Red blood cell glutathione peroxidase activity in multiple sclerosis

Summary Glutathione peroxidase (GSH-Px) activity of red blood cells of 23 multiple sclerosis (MS) patients (10 men and 13 women, aged 22–64 years) was examined and compared to the enzyme activity of 26 healthy persons (15 men and 11 women, aged 19–50 years). It was found that the mean GSH-Px activity was significantly higher (P<0.001) in red blood cells of MS patients (39.1±8.1 IU/g Hb) as compared to the group of healthy persons (25.9±5.2 IU/g Hb). There was no difference according to sexes in both the MS patients and the control group. The results are discussed based on the hypothesis that organic peroxides play a role in the etiology of multiple sclerosis.     

Lipid studies in the blood and brain in multiple sclerosis and motor neurone disease

The lipid patterns of plasma, red blood cells, and leucocytes from normal controls and from patients with multiple sclerosis and motor neurone disease have been studied by thin-layer chromatography. The fatty acid composition of cholesterol esters and lecithin in plasma from normal subjects and from patients with multiple sclerosis are reported. The fatty acid composition of lecithin of cerebral white matter from normal control subjects, from multiple sclerosis, and from motor neurone disease as well as of cholesterol esters in multiple sclerosis are recorded and compared. No significant abnormalities were found in the blood lipid profile in multiple sclerosis, but in motor neurone disease red cell sphingomyelin was slightly reduced and white cell lecithin slightly increased. The fatty acids of plasma cholesterol esters showed a slight decrease in palmitic and stearic acid in multiple sclerosis. The fatty acids in lecithin in multiple sclerosis, both in the apparently normal white matter and in the plaques, showed a slightly increased degree of saturation, while the loss of unsaturated acids was in oleic acids.     

Degradation of human myelin in vitro by leucocytes from patients with multiple sclerosis.

In order to study the possible autoimmune basis of multiple sclerosis (MS) a quantitative method has been used to investigate breakdown of human myelin in vitro. We found that serum from MS patients and controls was generally devoid of any myelin degradative activity. However, isolated peripheral blood mononuclear cells from 43% of MS patients showed significant myelin degradative activity as did those from 61.5% of patients with rheumatoid arthritis (RA). Myelin degradation by cells was found in only 13% of patients with other neurological diseases and in no healthy controls. It is proposed that this non-specific peripheral cellular immune degradative activity originates from cells activated within the central nervous system of MS patients or the joints of individuals with RA. As a result, activity in the blood only indirectly reflects the ongoing inflammatory response at the primary site, accounting for the lack of correlation between changes in the blood and the clinical status of the MS patient. We further propose that the lack of in vitro myelin degradative activity in cells recovered from the cerebrospinal fluid is due to autoaggressive cells being sequestered to the brain.     

In vivo activated T lymphocytes in the peripheral blood and cerebrospinal fluid of patients with multiple sclerosis

We found an increase in peripheral-blood lymphocytes bearing the T-cell-specific activation antigen Ta1 in 20 of 35 patients with progressive multiple sclerosis, 4 of 18 patients with stable or improving multiple sclerosis, 1 of 17 patients with other neurologic diseases, and 1 of 14 normal controls (P less than 0.0002, Fisher's exact test). No increases in two other markers of T-cell activation, T113 and the interleukin-2 receptor, were found. In the cerebrospinal fluid, patients with progressive multiple sclerosis (pleocytosis, 3.9 +/- 1.6 cells per cubic millimeter) had 42 +/- 3.0 per cent Ta1+ cells. In contrast, patients with other inflammatory central nervous system diseases (36 +/- 13 cells per cubic millimeter) had 9.6 +/- 1.8 per cent Ta1+ cells (P less than 0.01). In patients with other neurologic diseases without inflammation (0.7 +/- 0.16 cells per cubic millimeter), the percentage of Ta1+ cells was equivalent to that in patients with multiple sclerosis (39 +/- 5.4 per cent), although the absolute number was lower. There was a positive correlation between the presence of Ta1+ cells in the spinal fluid and blood of patients with other neurologic diseases, but not patients with multiple sclerosis. Less than 1 per cent of lymphocytes from the spinal fluid of patients with multiple sclerosis expressed interleukin-2 receptors, as compared with 9.8 per cent of cells from subjects with other inflammatory neurologic diseases (P less than 0.01). These results suggest that the T cells in the spinal fluid of patients with multiple sclerosis may be activated by a different mechanism or in a different temporal sequence from that in patients with other nervous system diseases. Furthermore, the increase in Ta1+ cells in the peripheral blood of patients with multiple sclerosis demonstrates systemic immune activation in the disease; monitoring such cells may provide an objective measure of abnormal immunologic activity.     

