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.

Probiotic effects of marine Debaryomyces hansenii CBS 8339 on innate immune and antioxidant parameters in newborn goats.

Several marine Debaryomyces hansenii strains have shown probiotic effects on aquatic animals, and D. hansenii-derived ß-glucans have recently provided immunostimulant effects on goat leukocytes. This study assessed the probiotic effects of live yeast D. hansenii CBS 8339 on newborn goats administered orally, and subsequently challenged in vitro with Escherichia coli. D. hansenii CBS 8339 demonstrated the capacity to survive gastrointestinal tract conditions (bile salts and acid pH tolerance) and adhere to goat intestine. Twelve Saanen × Nubian crossbred newborn goats (2.9 ± 0.47 kg) were fed with a controlled diet or D. hansenii (0.7 g/kg body weight per day)-supplemented milk for 30 days. Blood samples of newborn goats were taken at days 15 and 30, and peripheral blood leukocytes were isolated for bacterial challenge, and immunological and antioxidant analyses. Despite cell viability was higher in leukocytes of goat kids fed with the yeast supplement, protection against E. coli challenge was not significantly affected. On the other hand, at day 15, oral administration of D. hansenii enhanced respiratory burst and catalase activity and increased superoxide dismutase activity after challenge. In contrast, at day 30, administration of the yeast supplement increased peroxidase activity and enhanced nitric oxide production and catalase activity after challenge. Finally, the yeast-supplemented diet upregulated the expression of the receptor genes TLR (2, 4, 6), modulator genes Raf.1, Syk, and Myd88, transcription factor gene AP-1, and cytokine genes IL-1ß and TNF-α only at day 15 in leukocytes from unchallenged goat kids. These results demonstrated that a short time (15 days) of orally administering the probiotic D. hansenii CBS 8339 to newborn goats stimulated innate immune and antioxidant parameters and the expression of immune-related gene signaling pathways.

A Macrophage Response to Mycobacterium leprae Phenolic Glycolipid Initiates Nerve Damage in Leprosy.

Mycobacterium leprae causes leprosy and is unique among mycobacterial diseases in producing peripheral neuropathy. This debilitating morbidity is attributed to axon demyelination resulting from direct interaction of the M. leprae-specific phenolic glycolipid 1 (PGL-1) with myelinating glia and their subsequent infection. Here, we use transparent zebrafish larvae to visualize the earliest events of M. leprae-induced nerve damage. We find that demyelination and axonal damage are not directly initiated by M. leprae but by infected macrophages that patrol axons; demyelination occurs in areas of intimate contact. PGL-1 confers this neurotoxic response on macrophages: macrophages infected with M. marinum-expressing PGL-1 also damage axons. PGL-1 induces nitric oxide synthase in infected macrophages, and the resultant increase in reactive nitrogen species damages axons by injuring their mitochondria and inducing demyelination. Our findings implicate the response of innate macrophages to M. leprae PGL-1 in initiating nerve damage in leprosy.

DNAhsp65 Vaccine as Therapy against Paracoccidioidomycosis.

The conventional treatment for fungal diseases usually shows long periods of therapy and the high frequency of relapses and sequels. New strategies of the treatment are necessary. We have shown that the Mycobacterium leprae HSP65 gene can be successfully used as therapy against murine Paracoccidioidomycosis (PCM). Here, we described the methodology of DNAhsp65 immunotherapy in mice infected with the dimorphic fungus Paracoccidioides brasiliensis, one of PCM agent, evaluating cytokines levels, fungal burden, and lung injury. Our results provide a new prospective on the immunotherapy of mycosis.

Study of murine experimental Jorge Lobo's disease by analysis of peritoneal lavage cells and footpad histopathology: early versus chronic lesions.

