Neutrophils Contribute to the Protection Conferred by ArtinM against Intracellular Pathogens: A Study on Leishmania major

ArtinM, a D-mannose binding lectin from Artocarpus heterophyllus, has immunomodulatory activities through its interaction with N-glycans of immune cells, culminating with the establishment of T helper type 1 (Th1) immunity. This interaction protects mice against intracellular pathogens, including Leishmania major and Leishmania amazonensis. ArtinM induces neutrophils activation, which is known to account for both resistance to pathogens and host tissue injury. Although exacerbated inflammation was not observed in ArtinM-treated animals, assessment of neutrophil responses to ArtinM is required to envisage its possible application to design a novel immunomodulatory agent based on carbohydrate recognition. Herein, we focus on the mechanisms through which neutrophils contribute to ArtinM-induced protection against Leishmania, without exacerbating inflammation. For this purpose, human neutrophils treated with ArtinM and infected with Leishmania major were analyzed together with untreated and uninfected controls, based on their ability to eliminate the parasite, release cytokines, degranulate, produce reactive oxygen species (ROS), form neutrophil extracellular traps (NETs) and change life span. We demonstrate that ArtinM-stimulated neutrophils enhanced L. major clearance and at least duplicated tumor necrosis factor (TNF) and interleukin-1beta (IL-1β) release; otherwise, transforming growth factor-beta (TGF-β) production was reduced by half. Furthermore, ROS production and cell degranulation were augmented. The life span of ArtinM-stimulated neutrophils decreased and they did not form NETs when infected with L. major. We postulate that the enhanced leishmanicidal ability of ArtinM-stimulated neutrophils is due to augmented release of inflammatory cytokines, ROS production, and cell degranulation, whereas host tissue integrity is favored by their shortened life span and the absence of NET formation. Our results reinforce the idea that ArtinM may be considered an appropriate molecular template for the construction of an efficient anti-infective agent.     

Method for kinetic analysis of drug-induced cell cycle perturbations.

A method is described for quantitative study of the flux of cells through the cell cycle phases in in vitro systems perturbed by chemicals, such as chemotherapeutic agents. The method utilizes cell count and the flow cytometric technique of bromodeoxyuridine (BrdUrd) labeling, according to an optimized strategy. Cells are exposed to BrdUrd during the last minutes of drug treatment and fixed for analysis at 0, 1/3Ts, 2/3Ts, Ts, and Tc + TG1 recovery times, where Ts, TG1, Tc are the mean durations of phases S and G1 and of the whole cycle of control cells. As an example of application of the proposed procedure, a kinetic study of the effect of 1-(2-chloroethyl)-1-nitrosourea (CNU) on the L1210 cell cycle is described. Simple data analysis, requiring only a pocket calculator, showed that cells in phases G1 and G2M at the end of a 1 h treatment with 1 microgram/ml CNU were fully able to leave these phases but were destined to remain blocked in the following G2M phase (G1 for a minority of them). We also found that cells initially in S phase were slightly delayed in completing their S phase and that 50% of them remained temporarily blocked in the subsequent G2M phase, irrespective of their position in the S phase.     

The three-dimensional structure of the aspartyl protease from the HIV-1 isolate BRU.

The crystal structure of the aspartyl protease encoded by the gene pol of the human immunodeficiency virus (HIV-1, isolate BRU) has been determined to 2.7 A resolution. The enzyme, expressed as an insoluble denatured polypeptide in inclusion bodies of Escherichia coli has been renatured and crystallized. It differs by several amino acid replacements from the homologous enzymes of other HIV-1 isolates. A superposition of the C alpha-backbone of the BRU protease with that of the SF2 protease gives a roots mean square positional difference of 0.45 A. Thus, neither the denaturation/renaturation process nor the amino acid replacements have a noticeable effect on the three-dimensional structure of the BRU protease or on the detailed conformation of the catalytic site, which is very similar to that of other aspartyl proteases.     

Purification of Tissue Forms (Amastigotes) of Trypanosoma cruzi by Immunoaffinity Chromatography1

A rapid and simple method for the purification of amastigotes of Trypanosoma cruzi from spleens of infected mice is described. A protein A-Scpharose 4B immunoadsorbent column bound with antisera to epimastigotes of T. cruzi was used to purify the tissue forms of this parasite. Host cells and debris are not retained, and parasites can be eluted in high yields and purity. Studies of surface glycoproteins and glycolipids of the purified amastigotes with 18 lectins of various specificities revealed the presence on the parasites of receptors for N-acetylglucosamine, N-acetylgalactosamine, D-galactose, and D-mannose binding lectins.     

Development of calcium and secretory responses in the human promyelocytic leukemia cell line HL60

We have begun to characterize the development of the excitation-response coupling sequence in the human promyelocytic leukemia cell line HL60. Using the recently developed fluorescent calcium probe quin-2, it was found that DMSO induced myeloid differentiation of the HL60 cells is accompanied by the development of a calcium response to the addition of the chemotactic factors fMet-Leu-Phe and leukotriene B4. The characteristics (time course, concentration dependence, stereospecificity, and metabolic dependence) of the calcium response are extremely similar to those previously described in human neutrophils. These results imply that functional receptors for leukotriene B4 appear in HL60 cells upon the induction of differentiation and also lend strong support to the use of these HL60 cells as a model of human myeloid differentiation. We have also characterized the emergence of a secretory response to fMet-Leu-Phe and leukotriene B4 in cytochalasin B treated HL60 cells. In addition, it is found that differentiation was required for the calcium ionophore A23187 to express its secretory activity toward the HL60 cells. This last set of results implies that differentiation is accompanied by the coordinated appearance of surface receptors and cytoplasmic factors required for the expression of cellular responsiveness.     

Glioblastoma therapy by direct intralesional administration of I-131 radioiodine labeled antitenascin antibodies

Cell Biochemistry and Biophysics - Thirty patients with recurrent glioblastomas (29 brain, 1 spinal cord) received intralesional radioimmunotherapy aiming to control the progression of the tumor...     

The legacy of forest logging on organic matter inputs and storage in tropical streams

Riparian forests play an important role in stream ecosystems, as they support biodiversity, reduce water erosion, and provide litter that fuels aquatic biota. However, they are affected by great array of anthropogenic threats (e.g., fire, logging, and organic pollution), which alter species composition and their physical structure. Although forest recovery after disturbance such as logging can take decades, the legacy of forest clear-cut logging on key processes in tropical riparian ecosystems is mostly unknown. Here, we investigated how litter inputs (leaves, twigs, and reproductive parts) and storage, key processes for carbon and nutrient recycling and for forest and stream biota, are influenced by riparian vegetation undergoing succession (after 28 years from logging) through the comparison of reference and logged forest sites in the Cerrado biome. Litterfall was overall similar between forest types, but litterfall of twigs was twofold higher at logged than reference sites. Similarly, litter inputs from the bank to the stream (i.e., lateral inputs) and streambed storage were 50–60% higher at logged than reference sites. The higher litterfall observed in logged forests could be related to higher proportion of tree species that are characteristic of primary and secondary successional stages, including fast-growing and liana species, which often are more productive and common in anthropogenic areas. Our results showed that the legacy impact of clear-cut logging, even if residual woody vegetation is maintained in riparian buffers, can shift the type, quantity, and seasonality of litter subsidies to tropical streams. This knowledge should be considered within the context of management and conservation of communities and ecosystem processes in the forest-stream interfaces.