Inflammatory monocytes determine endothelial nitric-oxide synthase uncoupling and nitro-oxidative stress induced by angiotensin II.

Endothelial nitric-oxide synthase (eNOS) uncoupling and increased inducible NOS (iNOS) activity amplify vascular oxidative stress. The role of inflammatory myelomonocytic cells as mediators of these processes and their impact on tetrahydrobiopterin availability and function have not yet been defined. Angiotensin II (ATII, 1 mg/kg/day for 7 days) increased Ly6C(high) and CD11b(+)/iNOS(high) leukocytes and up-regulated levels of eNOS glutathionylation in aortas of C57BL/6 mice. Vascular iNOS-dependent NO formation was increased, whereas eNOS-dependent NO formation was decreased in aortas of ATII-infused mice as assessed by electron paramagnetic resonance (EPR) spectroscopy. Diphtheria toxin-mediated ablation of lysozyme M-positive (LysM(+)) monocytes in ATII-infused LysM(iDTR) transgenic mice prevented eNOS glutathionylation and eNOS-derived N(ω)-nitro-L-arginine methyl ester-sensitive superoxide formation in the endothelial layer. ATII increased vascular guanosine triphosphate cyclohydrolase I expression and biopterin synthesis in parallel, which was reduced in monocyte-depleted LysM(iDTR) mice. Vascular tetrahydrobiopterin was increased by ATII infusion but was even higher in monocyte-depleted ATII-infused mice, which was paralleled by a strong up-regulation of dihydrofolate reductase expression. EPR spectroscopy revealed that both vascular iNOS- and eNOS-dependent NO formation were normalized in ATII-infused mice following monocyte depletion. Additionally, deletion as well as pharmacologic inhibition of iNOS prevented ATII-induced endothelial dysfunction. In summary, ATII induces an inflammatory cell-dependent increase of iNOS, guanosine triphosphate cyclohydrolase I, tetrahydrobiopterin, NO formation, and nitro-oxidative stress as well as eNOS uncoupling in the vessel wall, which can be prevented by ablation of LysM(+) monocytes.

Urinary excretion of porphyrins is increased in patients with HIV-1 infection.

Urine concentrations of total porphyrins and of porphyrin precursors, delta-aminolaevulinic acid and porphobilinogen, were analysed in the first-morning urine samples of 36 people with established HIV-1 infection. For comparison, we also analysed the urine samples of 26 healthy HIV-seronegative people. In patients with HIV-1 infection concentrations of total porphyrins were found to be significantly higher than in healthy controls. Furthermore, there was a close correlation between concentrations of total porphyrins and porphobilinogen and urine neopterin concentrations in patients. Our data indicate an association between chronic immune activation and altered porphyrin metabolism. The data may provide a rationale for the observation of acute porphyria in patients with HIV-1 infection.

Studies on activation variables in multiple sclerosis.

The study aimed to evaluate the usefulness of some selected immune variables as markers of disease activation, progression and possible etiopathogenesis of multiple sclerosis (MS). Levels of neopterin, a factor known to be released from macrophages and monocytes at increased rates in cellular immune reactions were higher in cerebrospinal fluid (CSF) from patients with MS during exacerbations in comparison with remissions. The elevation in CSF during exacerbations was not reflected in serum. Expression of HLA class II antigens (DR) on activated T lymphocytes in CSF were encountered at elevated percentage in only 10% of patients with MS, against 81% of patients with acute aseptic meningoencephalitis (AM). In patients with AM, the DR expression on CSF T cells was found on both CD8+ and CD4+ cells. There was no correlation between percentage of DR+ T cells in CSF and disability, exacerbation/remission or recent onset of disease in patients with MS. Phenotypic characterization of mononuclear cells in CSF and peripheral blood revealed increased proportion of CD5+ cells in CSF compared to peripheral blood which in MS was not reflected by any changes in CD4+ or CD8+ cells, while in AM the increase of CD5+ cells in CSF was followed by increase of CD4+ cells. A population of CD5+, CD8-, CD4- cells might be postulated to occur in MS CSF. Levels of CD8+ cells in peripheral blood and CSF did not fluctuate in parallel with disease activity as measured as clinical exacerbation. OKB7+, OKM1+ and HLA-DR+ cells differed significantly between CSF and peripheral blood, indicative of a selective passage of cells into the central nervous system (CNS) - CSF compartment. Proliferating cells expressing transferrin receptors (OKT9) were generally few or absent in CSF of patients with MS. MS patients' bone marrow mononuclear cells showed higher spontaneous proliferation both in comparison with cells from bone marrow of control subjects and peripheral blood lymphocytes from MS patients. PHA response of bone marrow mononuclear cells from MS patients was also higher than that from controls. There was, however, no significant difference in proliferative response of peripheral blood lymphocytes between MS patients and controls. Seven of 11 MS patients showed morphological signs of activation in their bone marrow, without correlation to patients' clinical condition. Higher levels of undifferentiated or activated cells, measured as OKT10+ cells were found in peripheral blood of patients with MS compared to controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Interrelationships between serologic markers of immune activation and T lymphocyte subsets in HIV infection.

