Rapid apoptosis induced by Shiga toxin in HeLa cells

Apoptosis was induced rapidly in HeLa cells after exposure to bacterial Shiga toxin (Stx1 and Stx2; 10 ng/ml). Approximately 60% of HeLa cells became apoptotic within 4 h as detected by DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and electron microscopy. Stx1-induced apoptosis required enzymatic activity of the Stx1A subunit, and apoptosis was not induced by the Stx2B subunit alone or by the anti-globotriaosylceramide antibody. This activity was also inhibited by brefeldin A, indicating the need for toxin processing through the Golgi apparatus. The intracellular pathway leading to apoptosis was further defined. Exposure of HeLa cells to Stx1 activated caspases 3, 6, 8, and 9, as measured both by an enzymatic assay with synthetic substrates and by detection of proteolytically activated forms of these caspases by Western immunoblotting. Preincubation of HeLa cells with substrate inhibitors of caspases 3, 6, and 8 protected the cells against Stx1-dependent apoptosis. These results led to a more detailed examination of the mitochondrial pathway of apoptosis. Apoptosis induced by Stx1 was accompanied by damage to mitochondrial membranes, measured as a reduced mitochondrial membrane potential, and increased release of cytochrome c from mitochondria at 3 to 4 h. Bid, an endogenous protein known to permeabilize mitochondrial membranes, was activated in a Stx1-dependent manner. Caspase-8 is known to activate Bid, and a specific inhibitor of caspase-8 prevented the mitochondrial damage. Although these data suggested that caspase-8-mediated cleavage of Bid with release of cytochrome c from mitochondria and activation of caspase-9 were responsible for the apoptosis, preincubation of HeLa cells with a specific inhibitor of caspase-9 did not protect against apoptosis. These results were explained by the discovery of a simultaneous Stx1-dependent increase in endogenous XIAP, a direct inhibitor of caspase-9. We conclude that the primary pathway of Stx1-induced apoptosis and DNA fragmentation in HeLa cells is unique and includes caspases 8, 6, and 3 but is independent of events in the mitochondrial pathway.     

Protection of monkeys against Shiga toxin induced by Shiga toxin-liposome conjugates

BACKGROUND: We previously reported that the purified Shiga toxins (Stx) Stx1 and Stx2, when coupled with liposomes, induced substantial production of anti-Stx1 and anti-Stx2 IgG antibody, respectively, in mice. The levels of anti-Stx antibody in the sera of mice immune to Stx-liposome correlated well with the protection against subsequent challenge with Stx. Furthermore, mice immunized with a mixture of Stx1-liposome and Stx2-liposome were successfully protected against oral infection with cytotoxin-producing Escherichia coli O157:H7. METHODS: In this study, the induction of protection against Stx2 by Stx2-liposomes was evaluated in monkeys. RESULTS: Stx2-liposomes induced a substantial amount of anti-Stx2 IgG antibodies as well as Stx2 neutralizing antibodies in monkeys. Test monkeys were successfully protected against challenge with lethal doses of Stx2. Moreover, these monkeys showed no apparent symptoms, while nonimmunized control monkeys died within 4 days with hemorrhagic gastroenteritis and renal disorder. In addition, as shown by other cases involving antigen-liposome conjugates, Stx2-liposome did not induce anti-Stx2 IgE antibody production, though it stimulated the production of a substantial amount of anti-Stx2 IgG antibodies. CONCLUSION: These results suggest that Stx-liposome conjugates may serve as candidate vaccines to induce protection against death caused by cytotoxin-producing E. coli infection.     

Escherichia coli strains producing a novel Shiga toxin 2 subtype circulate in China

Shiga toxin (Stx) is the key virulence factor in Shiga toxin producing Escherichia coli (STEC), which can cause diarrhea and hemorrhagic colitis with life-threatening complications. Stx comprises two toxin types, Stx1 and Stx2. Several Stx1/Stx2 subtypes have been identified in E. coli, which are variable in sequences, toxicity and host specificity. Here, we report the identification of a novel Stx2 subtype, designated Stx2k, in E. coli strains widely detected from diarrheal patients, animals, and raw meats in China over time. Stx2k exhibits varied cytotoxicity in vitro among individual strains. The Stx2k converting prophages displayed considerable heterogeneity in terms of insertion site, genetic content and structure. Whole genome analysis revealed that the stx_2k-containing strains were genetically heterogeneous with diverse serotypes, sequence types, and virulence gene profiles. The nine stx_2k-containing strains formed two major phylogenetic clusters closely with strains belonging to STEC, enterotoxigenic E. coli (ETEC), and STEC/ETEC hybrid. One stx_2k-containing strain harbored one plasmid-encoded heat-stable enterotoxin sta gene and two identical copies of chromosome-encoded stb gene, exhibiting STEC/ETEC hybrid pathotype. Our finding enlarges the pool of Stx2 subtypes and highlights the extraordinary genomic plasticity of STEC strains. Given the wide distribution of the Stx2k-producing strains in diverse sources and their pathogenic potential, Stx2k should be taken into account in epidemiological surveillance of STEC infections and clinical diagnosis.     

