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Nitric oxide in tuberculosis and leprosy
Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is a global disease that kills about two million people each year. Leprosy is caused by Mycobacterium leprae, and primarily affects the skin and peripheral nervous system. About 10-20% of leprosy patients suffer from reactions associated with acute inflammation that can lead to rapid and severe nerve damage. Most individuals infected with Mtb or M leprae do not develop clinical disease, which indicates that human hosts have effective defence mechanisms. In macrophages activated by cytokines such as IFN-γ, inducible nitric oxide synthase (iNOS) catalyses the production of nitric oxide (NO) from L-arginine. In an inflammatory environment, NO reacts with the superoxide radical (O2-) to yield peroxynitrite, an unstable metabolite that can rapidly nitrosylate tyrosine residues on proteins to form the stabile end product nitrotyrosine (NT). Many studies using experimental models have indicated that NO is important in host response to M leprae and Mtb, but this is controversial in human disease. Thus, our aim was to investigate the presence and the role of NO in the human mycobacterial infectious diseases TB and leprosy.

Levels of the NO metabolites nitrite and nitrate were initially increased in urine from patients with reactional leprosy but were normalised by treatment with prednisolone, and this was associated with clinical improvement. Immunohistochemistry revealed local production of NO in skin biopsies from patients with borderline leprosy and reversal reactions, which was detected as reactivity to iNOS and NT in macrophage-rich granulomas. Ultrastructural studies showed NT-positive aggregations of neurofilaments in dermal nerves from leprosy patients. Patients with active tuberculosis had increased urinary levels of NO metabolites, which were normalised after anti-TB treatment. Household contacts of patients with tuberculosis had increased levels of NO metabolites in plasma and serum. Immunohistochemical examination of biopsies from patients with TB indicated local, iN OS-mediated generation ofNO in macrophage-rich granulomas. In an experimental model of TB, local production of NO in the lungs was substantial in the acute phase of infection, and immunoelectron microscopy detected NT in phagosomes containing Mtb and on the surface of the bacteria. In an in vitro model, NO and peroxynitrite killed Mtb H37Ra and induced upregulation of several bacterial proteins. Peroxynitrite also mediated tyrosine nitration of albumin associated with the surface of Mtb. In a randomised, double-blind trial in Ethiopia, arginine supplementation in patients receiving conventional chemotherapy increased sputum conversion and reduced the prevalence of cough in HIV -negative, smear-positive patients with active TB.

In conclusion, these results demonstrate that iNOS-mediated production of NO occurs in human tuberculosis and leprosy; NO and peroxynitrite can kill Mtb and modify protein expression in the bacteria; and arginine leads to clinical improvement in TB patients.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2002. , 106 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 749
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-26667Local ID: 11233ISBN: 91-7373-193-5 (print)OAI: oai:DiVA.org:liu-26667DiVA: diva2:247216
Public defence
2002-11-01, Berzeliussalen, Hälsouniversitetet, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-09-21Bibliographically approved
List of papers
1. Increased levels of nitric oxide metabolites in urine from leprosy patients in reversal reaction
Open this publication in new window or tab >>Increased levels of nitric oxide metabolites in urine from leprosy patients in reversal reaction
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1999 (English)In: Leprosy Review, ISSN 0305-7518, E-ISSN 2162-8807, Vol. 70, no 1, 52-55 p.Article in journal (Refereed) Published
Abstract [en]

We measured the metabolites of NO [nitrite (NŌ 2) and nitrate (NŌ 3)] in urine from Ethiopian patients suffering from leprosy. The urinary level of NŌ 2/NŌ 3 in a group of healthy Ethiopians was 1020 ± 471 μM (n = 22). Leprosy patients in reversal reaction had significantly higher levels of NŌ 2/NŌ 3 (1817 ± 492 μM, P < 0.001, n = 12) than both the control group and leprosy patients who were not in reversal reaction (1079 ± 446 μM, n = 12). We conclude that the reversal reaction in leprosy is associated with increased urinary levels of nitric oxide metabolites.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26097 (URN)10438255 (PubMedID)10556 (Local ID)10556 (Archive number)10556 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
2. Expression of inducible nitric oxide synthase and nitrotyrosine in borderline leprosy lesions
Open this publication in new window or tab >>Expression of inducible nitric oxide synthase and nitrotyrosine in borderline leprosy lesions
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2001 (English)In: British Journal of Dermatology, ISSN 0007-0963, E-ISSN 1365-2133, Vol. 145, no 5, 809-815 p.Article in journal (Refereed) Published
Abstract [en]