Relationships among four serologic activation markers and T cell subsets were measured in HIV-seropositive former blood donors (N = 64) and seronegative controls (N = 61). Significant correlations were observed for the HIV group in pairwise comparisons of soluble IL-2 receptor (sIL-2R), beta 2-microglobulin (beta 2M), neopterin (NEOP), and soluble CD8 (sCD8). CD4 cell levels (number/microliter) in the HIV group showed significant negative correlation with all four serologic markers; CD8 cell levels, in contrast, showed no significant correlation with any serologic activation marker measured. Significant correlations were observed, however, among various cell surface activation markers and serologic activation markers. Specifically, the proportion of CD8 cells expressing CD45RA showed significant negative correlations with NEOP and B2M levels, whereas the proportion of CD8 cells expressing HLA-DR showed significant positive correlations with B2M and sIL-2R levels. Further, the proportion of CD8 cells expressing CD38 showed significant positive correlations with all four serologic activation markers. These findings indicate that sIL-2R, B2M, NEOP, and sCD8 show similar quantitative changes and correlational relationships to CD4 cell destruction in HIV infection; they differ, however, in their relationships to proportional changes in activated CD8 cell subsets.

Neopterin augmentation of tumor necrosis factor production.

Gamma interferon (gamma-IFN), lipopolysaccharide (LPS)-gamma or interleukin-2 (IL-2)-induced tumor necrosis factor alpha (TNF alpha) production by both macrophages and peripheral blood mononuclear cells (PBMC), was increased in the presence of neopterin. Addition of neopterin caused an increased level of TNF alpha, but did not affect the kinetics of the TNF alpha production, which showed peak levels of cytotoxic activity 4 h after stimulatory treatment. Using anticytokine antibodies, we concluded that the neopterin effect was mainly gamma-IFN mediated, and only slightly affected by anti IL-2 receptor antibodies. The neopterin augmented TNF alpha production can be attributed to an immunological role for neopterin in the enhancement of cell-mediated immune (CMI) response.

Neopterin as a new biochemical marker in the clinical assessment of ulcerative colitis.

In previous publications we showed that neopterin, a pyrazino-pyrimidin compound, represents a biochemical marker for the assessment of cellular immune responses. We thought that the evaluation of this marker molecule might enable insight into the activity of cellular immune responses underlying ulcerative colitis (UC). Evaluation of urinary neopterin excretion in 25 consecutive untreated UC patients revealed striking correlations between neopterin levels and the severity of disease: elevated levels were observed in 9 out of 9 patients with moderately severe to severe, in 3 of 4 with mild and in none of 12 patients with quiescent disease. Further evidence for a correlation between disease activity and neopterin excretion was obtained on the basis of long-term follow-up studies performed in 4 cases. These studies indicated normalization of neopterin levels when clinical remission was achieved. Thereafter, the relative significance of neopterin excretion for determination of clinical stage was assessed by linear correlation analyses and was compared with conventional clinical parameters such as anemia, number of motions per day, raised temperature, ESR and extent of bowel involvement. The logarithm of neopterin excretion and the extent of bowel involvement were the two single parameters most closely related to the clinical stage of ulcerative colitis. We, therefore, conclude that evaluation of neopterin excretion in ulcerative colitis patients represents a new and useful tool for the clinical monitoring of disease activity.

Increased neopterin and interferon-gamma secretion and lower availability of L-tryptophan in major depression: further evidence for an immune response.