Comparison of methods to identify microglial cells and macrophages in the human central nervous system.

The macrophage markers non-specific esterase, alpha 1-antitrypsin, alpha 1-antichymotrypsin, and lysozyme were compared with conventional microglial and macrophage stains in the human central nervous system. In a series of specimens from cases of head trauma, conventionally fixed and embedded, the modified Weil-Davenport stain was unequivocally best for demonstrating reactive microglia. alpha 1-antichymotrypsin, however, was the most effective for showing macrophages in a series of specimens from patients with other conditions, which included inflammatory, neoplastic, and non-inflammatory diseases. The non-specific esterase reaction was unsatisfactory in tissues fixed in neutral formalin but was successful in fresh frozen tissue. In a series of specimens from cases of multiple sclerosis, non-specific esterase showed demyelination clearly and stained neuronal cytoplasm. It also stained macrophages but was less satisfactory for lipid-bearing phagocytes in multiple sclerosis than oil red 0.     

Investigation of in vivo activated T cells in multiple sclerosis and inflammatory central nervous system diseases

Monoclonal antibodies have recently been characterized which identify activated T cells at different stages of differentiation. We compared the expression of the late appearing activation antigen defined by monoclonal antibody TS2/7 with the expression of early appearing activation antigens in a group of patients with active multiple sclerosis, encephalitis, non-inflammatory other neurologic diseases, and normal controls. An increase in TS2/7 reactivity of peripheral blood T cells was found in MS patients compared to controls (P less than 0.001), however, there was no increase in the level of early activation antigens. This was in contrast to three patients with viral encephalitis, who had an increase in the early activation antigen 4F2, but minimal, if any, increase in the TS2/7 reactive antigen. This study demonstrates that in vivo, as in vitro, it is possible to identify multiple differentiation stages for activated T cells. Furthermore, the presence of activated T cells in the peripheral blood of multiple sclerosis patients suggests that there is systemic immune activation in MS, and could provide a means to monitor abnormal immunologic activity in MS when these cells are functionally characterized.     

Increased oxidative stress and altered levels of antioxidants in asthma

BACKGROUND: Reactive oxygen species might play an important role in the modulation of airway inflammation. There is evidence of an oxidant-antioxidant imbalance in asthma. Although several oxidants and antioxidants are likely to be involved, alterations in only limited parameters have been studied in isolation. OBJECTIVE: We investigated changes in a wide range of oxidants and antioxidants to create a comprehensive picture of oxidant-antioxidant imbalance. METHODS: In the peripheral blood of 38 patients with bronchial asthma and 23 control subjects, oxidative stress was measured in terms of superoxide anion generation by leukocytes, lipid peroxidation products, total nitrates and nitrites, total protein carbonyls, and total protein sulfhydrils in plasma. Antioxidant status was evaluated by measuring red blood cell superoxide dismutase and catalase activity, total blood glutathione, and glutathione peroxidase activity in red blood cells and leukocytes and total antioxidant capacity in plasma. RESULTS: Asthmatic patients showed increased superoxide generation from leukocytes, increased total nitrites and nitrates, increased protein carbonyls, and increased lipid peroxidation products and decreased protein sulfhydrils in plasma, indicating increased oxidative stress. They also showed increased superoxide dismutase activity in red blood cells and increased total blood glutathione and decreased glutathione peroxidase activity in red blood cells and leukocytes. Red blood cell catalase activity and the total antioxidant capacity of plasma were not altered. CONCLUSION: There are alterations in a wide array of oxidants and antioxidants, with balance shifting toward increased oxidative stress in asthma. Therapeutic augmentation of the antioxidant defenses might be beneficial.     