The murine model of Jorge Lobo's disease is characterized by histological alterations similar to those seen in human disease, including a large number of viable fungi. This study evaluated the immune response of mice with early and late macroscopic lesions (5 and 13 months post-inoculation [p.i.], respectively) by the analysis of peritoneal lavage cells and footpad (FP) histology. The FP of mice were inoculated with 1 × 10(6) fungi (viability index of 41%). At 5 and 13 months p.i., the granuloma mainly consisted of macrophages and multinucleated giant cells, but a larger number of neutrophils was observed at 5 months and lymphocytes at 13 months. The number of fungi in the FP and fungal viability were 1.8 ± 1.1 × 10(6) fungi/ml and 38.5% at 5 months p.i. and 30.8 ± 11.7 × 10(6) fungi/ml and 9% at 13 months (P < .05). Higher production of H2O2, O2(-), IL-10, and TNF-α were observed at 13 months (P < .05), but there was no significant difference in the production of NO, IL-2, IL-4, IL-12 and IFN-γ. The results showed significant differences between early and late lesions and support the use of BALB/c mice for evaluation of the different phases of infection.

Induction and treatment of anergy in murine leprosy.

Leprosy is a disease consisting of a spectrum of clinical, bacteriological, histopathological and immunological manifestations. Tuberculoid leprosy is frequently recognized as the benign polar form of the disease, while lepromatous leprosy is regarded as the malignant form. The different forms of leprosy depend on the genetic and immunological characteristics of the patient and on the characteristics of the leprosy bacillus. The malignant manifestations of lepromatous leprosy result from the mycobacterial-specific anergy that develops in this form of the disease. Using murine leprosy as a model of anergy in this study, we first induced the development of anergy to Mycobacterium lepraemurium (MLM) in mice and then attempted to reverse it by the administration of dialysable leucocyte extracts (DLE) prepared from healthy (HLT), BCG-inoculated and MLM-inoculated mice. Mice inoculated with either MLM or BCG developed a robust cell-mediated immune response (CMI) that was temporary in the MLM-inoculated group and long-lasting in the BCG-inoculated group. DLE were prepared from the spleens of MLM- and BCG-inoculated mice at the peak of CMI. Independent MLM intradermally-inoculated groups were treated every other day with HLT-DLE, BCG-DLE or MLM-DLE, and the effect was documented for 98 days. DLE administered at a dose of 1.0 U (1 × 10(6) splenocytes) did not affect the evolution of leprosy, while DLE given at a dose of 0.1 U showed beneficial effects regardless of the DLE source. The dose but not the specificity of DLE was the determining factor for reversing anergy.

Study of murine experimental Jorge Lobo's disease by analysis of peritoneal lavage cells and footpad histopathology: early versus chronic lesions

The murine model of Jorge Lobo's disease is characterized by histological alterations similar to those seen in human disease, including a large number of viable fungi. This study evaluated the immune response of mice with early and late macroscopic lesions (5 and 13 months post-inoculation [p.i.], respectively) by the analysis of peritoneal lavage cells and footpad (FP) histology. The FP of mice were inoculated with 1 × 106 fungi (viability index of 41%). At 5 and 13 months p.i., the granuloma mainly consisted of macrophages and multinucleated giant cells, but a larger number of neutrophils was observed at 5 months and lymphocytes at 13 months. The number of fungi in the FP and fungal viability were 1.8 ± 1.1 × 106 fungi/ml and 38.5% at 5 months p.i. and 30.8 ± 11.7 × 106 fungi/ml and 9% at 13 months (P < .05). Higher production of H2O2, O2−, IL-10, and TNF-α were observed at 13 months (P < .05), but there was no significant difference in the production of NO, IL-2, IL-4, IL-12 and IFN-γ. The results showed significant differences between early and late lesions and support the use of BALB/c mice for evaluation of the different phases of infection

Mycobacterial phenolic glycolipids with a simplified lipid aglycone modulate cytokine levels through Toll-like receptor 2.