Background In the response to T-helper cell (Th1)-type cytokines and interactions with pathogens, high levels of nitric oxide (NO) are produced by activated macrophages expressing the inducible NO synthase (iNOS). The role and importance of reactive nitrogen intermediates (RNIs) such as NO and peroxynitrite in the host response to diseases caused by intracellular pathogens such as Mycobacterium leprae and M. tuberculosis is unclear.

Objectives The aim of this study was to investigate the presence of local production of NO and peroxynitrite in borderline leprosy by using antibodies against iNOS and the product of peroxynitrite, nitrotyrosine (NT).

Methods We detected the presence of iNOS and NT in skin biopsies from borderline leprosy patients, with and without reversal reaction (RR), by immunohistochemistry (n = 26).

Results In general, the granulomas from borderline leprosy lesions with and without RR showed high and specific expression of iNOS and NT. Moreover, strong immunoreactivity to iNOS and NT was observed in granulomas surrounding and infiltrating dermal nerves. The expression of iNOS and NT was also strong in keratinocytes, fibroblasts and endothelial cells in close relation to the granulomatous reaction. In contrast, normal human skin showed no expression of iNOS and NT in these cells.

Conclusions We conclude that iNOS and NT are expressed in granulomas from borderline leprosy patients with and without RR and propose that RNIs might be involved in the nerve damage following RR in leprosy.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26100 (URN)10.1046/j.1365-2133.2001.04491.x (DOI)10559 (Local ID)10559 (Archive number)10559 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
3. High dose prednisolone treatment of leprosy patients undergoing reactions is associated with a rapid decrease in urinary nitric oxide metabolites and clinical improvement
Open this publication in new window or tab >>High dose prednisolone treatment of leprosy patients undergoing reactions is associated with a rapid decrease in urinary nitric oxide metabolites and clinical improvement
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2000 (English)In: Leprosy Review, ISSN 0305-7518, E-ISSN 2162-8807, Vol. 71, no 3, 355-362 p.Article in journal (Refereed) Published
Abstract [en]

Evidence is accumulating that nitric oxide (NO) produced by macrophages has a role in the pathogenesis of reactions in leprosy. We followed the urinary levels of the metabolites of NO [nitrite (NO 2 -) and nitrate (NO 3 -)] and the clinical response to prednisolone treatment in leprosy patients (n = 9) admitted to ALERT leprosy hospital Addis Ababa, Ethiopia, because of reversal reaction (RR) or erythema nodosum leprosum (ENL). In untreated reactional leprosy patients, the levels of urinary NO metabolites (1645 ± 454 μM, n = 9, ENL = 4, RR = 5) decreased significantly 2 weeks after high dose prednisolone treatment (1075 ± 414 μM, P < 0.05), and remained stable 4 (895 ± 385 μM, P < 0.02) and 6 weeks following treatment initiation (1048 ± 452 μM, P < 0.02). This decrease was also present when the reactional patients were subdivided according to the type of reaction (ENL, RR) and coincided with a clinical improvement. In patients showing a poor clinical response to steroids, no or minor effects on the urinary NO metabolite levels were observed. We conclude that there is a correlation between the decrease in urinary NO metabolites and a favourable clinical response after high dose prednisolone treatment of reactional leprosy patients.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26098 (URN)11105495 (PubMedID)10557 (Local ID)10557 (Archive number)10557 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
4. Nitrotyrosine localization to dermal nerves in borderline leprosy
Open this publication in new window or tab >>Nitrotyrosine localization to dermal nerves in borderline leprosy
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2004 (English)In: British Journal of Dermatology, ISSN 0007-0963, E-ISSN 1365-2133, Vol. 150, no 3, 570-574 p.Article in journal (Refereed) Published
Abstract [en]

Background  Nerve damage is a common and disabling feature of leprosy, with unclear aetiology. It has been reported that the peroxidizing agents of myelin lipids—nitric oxide (NO) and peroxynitrite—are produced in leprosy skin lesions.