There is now some evidence that major depression may be accompanied by an immune response. The latter condition is suggested by elevated secretion of neopterin and interferon-gamma (IFN gamma) and by lower L-tryptophan (L-TRP) plasma levels. This study investigated the plasma levels of neopterin, L-TRP, and the L-TRP/competing amino acids (CAA) ratio in 30 normal control subjects and 47 depressed subjects (16 minor depressed, 13 simple major depressed, and 18 melancholic subjects), and IFN gamma secretion by mitogen-stimulated peripheral blood mononuclear cells in 7 normal control subjects and 13 major depressed subjects. Plasma neopterin levels were significantly higher in depressed subjects than in normal controls; 61% of melancholic patients had increased neopterin levels (> or = 7 nmol/l) with a specificity of 90%. Patients with major depression had significantly lower L-TRP and L-TRP/CAA values compared with normal control subjects. The amino acid values were significantly and negatively correlated with plasma neopterin levels. Major depressed subjects exhibited significantly higher IFN gamma secretion than did normal control subjects. The results further support the hypothesis that major depression is accompanied by an immune response and that the lower L-TRP availability in that illness may be an epiphenomenon of immune activation.

Evaluation of commercially available assays of neopterin and beta 2-microglobulin for the assessment of disease progression in FIV-infected cats.

Serum or plasma samples from cats at different stages of feline immunodeficiency (FIV) infection and from uninfected cats were tested using immunoassays designed to detect human neopterin and beta 2-microglobulin (beta 2M). The results obtained from the anti-human neopterin assay did not correlate with infection status, time post-infection, fCD4 count or clinical picture. Feline samples gave negative results in the anti-human beta 2M assay. The assay kits used in this study are not suitable for the determination of the effect of FIV infection on immune activation markers in the cat.

Possible uses of urinary neopterin and biopterin measurement.

The measurement of urinary neopterin and biopterin may be an important non-specific diagnostic tool. Urinary biopterin has been found to be decreased in parkinsonian patients, and in hyperphenylalanemia as a variant of phenylketonuria. Urinary neopterin has been found to be elevated in numerous conditions. It has been shown to be significantly higher in AIDS patients than in ARC patients, and significantly higher in ARC patients than in controls. Urinary neopterin has been shown to be prognostically elevated in a wide range of neoplasias, including multiple myeloma, hematologic and gynecologic neoplasias. Neopterin is thought to be an in-vitro indicator of activation of the immune system.

Production and characterization of a monoclonal antibody against neopterin.

We have produced and characterized the first monoclonal antibody against neopterin (D-erythro-6-(1,2,3,-trihydroxypropyl)pterin). The antibody specifically recognizes neopterin in a modified RIA. The binding capacity in this assay is 34%, the sensitivity limit of inhibition is 0.9 nmol/l. Cross-reactivity exists with monapterin (L-threo(1,2,3,trihydroxypropyl)pterin) in 30%, with other pteridines cross-reactivity has been found in less than 5%.

[Immunoregulatory disorders and activation of the immune system in dilated cardiomyopathy]. / Narushenie immunoreguliatsii i aktivatsiia sistemy immuniteta pri dilatatsionnoi kardiomiopatii.

The level of immunoregulatory lymphocytic subpopulations (CD3-, CD4-, CD8-cells, CD4/SD8 ratio, IaDr-positive cells, SD16-cells), lymphocyte natural cytotoxicity (NCT), the concentration of serum interferon (IF), beta 2-microglobulin (beta 2-M) and neopterin were investigated in 30 patients with dilatative cardiomyopathy (DCMP). Immunoregulatory lymphocytic subpopulations were defined by indirect immunofluorescence using monoclonal antibodies to differentiating markers of BMA and OKT series. A decrease in SD4/SD8 ratio less than 1 was observed in 10 patients, an increase in SD4/SD8 ratio over 2.3 was observed in 5 patients. A significant rise of IaDr-positive cells, a decrease in NCT, an increase in the concentration of IF, beta 2-M and neopterin were observed. There was correlation of an increase in IF concentration, the number of IaDr-positive cells and an increase in the level of neopterin and beta 2-M. Signs of activation of the immune system were more frequently noted in patients with a decreased (less than 1) or increased CD4/CD8 ratio. The role of disorders of immunoregulation and activation of the immune system in DCMP pathogenesis was discussed.

Functional analysis of the effect of monoclonal antibodies on monkey liver phenylalanine hydroxylase.

An analysis of the effect of eleven monoclonal antibodies on the functional characteristics of monkey liver phenylalanine hydroxylase is presented. These eleven antibodies have been found to react with eight distinct regions on the phenylalanine hydroxylase protein. PH1 antibody inhibits enzyme activity, is dependent on phenylalanine for its binding, and appears to be related to structural changes occurring during phenylalanine activation of the enzyme activity. PH2 and PH3 antibodies stimulate enzyme activity, their binding is inhibited by lysolecithin and this group apparently is recognizing structures involved in lysolecithin activation of the enzyme activity. PH5, PH10, PH12 and PH6 recognise sites on phenylalanine hydroxylase affected by lysolecithin activation.