Phenolic glycolipids (PGLs) are virulence factors present in the cell walls of many pathogenic mycobacteria. PGLs have been implicated in various aspects of mycobacterial disease, but there are limited structure-activity data available for these molecules. We report here the preparation of seven synthetic PGL analogues, differing from the native compounds in the replacement of the complex phenolic lipid moiety with a p-methoxyphenyl group. The ability of these compounds to stimulate or inhibit the production of cytokines (TNF-α, IL-1ß, IL-6, MCP-1) and nitric oxide (NO) was then evaluated by ELISA-based assays. None of the compounds stimulated the production of these biological signalling molecules. In contrast, they each displayed concentration-dependent inhibitory activity, related to the methylation pattern of the molecule and mediated by Toll-like receptor 2. Additional studies revealed that native PGL-I from Mycobacterium leprae and a synthetic PGL-I analogue containing a simplified lipid domain had enhanced inhibitory activities relative to the corresponding analogues containing the p-methoxyphenyl aglycone; however, the natural lipid phenolthiocerol was only weakly active. These studies reveal that synthetic molecules of this type can be used as probes for PGL function. Moreover, their ease of synthesis relative to the natural glycolipids, as well as their more favourable aqueous solubility, should allow for more thorough structure-activity relationship studies.

Haemoglobins of Mycobacteria: structural features and biological functions.

The genus Mycobacterium is comprised of Gram-positive bacteria occupying a wide range of natural habitats and includes species that range from severe intracellular pathogens to economically useful and harmless microbes. The recent upsurge in the availability of microbial genome data has shown that genes encoding haemoglobin-like proteins are ubiquitous among Mycobacteria and that multiple haemoglobins (Hbs) of different classes may be present in pathogenic and non-pathogenic species. The occurrence of truncated haemoglobins (trHbs) and flavohaemoglobins (flavoHbs) showing distinct haem active site structures and ligand-binding properties suggests that these Hbs may be playing diverse functions in the cellular metabolism of Mycobacteria. TrHbs and flavoHbs from some of the severe human pathogens such as Mycobacterium tuberculosis and Mycobacterium leprae have been studied recently and their roles in effective detoxification of reactive nitrogen and oxygen species, electron cycling, modulation of redox state of the cell and facilitation of aerobic respiration have been proposed. This multiplicity in the function of Hbs may aid these pathogens to cope with various environmental stresses and survive during their intracellular regime. This chapter provides recent updates on genomic, structural and functional aspects of Mycobacterial Hbs to address their role in Mycobacteria.

Nanobiotechnological approaches to delivery of DNA vaccine against fungal infection.

Vaccines play an essential role in keeping humans healthy. Innovative approaches to their use include the utilization of plasmid DNA encoding sequences to express foreign antigens. DNAhsp65 from Mycobacterium leprae is suitable for this purpose due to its ability to elicit a powerful immune response. Controlled release systems represent a promising approach to delivering vaccines. In this work, we used liposomes or PLGA systems to deliver DNAhsp65 to treat the pulmonary fungal infection Paracoccidioidomycosis. Both formulations modulated a protective immune response and reduced the pulmonary fungal burden even in the groups receiving less than four times the amount of the DNAhps65 entrapped within the nanoparticles. Although both systems had the same effective therapeutic results, the advantage of the liposome formulation was that it was administered intranasally, which may be more easily accepted by patients. These systems are a great alternative to be considered as adjuvant vaccine therapy for systemic mycosis.

Nitric oxide metabolites in sera of patients across the spectrum of leprosy.

Leprosy, a chronic infectious disease, caused by Mycobacterium leprae infection, manifests itself as a clinical spectrum depending on the patients' immunological response, finally leading to peripheral nerve damage and deformities in the patients. Nitric oxide (NO) which is known to contribute to pathogenesis of several neurological diseases has been detected in tissues and urine of leprosy patients. This is the first study assessing NO as its stable end products, nitrites and nitrates, in sera of patients across the spectrum of the disease as a possible parameter of prognostic value. Comparison of NO metabolites showed a significant increase in multibacillary patients and patients with type I reactions as compared to healthy control individuals. These levels reduced significantly after treatment. This study has further borne out the utility and reliability of the cadmium-reduction method of estimation of NO metabolites--a relatively inexpensive procedure that lends itself to large-scale screening and follow-up of patients.

Leaves of Cassia tora as a novel cancer therapeutic--an in vitro study.