Angiotensin II type 1 and 2 receptors and lymphatic vessels modulate lung remodeling and fibrosis in systemic sclerosis and idiopathic pulmonary fibrosis

OBJECTIVE:

To validate the importance of the angiotensin II receptor isotypes and the lymphatic vessels in systemic sclerosis and idiopathic pulmonary fibrosis.

METHODS:

We examined angiotensin II type 1 and 2 receptors and lymphatic vessels in the pulmonary tissues obtained from open lung biopsies of 30 patients with systemic sclerosis and 28 patients with idiopathic pulmonary fibrosis. Their histologic patterns included cellular and fibrotic non-specific interstitial pneumonia for systemic sclerosis and usual interstitial pneumonia for idiopathic pulmonary fibrosis. We used immunohistochemistry and histomorphometry to evaluate the number of cells in the alveolar septae and the vessels stained by these markers. Survival curves were also used.

RESULTS:

We found a significantly increased percentage of septal and vessel cells immunostained for the angiotensin type 1 and 2 receptors in the systemic sclerosis and idiopathic pulmonary fibrosis patients compared with the controls. A similar percentage of angiotensin 2 receptor positive vessel cells was observed in fibrotic non-specific interstitial pneumonia and usual interstitial pneumonia. A significantly increased percentage of lymphatic vessels was present in the usual interstitial pneumonia group compared with the non-specific interstitial pneumonia and control groups. A Cox regression analysis showed a high risk of death for the patients with usual interstitial pneumonia and a high percentage of vessel cells immunostained for the angiotensin 2 receptor in the lymphatic vessels.

CONCLUSION:

We concluded that angiotensin II receptor expression in the lung parenchyma can potentially control organ remodeling and fibrosis, which suggests that strategies aimed at preventing high angiotensin 2 receptor expression may be used as potential therapeutic target in patients with pulmonary systemic sclerosis and idiopathic pulmonary fibrosis.     

Analysis of carrageenan-induced suppression of antibody response.

This paper describes the induction of immunosuppression by multiple injections of carrageenan-lambda (cgn) in BALB/c mice. While cgn alone induced non-specific suppression that persisted for up to 25 days, priming with sheep red blood cells (SRBC) of mice within 10 days of cgn treatment prolonged the suppressive state. Also, antigen (SRBC)-specific suppression was observed when these mice were given a secondary challenge 25-30 days afer priming. Thus multiple cgn treatment along with priming SRBC generated non-specific and specific suppressive states which were transferable by Thy-1+ Lyt-2+ splenocytes.     

Interferon production in multiple sclerosis

The interferon-synthesizing activity of the peripheral blood leukocytes and serum interferon in the blood of patients with multiple sclerosis were studied. A reverse relationship was found between antibody production (high titers of measles antibody) and the interferon-synthesizing activity of peripheral blood leukocytes in these patients. A 3-fold decline in serum interferon titers was observed in patients with multiple sclerosis as compared with the control group.     

Natural killer cell activity in patients with multiple sclerosis: interferon and plasmapheresis

Peripheral blood lymphocytes from patients with multiple sclerosis (MS) were studied for natural killer (NK) cell activity and reactivity to interferon. NK activity determined at the same time in a 4-hr chromium-51 release assay using K562 target cells was significantly lower in MS patients than in controls. In-vitro treatment of MS lymphocytes with interferon resulted in only a slight increase in NK activity, while NK activity of normal individuals was markedly augmented by interferon. Leukopheresis of MS patients gave a rapid decrease in cytotoxic activity, which returned to pretreatment levels by 24 hrs. These results are consistent with the hypothesis of an immune deficit in multiple sclerosis.     

