Involvement of the nitric oxide pathway in the anti-depressant-like effects of thalidomide in mice.

BACKGROUND: Thalidomide is a sedative/hypnotic agent that is currently used to treat patients suffering from multiple myeloma, myelodysplastic syndromes and erythema nodosum leprosum. Although previous studies have demonstrated that thalidomide possesses anti-depressant-like properties, the exact mechanism that thalidomide exerts this effect is not understood. In this study, we used two mouse models of depression and investigated the possible role of nitric oxide (NO), NO synthase (NOS) and inducible NOS (iNOS) in the ant-depressant-like effects of thalidomide. METHODS: Male mice were injected with different doses of thalidomide intraperitoneally. In order to assess the anti-depressant-like properties of thalidomide, the immobility time of mice was assessed in the forced swimming test (FST) and tail suspension test (TST). Locomotor activity was assessed using the open-field test. To assess the role of nitric oxide, N(G)-nitro-L-arginine methyl ester (L-NAME, non-specific NOS inhibitor), aminoguanidine (selective iNOS inhibitor) or L-arginine (NO precursor) were administered intraperitoneally along with specific doses of thalidomide. RESULTS: Thalidomide (10 mg/kg) significantly reduced immobility time in FST and TST. Aminoguanidine (50 mg/kg) and L-NAME (10 mg/kg) significantly augmented the anti-immobility effects of thalidomide (5 mg/kg). L-arginine (750 mg/kg) significantly inhibited the anti-immobility effects of thalidomide (10 mg/kg). None of the treatment groups demonstrated alteration of locomotor activity. CONCLUSION: Thalidomide exerts its anti-depressant-like effects through a mechanism dependent upon NO inhibition.

Detection of cytokines and nitric oxide synthase in skin lesions of Jorge Lobo's disease patients

Studies investigating the immunopathological aspects of Jorge Lobo's disease have shown that the inflammatory infiltrate consists mainly of histiocytes and multinucleated giant cells involving numerous yeast-like cells of Lacazia loboi, with the T lymphocytes more common than B lymphocytes and plasma cells. The quantification of cytokines in peripheral blood mononuclear cells culture supernatant has revealed alterations in the cytokines profile, characterized by predominance of a Th2 profile. In view of these findings and of the role of cytokines in cell interactions, the objective of the present study was to investigate the presence of the cytokines IL-10, TGF-ß1 and TNF-α, as well as iNOS enzyme in granulomas induced by L. loboi. Histological sections obtained from skin lesions of 16 patients were analyzed by immunohistochemistry for the presence of these cytokines and iNOS. The results showed that TGF-ß1 was the cytokine most frequently expressed by cells present in the inflammatory infiltrate, followed by IL-10. There was a minimum to discrete positivity of cells expressing TNF-α and iNOS. The results suggest that the presence of immunosuppressive cytokines in skin lesions of patients with the mycosis might be responsible for the lack of containment of the pathogen as demonstrated by the presence of numerous fungi in the granuloma.

Inducible nitric oxide synthase immunoreactivity in the granulomatous intestinal lesions of naturally occurring bovine Johne's disease.

Inducible nitric oxide synthase (iNOS) is important in the control of a number of intracellular pathogens, including mycobacteria, and is a marker of classic macrophage activation. In human granulomatous diseases such as leprosy, a spectrum of granulomatous lesions is described, ranging from the tuberculoid to lepromatous types. Tuberculoid granulomas are associated with enhanced iNOS production and improved clinical outcomes over the lepromatous types. The aim of this study is to determine whether an association exists between morphology of bovine Johne's disease granulomas and lesion macrophage effector functions. To accomplish this, we retrospectively evaluated 24 cases of bovine Johne's disease. In each case, we recorded the predominant granuloma morphology and evaluated iNOS immunoreactivity and bacterial burden by acid-fast stains and mycobacterial immunolabeling. The results of this study demonstrate that all cases had granulomas with features most similar to the lepromatous type. This morphology correlated with heavy bacterial burdens demonstrated by acid-fast staining and mycobacterial immunoreactivity. None of the cases had high expression of iNOS in mycobacterial-positive granulomas. When iNOS immunoreactivity was identified, it was usually located near the crypts and was distinct from the granulomatous foci.

