Immune Response to the Recombinant Apa Protein from Mycobacterium tuberculosis Expressed in Streptomyces lividans After Intranasal Administration in Mice. Induction of Protective Response to Tubercle Bacillus Aerosols Exposure.

Identifying and evaluating potential vaccine candidates has become one of the main objectives to combat tuberculosis. Among them, mannosylated Apa antigen from Mycobacterium tuberculosis and the non-mannosylated protein expressed in Escherichia coli, have been studied. Although both proteins can induce a protective response in mice, it has been considered that native protein can be dispensed. In this work, we study the protective response induced by Apa expressed in E. coli and in Streptomyces lividans. The latter, like native is secreted as a double band of 45/47 kDa, however, only its 47 kDa band is mannosylated. Both antigens and BCG were intranasal administrated in mice, and animals were then challenged by aerosol with M. tuberculosis H37Rv. The results showed that both, Apa from S. lividans and E. coli conferred statistically significantly protection to animals compared to controls. The cytokine immune response was studied by an immunoassay after animals' immunization, revealing that Apa from S. lividans induced a statistically significant proliferation of T cell, as well as the expression of IFN-γ, IL-1ß, IL-17 and IL-10. In contrast, non-proliferation was obtained with non-mannosylated protein, but induction of IL-12 and IL-17 was observed. Together, these results demonstrate that both proteins were able to modulate a specific immune response against M. tuberculosis, that could be driven by different mechanisms possibly associated with the presence or not of mannosylation. Furthermore, stimulation of cells from BCG-vaccinated animals with the proteins could be an important tool, to help define the use of a given subunit-vaccine after BCG vaccination.

Adoptive transfer of CD4(+)Foxp3(+) regulatory T cells to C57BL/6J mice during acute infection with Toxoplasma gondii down modulates the exacerbated Th1 immune response.

Infection of C57BL/6J mice with the parasite Toxoplasma gondii triggers a powerful Th1 immune response that is detrimental to the host. During acute infection, a reduction in CD4(+)Foxp3(+) regulatory T cells (Treg) has been reported. We studied the role of Treg during T. gondii infection by adoptive transfer of cells purified from transgenic Foxp3(EGFP) mice to infected wild type animals. We found a less severe weight loss, a significant delayed mortality in infected Treg-transferred mice, and reduced pathology of the small intestine that were associated with lower IFN-γ and TNF-α levels. Nevertheless, higher cyst number and parasite load in brain were observed in these mice. Treg-transferred infected mice showed reduced levels of both IFN-γ and TNF-α in sera. A reduced number of CD4(+) T cells producing IFN-γ was detected in these mice, while IL-2 producing CD4(+) T cells were restored to levels nearly similar to uninfected mice. CD25 and CD69 expression of CD4(+) T cells were also down modulated. Our data show that the low Treg cell number are insufficient to modulate the activation of CD4(+) T cells and the production of high levels of IFN-γ. Thus, a delicate balance between an optimal immune response and its modulation by Treg cells must exist.

The response of the fibrinolytic system to mycobacteria infection.

The human pathogen Mycobacterium tuberculosis binds to a variety of host cell proteins, including those of the fibrinolytic system. These observations prompted us to study the expression of components of this system in an animal model of progressive pulmonary tuberculosis. Lung homogenates from BALB/c mice infected with M. tuberculosis H37Rv were analyzed to determine the expression and enzymatic activity of plasmin/plasminogen and tissue plasminogen activator, as well as the mRNA levels for plasminogen, tissue and urokinase plasminogen activators. Plasminogen was also detected in infected lungs with immunohistochemistry. The results show that the expression of molecules of the fibrinolytic system increased gradually over the course of the infection, peaking during the chronic phase of the disease. Furthermore, in vitro experiments showed that both plasminogen activators were specifically induced after the stimulation of spleen cells from BCG-immunized mice with M. tuberculosis proteins. Together, these results show that molecules of the fibrinolytic system are up-regulated in the chronic phase of experimental tuberculosis and suggest that the mycobacterium itself could play an important role in the overexpression of molecules of the fibrinolytic system, contributing to chronic inflammation in tuberculosis.