(2''-5'') Oligo A synthetase in human polymorphonuclear cells increased activity in interferon treatment and in viral infections

The interferon (IFN)-induced enzyme 2-5A synthetase was found in human peripheral blood polymorphonuclear cells (PMNL). The average enzyme activity in a group of 15 patients with various viral infections was significantly higher (25-fold) than in healthy individuals. Eight patients with multiple sclerosis and six patients with bacterial infections were found to have normal 2-5A synthetase levels in the PMNL. Relationship of PMNL 2-5A synthetase levels to IFN was confirmed by finding enzyme increases in PMNL incubated in vitro with IFN, as well as in patients undergoing IFN therapy. These findings suggest that in PMNL, as in other cells, the level of 2-5A synthetase can be regulated by IFN and can be increased as a result of IFN information in diseases.     

IL-2 receptor and HLA class II antigens on cerebrospinal fluid cells of patients with multiple sclerosis and other neurological diseases

The presence of IL-2 receptor and HLA class II antigens as detected by monoclonal antibodies on mononuclear cells from both cerebro-spinal fluid (CSF) and peripheral blood was examined by cytofluorographic analysis in patients with multiple sclerosis (MS) and other neurological diseases. CSF as compared to blood was enriched in cells expressing IL-2 receptor and HLA class II molecules both in MS patients and in other inflammatory diseases of the central nervous system suggesting that activated T-cells concentrate within the central nervous system.     

A blood test for multiple sclerosis based on the adherence of lymphocytes to measles-infected cells.

The increased capacity of lymphocytes from patients with multiple sclerosis to adhere to human epithelial cells persistently infected with measles virus has provided an accurate blood test for multiple sclerosis. When lymphocytes from affected patients were mixed with measles-infected human epithelial cells, the lymphocytes formed rosettes around a mean (+/-S.E.) of 69.2 +/-1.7% of the measles-infected cells. In contrast, lymphocytes from controls, either healthy or with other neurologic and non-neurologic diseases, formed rosettes around a mean of only 28.2+/-2.1% of the measles-infected cells. Of greater importance was the complete absence of overlap between multiple sclerosis and control values, thus indicating the diagnostic potential of the rosetting phenomenon. The severity, duration and activity of the disease had no effect on the degree of rosette formation.     

Expression of interleukin 2 receptor and class II histocompatibility antigens on lymphocytes of the cerebrospinal fluid in patients with multiple sclerosis and other neurological diseases

The presence of IL2 receptor and HLA class II antigens on mononuclear cells from both cerebrospinal fluid (CSF) and peripheral blood was examined in patients with multiple sclerosis (MS) and other neurological diseases. Cytofluorographic analysis of mononuclear cells was performed by means of indirect immunofluorescence on a flow cytometer using a linear scale. CSF as compared to blood was enriched in cells expressing IL2 receptor and HLA class II molecules both in MS patients and in other inflammatory diseases of the central nervous system. The site of activation of these cells remains however questionable.     

Natural killer cell activity in multiple sclerosis patients treated with recombinant interferon-alpha 2

Natural killer (NK) cell activity was evaluated in multiple sclerosis (MS) patients during a phase II trial of recombinant interferon-alpha 2 (IFN). Spontaneous NK activity against the K562 myeloid target cell increased significantly during the first week of treatment in the IFN treatment group. However, NK activity was also increased in the placebo treatment group. Long-term administration of IFN caused a decline of NK activity, below the pretreatment values. This decline was not paralleled by a decline in the percentage of Leu-7 cells in the peripheral blood. The ability of IFN to enhance NK activity during treatment was also evaluated. Enhancement of NK activity by IFN was depressed for the duration of the study in the IFN treatment group. After treatment was stopped, IFN enhancement of NK activity returned to the pre-study value. These studies demonstrate that spontaneous and IFN enhanced NK activity are profoundly affected by the administration of recombinant IFN-alpha 2 in MS patients.     