Objectives  To investigate the localization of nitrotyrosine (NT)—a local end-product of peroxynitrite—in leprosy lesions where dermal nerves are affected by a granulomatous reaction.

Methods  We investigated by immunohistochemistry and immunoelectron microscopy the localization of the inducible NO synthase (iNOS) and NT in biopsies exhibiting dermal nerves from patients with untreated leprosy.

Results  There were abundant NT-positive and iNOS-positive macrophages in the borderline leprosy granulomas infiltrating peripheral nerves identified by light microscopy, S-100 and neurofilament immunostaining. Immunoelectron microscopy showed NT reactivity in neurofilament aggregates and in the cell wall of Mycobacterium leprae.

Conclusions  Our results suggest that NO and peroxynitrite could be involved in the nerve damage following borderline leprosy.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-22038 (URN)10.1046/j.1365-2133.2004.05764.x (DOI)1090 (Local ID)1090 (Archive number)1090 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
5. Effects of HIV co-infection and chemotherapy on the urinary levels of nitric oxide metabolites in patients with pulmonary tuberculosis
Open this publication in new window or tab >>Effects of HIV co-infection and chemotherapy on the urinary levels of nitric oxide metabolites in patients with pulmonary tuberculosis
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1999 (English)In: Scandinavian Journal of Infectious Diseases, ISSN 0036-5548, E-ISSN 1651-1980, Vol. 31, no 2, 123-126 p.Article in journal (Refereed) Published
Abstract [en]

The presence of nitric oxide (NO) and its role as a factor in host defence against intracellular pathogens in human macrophages is controversial. We measured the metabolites of NO (nitrite (NO 2 -) and nitrate (NO 3 -)) in urine from Ethiopian patients suffering from tuberculosis. The urinary level of NO 2 -/NO 3 - in a group of healthy Ethiopians was 1020 ± 471 μM (n = 22). Untreated HIV negative patients with active pulmonary tuberculosis (1574 ± 588 μM, p < 0.01, n = 12) and household contacts to tuberculosis patients (1949 ± 812 μM, p = 0.006; n = 7) had significantly higher levels of urinary NO 2 -/NO 3 -, than the control group. Untreated HIV positive patients with pulmonary tuberculosis did not have increased levels of urinary NO 2 -/NO 3 -, (1101 ± 614 μM, n = 6). Some of the HIV negative untreated patients with pulmonary tuberculosis (1710 ± 519 μM, n = 6) were followed up after treatment and showed a reduction in the levels of urinary NO 2 -/NO 3 - 1 week after treatment (945 ± 599 μM, p < 0.05). We conclude that HIV negative patients with active pulmonary tuberculosis have increased urinary levels of nitric oxide metabolites with a reduction following specific anti-tuberculous chemotherapy.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26096 (URN)10.1080/003655499750006137 (DOI)10555 (Local ID)10555 (Archive number)10555 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
6. Expression of inducible nitric oxide synthase and nitrotyrosine in granuloma-associated macrophages from patients with tuberculosis
Open this publication in new window or tab >>Expression of inducible nitric oxide synthase and nitrotyrosine in granuloma-associated macrophages from patients with tuberculosis
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

NO produced by the inducible nitric oxide synthase (iNOS) is important in host defense against M tuberculosis. We investigated the expression of iNOS and nitrotyrosine (Ntyr) in untreated patients with tuberculosis. Many iNOS/Ntyr reactive macrophages were observed in pleuro-pulmonary granulomas comrrming the presence of high-output NO-production in human tuberculosis.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-81719 (URN)
Available from: 2012-09-21 Created: 2012-09-21 Last updated: 2012-09-21Bibliographically approved
7. Expression of inducible nitric oxide synthase and nitrotyrosineduring the evolution of experimental pulmonary tuberculosis
Open this publication in new window or tab >>Expression of inducible nitric oxide synthase and nitrotyrosineduring the evolution of experimental pulmonary tuberculosis
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2001 (English)In: Experimental and Toxicological Pathology, ISSN 0940-2993, E-ISSN 1618-1433, Vol. 53, no 4, 257-265 p.Article in journal (Refereed) Published
Abstract [en]