Hanseníase dimorfa reacional: estudo comparativo, em biópsias cutâneas, entre reações tipo 1 ocorridas antes e durante a poliquimioterapia / Manifestations of reactional tuberculoid patterns in borderline leprosy: comparative study, in cutaneous biópsias, between occured reactions type 1 before and during the multidrugtherapy

A hanseníase dimorfa é a forma clínica mais freqüentemente associada à ocorrência de reações de hipersensibilidade mediada por células (reações tipo 1), que podem ocorrer antes, durante ou depois do tratamento específico. Há várias teorias a respeito da patogênese dessas reações, as quais estão diretamente ligadas ao dano neural e às seqüelas. Visando-se compreender melhor a fisiopatologia das reações tipo 1, foram estudadas 10 biópsias cutâneas de indivíduos com hanseníase dimorfatuberculóidereacional não tratados e 10 de indivíduos dimorfos em reação reversa (após o início do tratamento específico), comparando-se os parâmetros morfológicos e imunoistoquímicos. Observou-se, no grupo em tratamento, maior positividade das células para a enzima óxido nítricosintase induzível (iNOS) e menor quantidade de linfócitos T CD8+ (p<0,05).Não houve diferenças significativas na baciloscopia e positividade para antígenos micobacterianos nos dois grupos, e nem na quantidade de células IL-10+, apesar de ter sido observada correlação negativa entre esta citocinae a proporção CD4/CD8 nos pacientes em tratamento (p<0,05). Notou-se, também, tendência à redução do infiltrado específico (linfócitos T e B) e aumento do número de células citotóxicas inespecíficas (NK) no grupo em tratamento. Estes resultados são concordantes com trabalhos recentes, que sugerem que a reação tipo 1 representaria um desequilíbrio imunológico entre citocinas pró-inflamatórias e anti-inflamatórias. Na presença de muitos bacilos viáveis e no contexto de um paciente sem imunidade celular plena, haveria uma tendência à piora no sentido do pólo virchoviano (downgrading), porém o tratamento específico, reduzindo a carga bacilar, favoreceria uma melhora da imunidade efetiva, caracterizada por um quadro histológico mais tuberculóide, com posterior desvio progressivo da imunidade adquirida para a inespecífica (resposta Th3 ou reguladora), interrompendo a reação e levando à cura

Hanseniase dimorfa reacional: estudo comparativo, em biopsias cutaneas, entre reacao tipo 1 ocorridas antes e durante a poliquimioterapia / ?

A hanseniase dimorfa e a forma clinica mais frequentemente associada a ocorrencia de reaçoes de hipersensibilidade medida por celulas (reacao tipo 1), que podem ocorrer antes, durante ou depois do tratamento especifico. Ha varias teorias a respeito da patogenese dessas reacoes, as quais estao diretamente ligadas ao dano neural e as sequelas. Visando-se compreender melhor a fisiopatologia das reacoes tipo 1, foram estudadas 10 biopsias cutaneas de individuos com hanseniase dimorfa-tuberculoide reacional nao tratados e 10 de individuos dimorfos em reacao reversa (apos o inicio do tratamento especifico), comparando-se os parametros morfologicos e imunoistoquimicos. Observou-se no grupo em tratamento, maior positividade das celulas para a enzima oxida nitrico sintase induzivel (iNOS) e menor quantidade de linfocitos T CD8+(p menor 0,05). Nao houve diferenças significativas na baciloscopia e positividade para antigenos micobacterianos nos dois grupos, e nem na quantidade de celulas IL-10+, apesar de ter sido observada correlaçao negativa entre esta citocina e a proporçao CD4/CD8 nos pacientes em tratamento (p menor 0,05). Notou-se tambem, tendencia a reduçao do infiltrado especifico (linfocitos T e B) e aumento do numero de celulas citotoxicas inespecificas (NK) no grupo em tratamento. Estes resultados sao concordantes com trabalhos recentes, que surgerem que a reaçao tipo 1 representaria um desiquilibrio imunologico entre citocinas pro-inflamatorias e anti-inflamatorias...

