Our results showed alteration of the phenotypic profile of B cells isolated after animal treatment with different protease-resistant proteoforms. Only one of the mutants presented increased serum half-life, so resistance to these proteases is not the only feature involved in EcAII stability in vivo. The variants with enzymatic activity and cytotoxicity levels equivalent to or better than EcAII WT were submitted to in vivo assays. Error-prone polymerase chain reaction was used to produce variants of EcAII more resistant to proteolytic cleavage by AEP and CTSB. In this work, we show that ASNase resistance to CTSB and/or AEP influences the formation of anti-ASNase antibodies, one of the main causes of hypersensitivity reactions in patients. EcAII activity in vivo has been described to be influenced by the human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB) these hydrolases cleave and could expose epitopes associated with the immune response against EcAII. L-asparaginase (ASNase) from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukaemia (ALL). Although HNBT vapor does not activate T cells as CS does, exposure to both HNBT and CS suppressed proliferation and IL-2 release, a pivotal cytokine involved with T cell proliferation and tolerance, and this effect may be related to nicotine content in both products. CS and HNBT exposures decreased PMA-induced interleukin-2 (IL-2) secretion and impaired Jurkat proliferation, effects also seen in cells exposed to nicotine. While both CS and HBNT exposures increased cell death, CS led to a higher number of necrotic cells, increased the production of ROS, NO, inflammatory cytokines and MTs when compared to HNBT-exposed cells, and led to a higher expression of MTs in mice. MT expressions were quantified by immunohistochemistry in the lungs and liver of C57Bl/6 mice exposed to CS, HNBT or air (1 h, twice a day for five days: via inhalation). Levels of metals in the exposure chambers were quantified by inductively coupled plasma mass spectrometry.
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Cell viability, proliferation, reactive oxygen species (ROS) production, 8-OHdG, MAP-kinases and nuclear factor κB (NFκB) activation and metallothionein expression (MTs) were assessed by flow cytometry nitric oxide (NO) and cytokine levels were measured by Griess reaction and ELISA, respectively. Cells were exposed to air, conventional cigarettes or heatsticks of HNBT for 30 min and were stimulated or not with phorbol myristate acetate (PMA). As heat-not-burn tobacco products (HNBT) vaporize lower levels of combustible products, we here compared the effects of cigarette smoke (CS) and HNBT vapor on Jurkat T cells. Robust evidence addresses the immunotoxic effects of combustible tobacco products. Cigarette smoke (CS) affects immune functions, leading to severe outcomes in smokers.