Nitric oxide (NO) is a relevant antimycobacterial factor in mouse macrophages. NO is a product of inducible nitric oxide synthase (iNOS). NO toxicity is greatly enhanced by reacting with superoxide to form peroxynitrite that reacts with many biological molecules. Tyrosine is one of the molecules with which NO reacts and the product is nitrotyrosine (NT). The production of peroxynitrite and the nitrosylation of proteins might play a role in bacterial killing and also in mediating host injury. In this study, we used a well-characterized mouse model of pulmonary tuberculosis to examine the local kinetics of expression and cellular distribution of iNOS and NT at the cellular and subcellular level. The histopathological study showed two phases of the disease: early and late. The early phase was characterized by mononuclear inflammation and granuloma formation. During this phase, high percentages of activated macrophages were observed that were immunostained for iNOS and NT. Immuno-electronmicroscopy showed NT immunoreactivity in lysosomes and mycobacterial wall and cytoplasm. The concentration of iNOS mRNA and NO metabolites were also elevated. The late phase was characterized by progressive pneumonia with focal necrosis and a decrease of iNOS mRNA and NO metabolites. The strongest NT immunostained areas were the necrotic tissue. Macrophages became foamy cells with scarce iNOS immunostaining but strong NT immunoreactivity. At the ultrastructural level, these cells showed NT immunolabeling in cytoskeleton, mitochondria, lysosomes and cell membrane. NT was also located in bronchial epithelial cell mitochondria, in cell membranes and cytoplasm of endothelial cells and in actin bundles within smooth muscle cells. These results suggest an important role of NO in mycobacterial killing, particularly during the early phase of the infection. They also suggest an important participation by NO in tissue damage during the late phase of the disease.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26099 (URN)10.1078/0940-2993-00182 (DOI)10558 (Local ID)10558 (Archive number)10558 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
8. Arginine as an adjuvant to chemotherapy improves clinical outcome in active tuberculosis
Open this publication in new window or tab >>Arginine as an adjuvant to chemotherapy improves clinical outcome in active tuberculosis
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2003 (English)In: European Respiratory Journal, ISSN 0903-1936, E-ISSN 1399-3003, Vol. 21, no 3, 483-488 p.Article in journal (Refereed) Published
Abstract [en]

Nitric oxide (NO) is involved in the host defence against tuberculosis (TB). Patients with TB exhibit increased catabolism and reduced energy intake. Thus the hypothesis for this study was that restoring a relative deficiency in the amino acid arginine, the substrate for mycobactericidal NO production, would improve the clinical outcome of TB by increasing NO production.

In a randomised double-blind study, patients with smear-positive TB (n=120) were given arginine or placebo for 4 weeks in addition to conventional chemotherapy. Primary outcomes were sputum conversion, weight gain, and clinical symptoms after week 8. Secondary outcomes were sedimentation rate and levels of NO metabolites, arginine, citrulline, and tumour necrosis factor‐α.

Compared with the human immunodeficiency virus (HIV)−/TB+ placebo group, the HIV−/TB+ patients in the arginine group showed significant improvement, defined as increased weight gain, higher sputum conversion rate and faster reduction of symptoms, such as cough. The arginine level increased after week 2 in the HIV−/TB+ arginine group (100.2 µM (range 90.5–109.9) versus 142.1 µM (range 114.1–170.1)) compared with the HIV−/TB+ placebo group (105.5 µM (range 93.7–117.3) versus 95.7 µM (range 82.4–108.9)). HIV seroprevalence was 52.5%. No clinical improvement or increase in serum arginine was detected in arginine supplemented HIV+/TB+ patients compared with placebo.

Arginine is beneficial as an adjuvant treatment in human immunodeficiency virus-negative patients with active tuberculosis, most likely mediated by increased production of nitric oxide.

Keyword
arginine, human immunodeficiency virus, Mycobacterium tuberculosis, nitric oxide, tuberculosis, tumour necrosis factor-alpha
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-47812 (URN)10.1183/09031936.03.00090702 (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13Bibliographically approved

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