Pamela Cameron

Inhibition of lipopolysaccharide-induced macrophage IL-12 production by Leishmania mexicana amastigotes: the role of cysteine peptidases and the NF-kappaB signaling pathway.

Infection with lesion-derived Leishmania mexicana amastigotes inhibited LPS-induced IL-12 production by mouse bone marrow-derived macrophages. This effect was associated with expression of cysteine peptidase B (CPB) because amastigotes of CPB deletion mutants had limited ability to inhibit IL-12 production, whereas preincubation of cells with a CPB inhibitor, cathepsin inhibitor IV, was able to suppress the effect of wild-type amastigotes. Infection with wild-type amastigotes resulted in a time-dependent proteolytic degradation of IκBα and IκBβ and the related protein NF-κB. This effect did not occur with amastigotes of CPB deletion mutants or wild-type promastigotes, which do not express detectable CPB. NF-κB DNA binding was also inhibited by amastigote infection, although nuclear translocation of cleaved fragments of p65 NF-κB was still observed. Cysteine peptidase inhibitors prevented IκBα, IκBβ, and NF-κB degradation induced by amastigotes, and recombinant CPB2.8, an amastigote-specific isoenzyme of CPB, was shown to degrade GST-IκBα in vitro. LPS-mediated IκBα and IκBβ degradation was not affected by these inhibitors, confirming that the site of degradation of IκBα, IκBβ, and NF-κB by the amastigotes was not receptor-driven, proteosomal-mediated cleavage. Infection of bone marrow macrophages with amastigotes resulted in cleavage of JNK and ERK, but not p38 MAPK, whereas preincubation with a cysteine peptidase inhibitor prevented degradation of these proteins, but did not result in enhanced protein kinase activation. Collectively, our results suggest that the amastigote-specific cysteine peptidases of L. mexicana are central to the ability of the parasite to modulate signaling via NF-κB and consequently inhibit IL-12 production.

In Vitro Susceptibilities of Shigella flexneri and Streptococcus pyogenes to Inner Gel of Aloe barbadensis Miller

ABSTRACT Aloe barbadensis Miller (or Aloe vera) has widespread use in health products, and despite numerous reports on the whole plant, little work has been performed on the inner gel, which has been used extensively in these products. This report describes the in vitro susceptibilities of two bacteria to this component.

The role of intracellular Ca2+ in the regulation of proteinase- activated receptor-2 mediated nuclear factor kappa B signalling in keratinocytes

1 In this study, we examined the role of Ca2+ in linking proteinase‐activated receptor‐2 (PAR2) to the nuclear factor kappa B (NFκB) pathway in a skin epithelial cell line NCTC2544 stably expressing PAR2 (clone G). 2 In clone G, PAR2‐mediated NFκB luciferase reporter activity and NFκB DNA‐binding activity was reduced by preincubation with BAPTA‐AM but not BAPTA. Trypsin stimulation of inhibitory kappa B kinases, IKKα and IKKβ, was also inhibited following pretreatment with BAPTA‐AM. 3 BAPTA/AM also prevented PAR2‐mediated IKKα activation in cultured primary human keratinocytes. 4 The effect of BAPTA‐AM was also selective for the IKK/NFκB signalling axis; PAR2 coupling to ERK, or p38 MAP kinase was unaffected. 5 Pharmacological inhibition of the Ca2+‐dependent regulatory protein calcineurin did not inhibit trypsin‐stimulated IKK activity or NFκB‐DNA binding; however, inhibition of Ca2+‐dependent protein kinase C isoforms or InsP3 formation using GF109203X or the phospholipase C inhibitor U73122, respectively, reduced both IKK activity and NFκB‐DNA binding. 6 Mutation of PAR2 within the C‐terminal to produce a mutant receptor, which does not couple to Ca2+ signalling, but is able to activate ERK, abrogated NFκB‐DNA binding and IKK activity stimulated by trypsin. 7 These results suggest a predominant role for the InsP3/Ca2+ axis in the regulation of IKK signalling and NFκB transcriptional activation.

Verotoxin activates mitogen-activated protein kinase in human peripheral blood monocytes: role in apoptosis and proinflammatory cytokine release

In this study, we examined the role of mitogen‐activated protein (MAP) kinases in the effects of verotoxins (VTs), from Escherichia coli O157:H7, upon both apoptosis and the release of tumour necrosis factor alpha (TNF‐α) and granulocyte–macrophage colony‐stimulated factor (GM‐CSF) from human monocytes. Both VT1 and VT2 stimulated a weak, transient increase in c‐Jun‐N‐terminal kinase (JNK) activity and a strong activation of both p38 mitogen‐activated protein kinase (MAP kinase) and extracellular‐regulated kinase (ERK) activity in human monocytes, which was sustained in the case of p38 MAP kinase. Stimulation of human monocytes with VT2 (100 ng ml−1) did not result in an increase in apoptosis; however, the toxin stimulated the release of both TNF‐α and GM‐CSF. Pretreatment of human monocytes with the p38 MAP kinase inhibitor SB203580, at concentrations from 100 nM to 10 μM, significantly decreased the VT1‐ and VT2‐induced TNF‐α and GM‐CSF release from monocytes. In contrast, inhibition of MEK1 with PD98059 only significantly decreased GM‐CSF release. Pretreatment of monocytes with SP600125 inhibited both GM‐CSF and TNF‐α production; however, significant effects upon p38 MAP kinase and ERK activation were observed. Taken together, these results suggest a role for p38 MAP kinase and ERK in cytokine generation in response to the verotoxins. A role for JNK remains undetermined.

Essential role for verotoxin in sustained stress-activated protein kinase and nuclear factor kappa b signaling, stimulated by escherichia coli o157:H7 in vero cells

ABSTRACT The effects of Escherichia coli O157:H7 (strains E30480 and PM601) and the associated verotoxins (VTs), VT1 and VT2, on stress-activated protein kinase and nuclear factor kappa B (NF-κB) signaling were investigated with Vero cells, which are extremely sensitive to the cytotoxic effects of E. coli O157:H7 in vitro. Cell-free supernatants prepared from E30480 and PM601 cultures and purified VT1 and VT2 stimulated a strong and prolonged (>4-h) activation of both c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase. However, JNK activity stimulated in response to E30480 supernatants was substantially reduced following pretreatment with anti-VT1 and anti-VT2 antibodies, while a VT1 and VT2 gene knockout mutant of PM601 was unable to stimulate JNK activity. E30480 supernatants also caused a sustained activation of NF-κB DNA binding, degradation of inhibitory kappa B alpha (IκBα), and an increase in inhibitory kappa B kinase α activity, although PM601 supernatants and VT1 and VT2 had no effect. However, preincubation with VTs prolonged the transient activation of NF-κB and IκBα degradation stimulated by either tumor necrosis factor alpha or interleukin 1β, while preincubation with anti-VT antibodies prevented the prolonged loss of IκBα and partially reduced DNA binding in response to E30480 supernatants. These results strongly suggest that in Vero cells, VT plays an essential role in sustained JNK and NF-κB signaling in response to E. coli O157:H7 and that this action may underpin their cell-selective cytotoxic effects. These studies also suggest that another component released by strain E30480 contributes to the early activation of JNK and NF-κB.