Cassia tora Linn (Leguminacea) is a medicinal plant traditionally used as laxative, for the treatment of leprosy and various skin disorders. Preliminary phytochemical analysis of leaf showed the presence of polyphenols (3.7 mg gallic acid equivalent per gram dried leaves). The presence of phenolic compound prompted us to evaluate its antioxidant and antiproliferative potential. In the present study C. tora methanolic leaf extract (CTME) was evaluated for its nitric oxide scavenging activity and reducing power assays using Rutin and BHT as standards. The extract was studied for its lipid peroxidation inhibition assay using rat liver and brain. In all assays, a correlation existed between concentration of extract and percentage inhibition of free radical, reducing power and inhibition of lipid peroxidation. The antiproliferative activity of CTME with Cisplatin, anticancer drug was studied using human cervical cancer cells (HeLa). Proliferation of HeLa was measured by MTT assay, cell DNA content by modified diphenylamine method and apoptosis by Caspase 3 activity. The plant extract induced a marked concentration dependent inhibition on proliferation, reduced DNA content and apoptosis in HeLa. These results clearly indicate that C. tora is effective against free radical mediated diseases.

Identification of a repressor of a truncated denitrification pathway in Moraxella catarrhalis.

Growth of Moraxella catarrhalis in a biofilm resulted in marked upregulation of two open reading frames (ORFs), aniA and norB, predicted to encode a nitrite reductase and a nitric oxide reductase, respectively (W. Wang, L. Reitzer, D. A. Rasko, M. M. Pearson, R. J. Blick, C. Laurence, and E. J. Hansen, Infect. Immun. 75:4959-4971, 2007). An ORF designated nsrR, which was located between aniA and norB, was shown to encode a predicted transcriptional regulator. Inactivation of nsrR resulted in increased expression of aniA and norB in three different M. catarrhalis strains, as measured by both DNA microarray analysis and quantitative reverse transcriptase PCR. Provision of a wild-type nsrR gene in trans in an nsrR mutant resulted in decreased expression of the AniA protein. DNA microarray analysis revealed that two other ORFs (MC ORF 683 and MC ORF 1550) were also consistently upregulated in an nsrR mutant. Consumption of both nitrite and nitric oxide occurred more rapidly with cells of an nsrR mutant than with wild-type cells. However, growth of nsrR mutants was completely inhibited by a low level of sodium nitrite. This inhibition of growth by nitrite was significantly reversed by introduction of an aniA mutation into the nsrR mutant and was completely reversed by the presence of a wild-type nsrR gene in trans. NsrR regulation of the expression of aniA was sensitive to nitrite, whereas NsrR regulation of norB was sensitive to nitric oxide.

Scavenging of reactive nitrogen species by mycobacterial truncated hemoglobins.

Tuberculosis and leprosy are among the most challenging infectious threats to human health. The ability of mycobacteria to persist in vivo in the presence of reactive nitrogen and oxygen species implies the presence in these bacteria of effective detoxification (pseudoenzymatic) systems. Mycobacterium tuberculosis and Mycobacterium leprae truncated hemoglobins (trHbs) belonging to group I (or N; trHbN) and group II (or O; trHbO) have recently been implicated in the scavenging of nitrogen monoxide (*NO) and peroxynitrite (ONOO-/HOONO). Furthermore, M. leprae trHbO was found to act as an efficient scavenger of the strong oxidant trioxocarbonate(*1-) (CO3*-) following the reaction of peroxynitrite with carbon dioxide (CO2). Here, mechanisms for scavenging of reactive nitrogen species by mycobacterial trHbs are reviewed, and detailed protocols for assessing pseudoenzymatic kinetics are provided.

Nitric oxide scavenging by Mycobacterium leprae GlbO involves the formation of the ferric heme-bound peroxynitrite intermediate.

