Guinea pig model of Mycobacterium tuberculosis latent/dormant infection.

Although guinea pigs are considered one of the best animal models of tuberculosis, little data exist describing latent or dormant tuberculosis infection in these animals. Here we address this issue using a streptomycin auxotrophic mutant of Mycobacterium tuberculosis. This mutant grows unimpaired in the presence of streptomycin but in its absence shifts to latency/dormancy (lack growth and over-expression of alpha-crystallin). To establish infection animals are inoculated with the mutant followed by daily administration of streptomycin (three weeks), which allows initial microbial multiplication in the animal's tissues. Withdrawal of streptomycin establishes latency/dormancy and few viable organisms are recovered from the animals' lungs and spleen six months later. During the infectious process guinea pigs steadily gained weight and presented no clinical signs (scuff fur and lethargy) of disease. Histopathology of organs mimicked tuberculous lesions in humans and PBMC from infected animals strongly responded to stimulation with PPD. Finally, tuberculin skin test (a hallmark of latent infection diagnosis) performed in infected animals was strongly positive (>or=15 mm induration). These results point to an interesting and reliable model of latent/dormant tuberculosis infection in guinea pigs.

Anti-Leishmania activity of new ruthenium(II) complexes: Effect on parasite-host interaction.

Leishmaniasis is a parasitic disease caused by protozoa of the genus Leishmania. The many complications presented by the current treatment - including high toxicity, high cost and parasite resistance - make the development of new therapeutic agents indispensable. The present study aims to evaluate the anti-Leishmania potential of new ruthenium(II) complexes, cis­[RuII(η2-O2CR)(dppm)2]PF6, with dppm=bis(diphenylphosphino)methane and R=4-butylbenzoate (bbato) 1, 4-(methylthio)benzoate (mtbato) 2 and 3-hydroxy-4-methoxybenzoate (hmxbato) 3, in promastigote cytotoxicity and their effect on parasite-host interaction. The cytotoxicity of complexes was analyzed by MTT assay against Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, Leishmania (Leishmania) infantum promastigotes and the murine macrophage (RAW 264.7). The effect of complexes on parasite-host interaction was evaluated by in vitro infectivity assay performed in the presence of two different concentrations of each complex: the promastigote IC50 value and the concentration nontoxic to 90% of RAW 264.7 macrophages. Complexes 1-3 exhibited potent cytotoxic activity against all Leishmania species assayed. The IC50 values ranged from 7.52-12.59µM (complex 1); 0.70-3.28µM (complex 2) and 0.52-1.75µM (complex 3). All complexes significantly inhibited the infectivity index at both tested concentrations. The infectivity inhibitions ranged from 37 to 85%. Interestingly, the infectivity inhibitions due to complex action did not differ significantly at either of the tested concentrations, except for the complex 1 against Leishmania (Leishmania) infantum. The infectivity inhibitions resulted from reductions in both percentage of infected macrophages and number of parasites per macrophage. Taken together the results suggest remarkable leishmanicidal activity in vitro by these new ruthenium(II) complexes.

Evaluation of the effect of treatment of latent tuberculosis infection on QuantiFERON-TB gold assay results.

To evaluate the utility of the QuantiFERON-TB Gold assay for monitoring latent tuberculosis treatment efficacy, the assay was performed serially for healthcare workers receiving isoniazid therapy. After 9 months of isoniazid therapy, all of these healthcare workers remained QuantiFERON-TB Gold positive, and cellular proliferation assays revealed persistently strong purified protein derivative responses. These results do not support the use of the QuantiFERON-TB Gold assay to monitor therapy.

Identification of Mycobacterium tuberculosis ornithine carboamyltransferase in urine as a possible molecular marker of active pulmonary tuberculosis.