The distribution of interleukin-2 receptor bearing lymphocytes in multiple sclerosis: evidence for a key role of activated lymphocytes.

The identification of T cells in the brain using monoclonal antibodies has suggested a role for T cells in the pathogenesis of multiple sclerosis (MS). In the present study the monoclonal antibody anti-Tac, shown to react with interleukin-2 (IL-2) receptors expressed on activated T cells, was used to determine levels of recently activated T cells in blood, cerebrospinal fluid (CSF) and brain sections from MS patients at different stages of disease. The CSF of MS patients contained much higher numbers of IL-2 receptor positive lymphocytes (up to 67%) than blood cells from the same patients, or the CSF of patients with non-inflammatory neurological diseases. In histological sections of the brain of MS patients with active disease, perivascular lymphocytes expressing IL-2 receptors were detected, as were lymphocytes containing IL-2. In contrast, these were absent in brain sections from patients with chronic MS, secondary demyelination or from normal controls. These observations in CSF and brain suggest that in multiple sclerosis, T-cell activation is occurring within the CNS and not in peripheral lymphoid tissue.     

Induction of antiidiotypic immune response with autologous T-cell vaccine in patients with multiple sclerosis

Patients with different forms of multiple sclerosis were treated with a vaccine consisting of myelin-reactive T cells. It was found that after this treatment, lymphocytes from patients acquired the capacity to generate antiidiotypic proliferative response directed towards myelin-reactive T cells. The serum concentration of IFN-γ decreased about 2-fold 1.5–2.0 years after the start of vaccine therapy, whereas the concentration of IL-4 increased 2–3 fold. Myelin-reactive proliferative activity of peripheral blood mononuclear cells also decreased. The results of the 2-year follow-up study revealed no side effect of T-cell vaccination in patients with cerebrospinal form of multiple sclerosis and demonstrated its possible clinical efficiency in the treatment of this disease at early stages. Translated from Kletochnye Tehnologii v Biologii i Medicine, No. 3, pp. 145–150, August, 2008     

The role of regulatory T cells in the development of autoimmune process in multiple sclerosis

In the maintenance of immunological tolerance important role belongs to the recently discovered population of regulatory T-cells CD4+CD25+FoxP3 +. These cells have potential in suppressing pathologic immune responses observed at various autoimmune diseases including multiple sclerosis. We have shown a reduction in the number and functional activity of T-reg in peripheral blood of patients with multiple sclerosis in the acute stage, the increase in their number during remission, duration of the relationship of the autoimmune process and the degree of disability of patients with the contents of T-reg. The possibility of using the grown ex vivo T-reg for the correction of immunopathological process in multiple sclerosis.     

Passive hemagglutination and hemolysis tests for the detection of anti-DNA antibody.

Passive hemagglutination (PHA) and hemolysis (PHL) tests using chromium chloride-treated sheep red blood cells were developed to detect and measure the anti-DNA antibodies. Sonication of native DNA was found to prevent the incidence of non-specific agglutination. Sheep red cells were coated with double-stranded DNA (dsDNA) which had been sonicated and treated with nuclease S1 to digest the single-stranded regions in the DNA. The specificities for dsDNA-coated cells were checked by inhibition studies in PHA test and plaque assay. In clinical studies fairly close correlations were found between the antibodies to DNA and the activity of the disease in patients with systemic lupus erythematosus (SLE). Complement-fixing antibodies were detected in most of SLE patients with active lupus nephritis, but rarely in those in remission. Anticomplementary activity seemed to be negligible in PHL test. These tests are simple and may be useful to the diagnosis and the management of SLE.