Ferrous oxygenated (Fe(II)O2) hemoglobins (Hb's) and myoglobins (Mb's) have been shown to react very rapidly with NO, yielding NO3(-) and the ferric heme-protein derivative (Fe(III)), by means of the ferric heme-bound peroxynitrite intermediate (Fe(III)OONO), according to the minimum reaction scheme: Fe(II)O2 + NO (k(on))--> Fe(III)OONO (h)--> Fe(III) + NO3(-). For most Hb's and Mb's, the first step (indicated by k(on)) is rate limiting, the overall reaction following a bimolecular behavior. By contrast, the rate of isomerization and dissociation of Fe(III)OONO (indicated by h) is rate limiting in NO scavenging by Fe(II)O2 murine neuroglobin, thus the overall reaction follows a monomolecular behavior. Here, we report the characterization of the NO scavenging reaction by Fe(II)O2 truncated Hb GlbO from Mycobacterium leprae. Values of k(on) (=2.1x10(6) M(-1) s(-1)) and h (=3.4 s(-1)) for NO scavenging by Fe(II)O2 M. leprae GlbO have been determined at pH 7.3 and 20.0 degrees C, the rate of Fe(III)OONO decay (h) is rate limiting. The Fe(III)OONO intermediate has been characterized by optical absorption spectroscopy in the Soret region. These results have been analyzed in parallel with those of monomeric and tetrameric globins as well as of flavoHb and discussed with regard to the three-dimensional structure of mycobacterial truncated Hbs and their proposed role in protection from nitrosative stress.

Application of a viability-staining method for Mycobacterium leprae derived from the athymic (nu/nu) mouse foot pad.

Mycobacterium leprae cannot be cultured, so ascertaining viability of the organism remains a major obstacle, impeding many avenues of investigation. This study tested a two-colour, Syto9 and propidium iodide, fluorescence assay, which scores for membrane damage in individual bacilli, to determine if a rapid direct-count viability-staining technique can be reliably applied to M. leprae. A variety of experimental conditions were employed to validate this technique. This technique was also used to correlate the viability of M. leprae with the course of athymic mouse foot pad infection to optimize the provision of viable M. leprae as a research reagent. The data show that in untreated suspensions of M. leprae there is a good correlation between the metabolic activity of leprosy bacilli and their membrane damage. Fixation of M. leprae with ethanol, paraformaldehyde and gluteraldehyde completely suppressed their metabolic activity but showed little effect on their membrane integrity. The present study also showed that the metabolic activity of M. leprae declines more than the extent of membrane damage at 37 degrees C within 72 h, but that they are not significantly affected at 33 degrees C. Irradiation at 10(4) Gy showed high numbers of dead bacilli by the staining method. The results show that the reliability of metabolic-activity data as well as viability-staining data is dependent on the method by which M. leprae is killed. This staining method helped us predict reliably that the smaller M. leprae-infected athymic mouse foot pad seen early in infection, between 4 and 5 months, yields markedly better quality leprosy bacilli than older, larger foot pad infections, as defined by their metabolic activity and membrane integrity.

Apoptosis and nitric oxide release induced by thalidomide, gossypol and dexamethasone in cultured human chronic myelogenous leukemic K-562 cells.

The effects of different dosages of thalidomide, gossypol and dexamethasone on the levels of apoptosis and nitric oxide (NO) production were studied in a human chronic myelogenous leukemic cell line, K-562. Increases in the levels of apoptosis were induced by both 15 and 30 microM of thalidomide but only 15 microM significantly increased NO production. All dosages of gossypol used increased the production of NO and all dosages except 200 microM raised the level of apoptosis. After dexamethasone treatment the level of NO either decreased or stayed constant. Thus, some dosages of thalidomide and gossypol concomitantly raise the levels of apoptosis and NO production, but dexamethasone, though it induced apoptosis, had no significant effect on NO production.

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.

IL-12 and IL-18 synergistically induce the bactericidal activity of murine peritoneal cells against M. leprae.

We examined the effect of IL-12 and IL-18 on bactericidal activities of mouse peritoneal cell (PC) against Mycobacterium leprae (M. leprae). We demonstrated that IL-12 and IL-18 synergistically induced the NO-dependent bactericidal activity of PC by stimulating Natural Killer (NK) cells and T-cells through IFN-gamma production. IL-12 and IL-18 induced host cell death through NK-cells and T-cells. Therefore. IL-12 and IL-18 play an important role on direct killing of intracellular M. leprae and on indirect killing of them through inducing host cell death.