TcSERPIN, an inhibitor that interacts with cocoa defense proteins and has biotechnological potential against human pathogens.

In plants, serpins are a superfamily of serine and cysteine protease inhibitors involved in stress and defense mechanisms, with potential for controlling agricultural pests, making them important biotechnological tools. The objective of this study was to characterize a serpin from Theobroma cacao, called TcSERPIN, to identify its endogenous targets and determine its function and biotechnological potential. TcSERPIN has 390 amino acid residues and shows conservation of the main active site, RCL. Cis-elements related to light, stress, hormones, anaerobic induction, cell cycle regulation and defense have been identified in the gene's regulatory region. TcSERPIN transcripts are accumulated in different tissues of Theobroma cacao. Furthermore, in plants infected with Moniliophtora perniciosa and Phytophthora palmivora, the expression of TcSERPIN was positively regulated. The protein spectrum, rTcSERPIN, reveals a typical ß-sheet pattern and is thermostable at pH 8, but loses its structure with temperature increases above 66°C at pH 7. At the molar ratios of 0.65 and 0.49, rTcSERPIN inhibited 55 and 28% of the activity of papain from Carica papaya and trypsin from Sus scrofa, respectively. The protease trap containing immobilized rTcSERPIN captured endogenous defense proteins from cocoa extracts that are related to metabolic pathways, stress and defense. The evaluation of the biotechnological potential against geohelminth larvae showed that rTcSERPIN and rTcCYS4 (Theobroma cacao cystatin 4) reduced the movement of larvae after 24 hours. The results of this work show that TcSERPIN has ideal biochemical characteristics for biotechnological applications, as well as potential for studies of resistance to phytopathogens of agricultural crops.

Evaluation of in situ expression of effector and regulatory cytokines, TLR, galectins and matrix metalloproteinases in oral manifestations of paracoccidioidomycosis.

BACKGROUND: Although the pathophysiology of paracoccidioidomycosis (PCM) is not completely understood, the study of immune response against fungus has provided insight into understanding the natural course of the disease and its clinical manifestations, hence contributing to the development of preventive measures and treatment proposals. The aim of this study was to evaluate the histopathological and immunological aspects involved in the role of different effector and regulatory responses, as well as the correlation between the TLRs, Galectins, Matrix Metalloproteinases and cytoplasmic proteases of mast cells in this infection. METHODS: Sixteen biopsy specimens with oral lesions of chronic PCM, as well as 13 sections of normal oral mucosa were analyzed. Histopathological and immunological aspects involved in the role of different effector and regulatory responses were evaluated. Indirect immunohistochemistry was performed for IL-17, IL-10, IL-4, TGF-ß, FoxP3, Gal-1, Gal-3, Gal-9, TLR-2, TLR-4, MMP-3 and MMP-9, as well as for chymase and tryptase for mast cells identification. Fibrosis was quantified using Picrosirius. RESULTS: There was a significant increase in the area of fibrosis and in the number of cells expressing IL-10, IL-4, IL-17, FoxP3, Gal-3, TLR-2, MMP3 and MMP9 in patients with PCM in comparison with patients in the group control. There was no difference in the expression of TGF-ß, TLR-4, Gal-1 or Gal-9. Mast cells number was found to be significantly lower in oral chronic PCM when compared to control samples after quantification of mast cells and expression of chymase and tryptase. PCM granulomas were classified to the morphological aspects in organized ou non-organized. Expression of IL-4 in non-organized granulomas was significantly higher. CONCLUSION: The proteins studied herein appear to play an important role in the development and maintenance of oral lesions of PCM, as well as in the processes of development and progression of lesions caused by the fungus and by the immune response associated with the infection.

TGF-beta and CD23 are involved in nitric oxide production by pulmonary macrophages activated by beta-glucan from Paracoccidioides brasiliensis.

Pulmonary macrophages (PM), which are CD11b/CD18(+) and CD23(+), may be involved in the onset of inflammatory events caused by Paracoccidioides brasiliensis in the lungs. In the present study, we measured the nitric oxide (NO) and interleukin in PM production after intratracheal (i.t.) inoculation of an enriched beta-glucan cell wall fraction from P. brasiliensis (Fraction F1). BALB/c and C57/BL6 (B6) mice were i.t. treated with Fraction F1, and their PM were restimulated in vitro with LPS and interferon-gamma up to 14 days after treatment. Macrophages BALB/c mice produced less NO than PM from B6 mice. The lower NO production was caused by higher production of TGF-beta by pulmonary macrophages of BALB/c and was abrogated by anti-TGF-beta MoAb in vitro and in vivo. Other interleukins such as IL-10, IL-4 and a combination of IL-1, TNF-alpha and IL-6 were not involved in NO production induced by Fraction F1. Expression of CD11b increases and expression of CD23 decreases on PM of BALB/c mice after in vivo treatment whereas PM of B6 mice do not show a variation of their phenotype. Moreover, the ability of pulmonary macrophages to induce lymphocyte proliferation was reduced in mixed cultures of CD11b(+) or CD23(+) macrophages but was restored when lymphocytes were cultivated in the presence of NO inhibitor (L-NMMA). Thus, the results presented herein indicate that in BALB/c but not in B6 mice TGF-ss is strongly induced by Fraction 1 in PM in vivo and suppresses NO production. Low NO production by PM is associated with a change in CD11b/CD23 expression and with a high lymphocyte proliferative response. Thus, CD11b(+)/CD23(+) PM modulate NO and TGF-beta production in the pulmonary microenvironment.