Hanseníase dimorfa reacional: estudo comparativo, em biópsias cutâneas, entre reações tipo 1 ocorridas antes e durante a poliquimioterapia

A hanseníase dimorfa é a forma clínica mais freqüentemente associada à ocorrência de reações de hipersensibilidade mediada por células (reações tipo 1), que podem ocorrer antes, durante ou depois do tratamento específico. Há várias teorias a respeito da patogênese dessas reações, as quais estão diretamente ligadas ao dano neural e às seqüelas. Visando-se compreender melhor a fisiopatologia das reações tipo 1, foram estudadas 10 biópsias cutâneas de indivíduos com hanseníase dimorfatuberculóidereacional não tratados e 10 de indivíduos dimorfos em reação reversa (após o início do tratamento específico), comparando-se os parâmetros morfológicos e imunoistoquímicos. Observou-se, no grupo em tratamento, maior positividade das células para a enzima óxido nítricosintase induzível (iNOS) e menor quantidade de linfócitos T CD8+ (p<0,05).Não houve diferenças significativas na baciloscopia e positividade para antígenos micobacterianos nos dois grupos, e nem na quantidade de células IL-10+, apesar de ter sido observada correlação negativa entre esta citocinae a proporção CD4/CD8 nos pacientes em tratamento (p<0,05). Notou-se, também, tendência à redução do infiltrado específico (linfócitos T e B) e aumento do número de células citotóxicas inespecíficas (NK) no grupo em tratamento. Estes resultados são concordantes com trabalhos recentes, que sugerem que a reação tipo 1 representaria um desequilíbrio imunológico entre citocinas pró-inflamatórias e anti-inflamatórias. Na presença de muitos bacilos viáveis e no contexto de um paciente sem imunidade celular plena, haveria uma tendência à piora no sentido do pólo virchoviano (downgrading), porém o tratamento específico, reduzindo a carga bacilar, favoreceria uma melhora da imunidade efetiva, caracterizada por um quadro histológico mais tuberculóide, com posterior desvio progressivo da imunidade adquirida para a inespecífica (resposta Th3 ou reguladora), interrompendo a reação e levando à cura

Nitrotyrosine localization to dermal nerves in borderline leprosy.

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.

DDT inhibits the functional activation of murine macrophages and decreases resistance to infection by Mycobacterium microti.

DDT is still widely used in several parts of the world to control malaria, typhoid and dengue vectors, even though its use was banned in many countries based on toxicity data in wild life species. DDT has been shown to have immunotoxic effects in mice and to increase susceptibility to intracellular pathogens such as Mycobacterium leprae. However, little is known about the mechanisms underlying this effect. Activated macrophages play an important defensive role against intracellular pathogens, therefore our objective was to evaluate the effect of in vitro exposure to technical grade DDT (a mixture of three forms: 1,1,1-thricloro-2,2-bis(p-chlorophenyl)ethane (p,p'-DDT) (85%), o,p'-DDT (15%) and o,o'-DDT (trace amounts)), p,p'-DDT, 1,1-dicloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane on the functional activation of J774A.1 macrophages and their capability to limit growth of intracellular pathogens, using Mycobacterium microti as a model. We evaluated cytotoxicity and the effect on cell proliferation of 2.5, 5.0 and 10 microg/ml of DDT compounds. Functional macrophage activity (NO(*) and O(2)(-) production, and mRNA expression of TNF-alpha, IL-1beta and iNO synthase) and the ability of treated cells to limit infection by M. microti in IFN-gamma-activated macrophages were evaluated in cells exposed to 2.5 microg/ml of DDT compounds. Doses of 5 and 10 microg/ml induced direct cytotoxic effects precluding meaningful analysis of the above parameters, whereas 2.5 microg/ml of all DDT compounds inhibited macrophage activity and reduced their ability to limit the intracellular growth of M. microti without inducing cytotoxicity. Technical grade DDT and p,p'-DDE were the more potent compounds. Therefore, exposure to DDT compounds could represent an important risk for infection development by those intracellular pathogens against which NO(*) and/or O(2)(-) production represent the main immune protective mechanism.