The role of neopterin as a monitor of cellular immune activation in transplantation, inflammatory, infectious, and malignant diseases.

The accumulated knowledge about the organization and function of the human immune system contributes to a better understanding of the pathogenesis of most diverse disorders and is opening new avenues for therapeutic regimens. To gain further insight into the complex interactions within the components of the immune system, it has become increasingly necessary to develop rapid and simple methods to monitor the status of the immune system in patients. The determination of neopterin concentrations in human body fluids allows to investigate sensitively the cell-mediated immune status to be investigated with considerable sensitivity. In recent years it was shown that production and release of neopterin is inducible in human monocytes/macrophages by interferon gamma. Increased neopterin levels indicate endogenous formation of gamma interferon, and monitoring of neopterin levels therefore permits the activation status of the cell-mediated immune system to be examined. Neopterin concentrations in serum and in urine increase in parallel to the clinical course of infections with viruses, intracellular bacteria, and parasites. In patients with human immunodeficiency virus infection neopterin concentration in serum and urine is a significant predictor of disease progression, the statistical power being similar to CD4+ T-cell numbers. In patients with autoimmune disorders, neopterin levels correlate with the extent and the activity of the disease. Neopterin concentrations are also sensitive indicators of immunological complications in allograft recipients. In certain malignant diseases neopterin concentrations correlate with the stage of the disease and bear prognostic information. Results of neopterin measurements agree with the important role that the cellular immune system plays in these disorders.

Neopterin measured in serum and tissue culture supernates by a competitive enzyme-linked immunosorbant assay.

In this solid-phase competitive enzyme-linked immunosorbant assay for neopterin (a product of activated macrophages) in serum or supernates of cell cultures, we incubate antiserum to neopterin with standards or samples in the presence of solid-phase-bound conjugate of carrier-neopterin. Incubation with second antibody labeled with peroxidase then follows, before reaction with substrate. Analytical recovery, precision, and sensitivity of this method are similar to those of other immunoassays. The assay range is between 0.4 and 100 micrograms of neopterin per liter. Results obtained by this method compared with those from a conventional radioimmunoassay gave a correlation coefficient of 0.974.

Neopterin measurement provides evidence of altered cell-mediated immunity in patients with depression, but not with schizophrenia.

Neopterin is a validated marker of the activation of cell-mediated immunity in a variety of disease states. We measured neopterin and creatinine concentrations in the plasma and urine of 22 schizophrenic and 26 depressed patients admitted acutely to hospital, and compared results with those in a large group of normal controls. Neopterin/creatinine ratios were normal in the schizophrenic patients, but significantly elevated in the plasma of depressed patients. In each diagnostic group, the use of psychotropic drugs before admission had no effect on the neopterin ratios observed. Our findings indicate altered cell-mediated immunity in depression.

Cerebrospinal fluid and serum neopterin levels in patients with Lyme neuroborreliosis.

Elevated (greater than 3.0 nmol/l) cerebrospinal fluid neopterin concentrations were observed in 20 of 21 patients with Lyme neuroborreliosis compared with three of 11 control patients with headache, back pain or psychoneurotic disorders. Neopterin concentrations were correlated to mononuclear cell counts and protein concentrations in the cerebrospinal fluid (CSF). Following antibiotic treatment, CSF neopterin levels decreased. Serum neopterin levels were not significantly raised in patients with neuroborreliosis when compared to control subjects. Neopterin levels as well as cell count and protein concentration in the CSF are valuable inflammation markers of disease activity in Lyme neuroborreliosis.

Serum concentrations of interferon gamma, interleukin-6 and neopterin in patients with infectious mononucleosis and other Epstein-Barr virus-related lymphoproliferative diseases.

Serum levels of interferon gamma, interleukin-6 and neopterin were determined in 15 patients with different forms of Epstein-Barr virus-associated diseases: acute self-limiting infectious mononucleosis, chronic active infectious mononucleosis and X-linked lymphoproliferative syndrome. In patients with acute type of infection, neopterin, interferon gamma and interleukin-6 were elevated in nearly all patients. In contrast, the situation was less clear-cut in the other EBV-associated diseases; particularly interleukin-6 was undetectable in most cases. The results suggest that concomitant measurement of these diverse immune activation markers may provide interesting insights into the interactions between the virus and the host, and may also lead to therapeutic consequences.