C stimulation, followed by IP(1) generation, and nitric-oxide synthase activation. Conversely, the increase in perfusion pressure induced by micromolar concentrations of phenylephrine is attributable to the stimulation of alpha(1A)-adrenoceptors.     

A Homozygous Mutation in LYRM7/MZM1L Associated with Early Onset Encephalopathy,Lactic Acidosis,and Severe Reduction of Mitochondrial Complex III Activity

Mutations in nuclear genes associated with defective complex III (cIII) of the mitochondrial respiratory chain are rare, having been found in only two cIII assembly factors and, as private changes in single families, three cIII structural subunits. Recently, human LYRM7/MZM1L, the ortholog of yeast MZM1, has been identified as a new assembly factor for cIII. In a baby patient with early onset, severe encephalopathy, lactic acidosis and profound, isolated cIII deficiency in skeletal muscle, we identified a disease‐segregating homozygous mutation (c.73G>A) in LYRM7/MZM1L, predicting a drastic change in a highly conserved amino‐acid residue (p.Asp25Asn). In a mzm1Δ yeast strain, the expression of a mzm1^D25N mutant allele caused temperature‐sensitive respiratory growth defect, decreased oxygen consumption, impaired maturation/stabilization of the Rieske Fe–S protein, and reduced complex III activity and amount. LYRM7/MZM1L is a novel disease gene, causing cIII‐defective, early onset, severe mitochondrial encephalopathy.     

Immunohistochemistry analysis of bone marrow biopsies in multiple sclerosis patients undergoing autologous haematopoietic stem cells transplantation

Objective

Recently autologous haematopoietic stem cell transplantation (AHSCT) has been introduced for the treatment of severe forms of multiple sclerosis (MS). As little data are available on bone marrow (BM) of MS patients undergoing AHSCT, we investigated the morphological and phenotypic characteristics of MS BM.

Methods

BM biopsies of 14 MS patients screened for AHSCT and 10 control patients were evaluated to assess cellularity, morphology, immunological profile and bone marrow microenvironment. Immunohistochemistry analysis was performed to evaluate the expression of CD3, CD4, CD8, CD20, CD68, CD45, MMP-9.

Results

8 out of 14 MS (57%) patients showed a reduction of age-related bone marrow cellularity, possibly due to previous immunosuppressive therapies. There were no differences in the T CD3+ lymphocyte expression rate amongst MS and the control patients, the CD4/CD8 ratio (2:1) was maintained as was the rate of B lymphocytes. We found an increased, although not significant, MMP-9 expression (9.2%) in the bone marrow of MS patients, when compared to the control patients (6.3%).

Conclusion

The BM of MS patients showed a reduced cellularity and CD45+ cells content in comparison to the controls. A slightly increased expression of MMP-9 was also shown, possibly confirming an involvement of this compartment in the pathogenesis of the disease.     

Thyrotropin stimulates production of procoagulant and vasodilative factors in human aortic endothelial cells.

vasodilative Thyroid diseases have been associated with pathophysiological changes in the vasculature that may result from altered thyroid hormone production or to direct effect of elevated thyrotropin (TSH) levels on smooth muscle cells. A direct effect of TSH on vascular endothelium has not been considered. In the present study a strain of human aortic endothelial cells has been stimulated with TSH, and vascular parameters correlated with the atherosclerotic process have been analyzed. Addition of TSH induced an increase of cyclic AMP (cAMP) concentration in human aortic endothelial cells. Furthermore it induced a decrease of endothelin (from 30 +/- 2.5 to 13 +/- 1 fmol/mL) and of tissue plasminogen activator secretion (from 2,800 +/- 200 to 1,600 +/- 150 ng/mL). On the other hand, it increased nitric oxide (from 148 +/- 8 to 211 +/- 12 microM). TSH did not affect plasminogen activator inhibitor 1. Similar results were obtained when immunoglobulin Gs (IgGs) from Graves' disease patients were used. In conclusion, our findings suggest that TSH and IgGs from Graves' disease patients could stimulate endothelial cells, increasing the secretion of procoagulant and vasodilative factors, and that cAMP is involved in the transduction pathway. These findings are consistent with modifications of the fibrinolytic system reported in hypothyroidism and in Graves' disease. On the other hand, the increase of vascular resistance found in patients with hypothyroidism may be due to the altered thyroid hormone production and not to TSH directly, or to a different effect of TSH on peripheral vessels.