Although the antigen detection assay has the potential to discriminate active tuberculosis from latent infection, development of such a test for the accurate diagnosis of this serious disease has only recently become a matter of interest. Here we present evidence that a Mycobacterium tuberculosis protein (ornithine carboamyltransferase, coded for by MT_1694; Rv1656 [argF]) is an interesting candidate molecule for this test development. The protein was initially discovered by mass spectroscopy in urine of patients with pulmonary tuberculosis and shown by Western blot analysis to be present in M. tuberculosis crude cell extract as well as in the culture supernatant ("secreted" protein). In addition, a recombinant ornithine carboamyltransferase (rMT1694) produced in Escherichia coli was recognized by immunoglobulin G (IgG) antibodies from patients with active tuberculosis but not by IgG from uninfected healthy subjects. Moreover, rMT1694 was strongly recognized by peripheral blood mononuclear cells from both healthy tuberculin purified protein derivative (PPD)-positive individuals and patients with pulmonary tuberculosis. More importantly, a capture enzyme-linked immunosorbent assay formatted with rabbit IgG antibodies specific to rMT1694 was able to identify the presence of this antigen in urine samples from 6 of 16 patients with pulmonary tuberculosis and in none of 16 urine samples collected from healthy PPD(+) controls. These results indicate that an improved antigen detection assay based on M. tuberculosis ornithine carboamyltransferase may represent an important new strategy for the development of a specific and accurate diagnostic test for tuberculosis.

A new approach for quantifying furanodiene and curzerene: a case study on the essential oils of Eugenia uniflora L., Myrtaceae (pitangueira) leaves

The essential oil obtained from the leaves of Eugenia uniflora L., Myrtaceae, which grows in the Brazilian savannah, was studied by gas chromatography mass spectrometry (GC-MS). Furanodiene (1.2 percent) was thermally rearranged to curzerene (85.1 percent) to produce a combined content of 86.3 percent. GC analysis carried out under mild conditions (with a constant temperature of 100 ºC) showed that the furanodiene concentration was three-fold greater than the curzerene concentration, i.e., the essential oil contained 64.7 percent furanodiene and 21.6 percent curzerene. Germacrene B also rearranged to γ-elemene and the concentration of both was 2.3 percent. Special care should be taken when conventional gas chromatography analysis is used for quantifying compounds that can rearrange at high temperatures.

Comparative effect of ion calcium and magnesium in the activation and infection of the murine macrophage by Leishmania major.

Amastigotes of Leishmania major have a great ability to evade destruction in host cells. This study investigated the activation in resident, inflammatory macrophages and J774 cells in vitro treated with lipopolysaccharide (LPS), soluble Leishmania antigen (SLA), calcium ionophore (CaI) and magnesium (Mg2+) alone or combined. An increase in nitric oxide (NO) production was observed in J774 or inflammatory macrophages treated with LPS alone or in combination with SLA and CaI. The same treatments did not affect the NO release by resident macrophages. There was no interference in uptake of L. major but CaI decreased intracellular proliferation of the parasite. This study demonstrated the importance of CaI in decreasing L. major proliferation inside murine macrophages while Mg2+ seemed to increase parasite proliferation. These finding may help to understand the events involved in host cells' clearance of this pathogen.

Comparative effect of ion calcium and magnesium in the activation and infection of the murine macrophage by Leishmania major

Amastigotes of Leishmania major have a great ability to evade destruction in host cells. This study investigated the activation in resident, inflammatory macrophages and J774 cells in vitro treated with lipopolysaccharide (LPS), soluble Leishmania antigen (SLA), calcium ionophore (CaI) and magnesium (Mg2+) alone or combined. An increase in nitric oxide (NO) production was observed in J774 or inflammatory macrophages treated with LPS alone or in combination with SLA and CaI. The same treatments did not affect the NO release by resident macrophages. There was no interference in uptake of L. major but CaI decreased intracellular proliferation of the parasite. This study demonstrated the importance of CaI in decreasing L. major proliferation inside murine macrophages while Mg2+ seemed to increase parasite proliferation. These finding may help to understand the events involved in host cells' clearance of this pathogen..