Adenoma pleomorfo parotídeo gigante: a propósito de un caso clínico / Giant parotid pleomorphic adenoma: a case report

El adenoma pleomorfo es el tumor benigno más frecuente de las glándulas salivales, con mayor predilección por la glándula parótida. Se presenta un caso clínico de paciente femenino de 53 años de edad, con aumento de volumen en región parotídea y geniana derecha de15 × 12 centímetros, de ocho años de evolución, la tomografía simple de la región presenta tumoración parotídea bien delimitada, la cual afecta lóbulo superficial y profundo de la glándula parótida derecha, la biopsia incisional confi rmó el diagnóstico histopatológico de adenoma pleomorfo por lo cual se realiza parotidectomía total sin preservación del nervio facial.

Pleomorphic adenoma is the most common benign tumor of the salivaryglands, with greater predilection for the parotid gland. We presentthe case of a 53-year-old female patient with a 15 x 12 cm increasein volume in the parotid and right genial region with eight years ofevolution. A simple CT scan of the region revealed a well-defi ned parotidtumor aff ecting the superfi cial and deep lobe of the right parotidgland. An incisional biopsy confi rmed the histopathological diagnosisof pleomorphic adenoma, for which reason a total parotidectomy wasperformed without preservation of the facial nerve.

The PGRS domain of Mycobacterium tuberculosis PE_PGRS Rv1759c antigen is an efficient subunit vaccine to prevent reactivation in a murine model of chronic tuberculosis.

A third of the world population is latently infected with Mycobacterium tuberculosis and many cases of active tuberculosis arise from latent bacilli reactivation. Thus, it is important to design new vaccines to prevent reactivation. Using an experimental model of chronic tuberculosis in B6D2F1 mice, we observed constant expression of Rv1759c antigen, a member of the PE_PGRS gene family, on the cell wall of phagocytosed mycobacteria by activated macrophages located in lung granulomas. This antigen induced production of IFN-gamma after stimulation of cell suspensions from mediastinal lymph nodes. It was notorius that chronic infected mice immunized with this antigen and treated with corticosterone to induce reactivation showed not change in colony forming units (CFU), compared with the significant bacilli increase in non-vaccinated mice treated with corticosterone. These results suggest that this antigen could play an important role in the immune response that maintains latent infection, and could therefore, be a good candidate as a new subunit vaccine to prevent disease reactivation.

The PE-PGRS glycine-rich proteins of Mycobacterium tuberculosis: a new family of fibronectin-binding proteins?

A clone was isolated by screening of a cosmid library of Mycobacterium tuberculosis with an oligonucleotide designed from the N-terminal sequence of a previously reported proline-rich protein. Characterization of the 4481 bp insert showed the presence of polymorphic CG-repetitive sequences (PGRSs) with an ORF of 2.7 kb, encoding a 81.3 kDa protein (PE-PGRS81). Southern blot analysis and BLAST-p searches revealed several homologous sequences in the genome of M. tuberculosis. The deduced amino acid sequence was highly similar to a stretch of about 98 residues in the N-terminus present in several members of the PE-PGRS family available in the GenBank database, including 100% identity with the partial amino acid sequence of the potential protein encoded by orf3' as well as with the Rv0278c sequence. A neighbour-joining analysis of the 99 PE-PGRS sequences available in the database indicated that PE-PGRS81 is included in a group where its closest relatives are the sequences orf3', Rv0278c, Rv0279c, Rv1759c, Rv3652 and Rv0747. Probing with the complete coding regions of PE-PGRS81 and Rv1759c in Southern blot assays, on samples of genomic DNA from M. tuberculosis H37Rv, Mycobacterium bovis BCG and M. tuberculosis clinical isolates, showed a complex hybridization pattern for all strains. This shows the existence of intrastrain PGRS variability as reported for other PGRS members. In contrast, probing with the short conserved N-terminal region of Rv1759c reduced the hybridization to a single band. This marker allowed identification of M. tuberculosis clinical strains that lack Rv1759c. A recombinant C-terminal fragment of Rv1759c showed fibronectin-binding properties and was recognized by sera from patients infected with M. tuberculosis, suggesting that at least this member of the PE-PGRS is expressed in tuberculosis infection.