IFN-gamma and NO in mycobacterial disease: new jobs for old hands.

Granulomatous disease following exposure to Mycobacterium tuberculosis, Mycobacterium leprae or Mycobacterium avium is correlated with strong inflammatory and protective responses. The mouse model of mycobacterial infection provides an excellent tool with which to examine the inter-relationship between protective cell-mediated immunity and tissue-damaging hypersensitivity. It is well established that T cells and interferon (IFN)-gamma are necessary components of anti-bacterial protection. We propose that IFN-gamma also modulates the local cellular response by downregulating lymphocyte activation and by driving T cells into apoptosis, and that the events that limit excessive inflammation are largely mediated by IFN-gamma-induced nitric oxide (NO). In several murine models of mycobacterial infection, the absence of IFN-gamma and/or NO results in dysregulated granuloma formation and increased lymphocytic responses, which, in the case of M. avium infection, even leads to reduced bacterial growth.

Profiles of the mRNA expression by macrophages infected with Mycobacterium leprae and Mycobacterium avium complex.

In the present study, we examined profiles of the interaction of Mycobacterium leprae and Mycobacterium avium complex (MAC) with murine peritoneal macrophages (M phi s) in terms of up-regulation of M phi expression of proinflammatory and immunosuppressing cytolines (CKs) after infection. First, the reverse transcription polymerase chain reaction (RT-PCR) assay revealed that both MAC and M. leprae infections up-regulated M phi mRNA expression IL-12, TNF-alpha, IL-10, and transformating growth factor-beta (TGF-beta), except that M. leprae-infected M phi s showed no increase in the IL-12 mRNA expression. Second, the RT-PCR assay also showed some differences between M. leprae- and MAC-infected M phi s with respect to the modes of IL-10 and inducible nitric oxide synthase (iNOS) mRNA expression. That is MAC, but not M. leprae, infection caused a prolonged increase in the expression of IL-10 and iNOS mRNAs. Third, a ribonuclease protection assay revealed that M phi s co-infected with MAC and M. leprae showed the Il-12, TNF-alpha and IL-10 mRNA expression in an intermediate mode of those of M phi s infected with either M. leprae or MAC alone. This implies that the CK expression of M. leprae-infected M phi s may be modified by co-infection with MAC. These findings may suggest differential interactions of M. leprae and MAC organisms with murine peritoneal M phi s in terms of the activation of signal transduction pathways for expression of some kinds of immunoregulatory cytokines and immunoprotective enzymes.

Expression of inducible nitric oxide synthase and nitrotyrosine in borderline leprosy lesions.

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.

Immunohistochemical analysis of cellular infiltrate and gamma interferon, interleukin-12, and inducible nitric oxide synthase expression in leprosy type 1 (reversal) reactions before and during prednisolone treatment.

The effects of prednisolone treatment on the cellularity and cytokine (gamma interferon, interleukin-12, and inducible nitric oxide synthase) profiles of leprosy skin type 1 (reversal) reactions were studied using immunohistochemistry. Skin biopsies were taken from 15 patients with leprosy type 1 (reversal) reactions at days 0, 7, 28, and 180 after the start of steroid treatment. Prednisolone treatment had little effect at day 7, but by day 28 significant decreases were found in cytokine levels. Some patients maintained cytokine production at days 28 and 180. These results illustrate the strong Th1 profile of type 1 reactional lesions, the slow response to steroid therapy, and continuing activity at 180 days.

Pentoxifylline downregulates nitric oxide and tumor necrosis factor-alpha induced by mycobacterial lipoarabinomannan in a macrophage cell line.

Pentoxifylline (PTX), a phosphodiesterase inhibitor, is known to downregulate tumor necrosis factor-alpha (TNF-alpha) secretion induced by lipopolysacchride (LPS) and gamma interferon (IFN-gamma). We have had limited success in treating leprosy reactions, including erythema nodosum leprosum (ENL), in which TNF-alpha has been identified as a major proinflammatory cytokine. PTX inhibited production of NO (IC50 approximately equal to 1.0 mg/ml) and TNF-alpha (IC50 approximately equal to 0.05 mg/ml) in a dose-dependent fashion. As little as 0.5 mg/ml of PTX decreased NO production and 0.01 mg/ml of PTX inhibited TNF-alpha production. Western blot analyses demonstrated that iNOS was suppressed by PTX. Northern blot analyses showed significant reduction of TNF-alpha mRNA. We conclude that PTX is an effective inhibitor of lipoarabinomannan (LAM)-induced TNF-alpha production at both the product and transcriptional levels in our macrophage cell line. PTX also showed moderate inhibition of NO at the product level as well as translation of iNOS.

Role of inducible nitric oxide synthase in resistance to Mycobacterium leprae in mice.

The manifestation of leprosy in humans is largely determined by host immunity to Mycobacterium leprae and is a model for immunoregulation in a human disease. However, animal models available for exploration of the leprosy spectrum are inadequate. This study explored M. leprae infection in mice deficient in inducible nitric oxide synthase, and this report describes elements resembling borderline tuberculoid leprosy in humans.

Post-traumatic migration and emergence of a novel cell line upon the ependymal surface of the third cerebral ventricle in the adult mammalian brain.

This investigation describes the migration and emergence of significant numbers of what appear to be neuron-like cells upon the surface of the median eminence of the adult rodent neurohypophyseal system of the endocrine hypothalamus following the trauma of hypophysectomy. These cells appear to migrate through the neuropil of the underlying median eminence and emerge in large numbers upon the surface of the third cerebral ventricle within 7 days following hypophysectomy (axotomy) of supraoptic (SON) and paraventricular neurites (PVN) of the adult neurohypophyseal system. Previous investigations have demonstrated regeneration of the neural stem and neural lobe in a variety of mammalian species (Adams et al., J Comp Neurol, 1969;135:121-144; Beck et al., Neuroendocrinology, 1969;5:161-182; Scott et al., Exp Neurol, 1995;131-1:23-39; Scott and Hansen, Vir Med 1997;124:249-261). It also has been demonstrated that the process of regeneration is invariably accompanied by the up-regulation of nitric oxide synthase (NOS), the enzyme that catalyzes arginine to nitric oxide (NO) and that both neurohypophyseal regeneration, as well as migration and emergence of neuron-like cells upon the surface of the adjacent third cerebral ventricle, is associated with the up-regulation of NOS and increased expression of NO. It also has been amply demonstrated that this entire process of neurohypophyseal regeneration and cell migration is completely inhibited by the introduction of the antagonist of nitric oxide, namely, nitroarginine (Scott et al., Exp Neurol, 1995;131-1:23-39; Scott and Hansen, Vir Med, 1997;124:249-261). The emergence and migratory dynamics of this novel cell line upon the floor of the rodent third cerebral ventricle are discussed with respect to the role of the ubiquitous free radical NO and the implications and potential clinical applications of neuronal migration following trauma in the human central nervous system (CNS).

Immunocytochemical localization of inducible nitric oxide synthase and transforming growth factor-beta (TGF-beta) in leprosy lesions.

Inducible nitric oxide synthase (iNOS) and TGF-beta were localized by immunocytochemistry in skin lesions from patients across the leprosy spectrum, and from patients undergoing reversal reaction. iNOS expression was highest at the tuberculoid pole of the spectrum, and increased during reversal reaction. TGF-beta was observed throughout the leprosy spectrum, but was highest at the lepromatous pole. Levels of TGF-beta decreased during reversal reaction. Reduced levels of TGF-beta may contribute to unregulated inflammatory responses during reactional episodes.