Characterization of CTX‐M‐producing Escherichia coli by repetitive sequence‐based PCR and real‐time PCR‐based replicon typing of CTX‐M‐15 plasmids

The emergence of extended‐spectrum β‐lactamase (ESBL)‐producing Enterobacteriaceae is a major global concern. CTX‐M is the dominating ESBL type worldwide, and CTX‐M‐15 is the most widespread CTX‐M type. The dissemination of CTX‐M appears to be in part due to global spread of the Escherichia coli clone O25b‐ST131. However, the gene‐encoding CTX‐M is mainly located on mobile genetic elements, such as plasmids, that also promote the horizontal dissemination of the CTX‐M genes. In this study, 152 CTX‐M‐producing E. coli isolated in 1999–2008 in Örebro County, Sweden, were typed using a commercial repetitive sequence‐based PCR (the DiversiLab system), and the prevalence of ST131 was investigated by pabB PCR. Real‐time PCR‐based plasmid replicon typing was performed on 82 CTX‐M‐15‐producing E. coli isolates. In general, the CTX‐M‐producing E. coli population was genetically diverse; however, ST131 was highly prevalent (27%), and the dominating clone in our area. The bla_CTX‐M‐15 gene was mainly located on IncF plasmids (69%), but a relatively high proportion of IncI1 plasmids (29%) were also detected among E. coli with diverse rep‐PCR patterns, indicating that horizontal transmission of IncI1 plasmids carrying bla_CTX‐M‐15 may have occurred between different E. coli strains.     

Shiga toxin 1 induces apoptosis in the human myelogenous leukemia cell line THP-1 by a caspase-8-dependent, tumor necrosis factor receptor-independent mechanism

Shiga toxins (Stxs) induce apoptosis in a variety of cell types. Here, we show that Stx1 induces apoptosis in the undifferentiated myelogenous leukemia cell line THP-1 in the absence of tumor necrosis factor alpha (TNF-alpha) or death receptor (TNF receptor or Fas) expression. Caspase-8 and -3 inhibitors blocked, and caspase-6 and -9 inhibitors partially blocked, Stx1-induced apoptosis. Stx1 induced the mitochondrial pathway of apoptosis, as activation of caspase-8 triggered the (i) cleavage of Bid, (ii) disruption of mitochondrial membrane potential, and (iii) release of cytochrome c into the cytoplasm. Caspase-8, -9, and -3 cleavage and functional activities began 4 h after toxin exposure and peaked after 8 h of treatment. Caspase-6 may also contribute to Stx1-induced apoptosis by directly acting on caspase-8. It appears that functional Stx1 holotoxins must be transported to the endoplasmic reticulum to initiate apoptotic signaling through the ribotoxic stress response. These data suggest that Stxs may activate monocyte apoptosis via a novel caspase-8-dependent, death receptor-independent mechanism.     

Shiga toxin-producing Escherichia coli-associated kidney failure in a 40-year-old patient and late diagnosis by novel bacteriologic and toxin detection methods

Infection with Shiga toxin-producing Escherichia coli (STEC) is the most common cause of kidney failure in children. High morbidity is also associated with infections in the elderly. We describe STEC-associated kidney failure in a 40-year-old patient, including the methods used to identify STEC a month after disease onset.     

Cdkn2a suppresses metastasis in squamous cell carcinomas induced by the gain‐of‐function mutant p53R172H

p53 (TP53) is the most frequently mutated gene in squamous cell carcinomas (SCCs) of the skin and head and neck. Certain p53 mutations are oncogenic and promote invasion and metastasis in SCCs. However, it is unclear how the oncogenic function of mutant p53 is modulated by other molecular alterations that co‐exist in SCCs. Here, we show that deletion of the p53 gene and activation of an endogenous p53^R172H gain‐of‐function mutation in the skin induce carcinomas with similar kinetics and penetrance. Deletion of p53 induced primarily well‐differentiated SCCs. However, most of the tumours induced by p53^R172H were poorly differentiated SCCs, the only metastatic tumours in this model. These tumours expressed higher levels of cyclin D1 than the well‐differentiated SCCs and spindle carcinomas that developed in these mice. Unexpectedly, metastasis was not observed in mice that developed spindle carcinomas, which expressed high levels of the tumour suppressors p16^Ink4a and p19^Arf, encoded by Cdkn2a, a gene frequently deleted in human SCCs. Remarkably, deletion of the Cdkn2a gene in p53^R172H‐induced SCCs promoted a dramatic increase in metastasis rates and a shorter survival in mice that developed these tumours, compared with those observed in mice with tumours in which Cdkn2a was deleted in the presence of a p53 loss‐of‐function mutation or wild‐type p53. Accordingly, the survival of patients with head and neck SCCs bearing co‐occurring high‐risk p53 mutations and CDKN2A homozygous deletions was much shorter than that of patients with tumours in which high‐risk p53 mutations did not contain CDKN2A homozygous deletions, or that of patients with tumours in which homozygous CDKN2A deletions co‐existed with either low‐risk p53 mutations or potential loss‐of‐function mutations in p53. These findings genetically identify a population of SCC patients with worst outcomes and will help to predict outcomes according to the p53 status and alterations in CDKN2A. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Purvalanol A,a CDK inhibitor,effectively suppresses Src‐mediated transformation by inhibiting both CDKs and c‐Src

The nonreceptor tyrosine kinase c‐Src is frequently over‐expressed or hyperactivated in various human cancers and contributes to cancer progression in cooperation with up‐regulated growth factor receptors. However, Src‐selective anticancer drugs are still in clinical trials. To identify more effective inhibitors of c‐Src‐mediated cancer progression, we developed a new screening platform using Csk‐deficient cells that can be transformed by c‐Src. We found that purvalanol A, developed as a CDK inhibitor, potently suppressed the anchorage‐independent growth of c‐Src‐transformed cells, indicating that the activation of CDKs contributes to the c‐Src transformation. We also found that purvalanol A suppressed the c‐Src activity as effectively as the Src‐selective inhibitor PP2, and that it reverted the transformed morphology to a nearly normal shape with less cytotoxicity than PP2. Purvalanol A induced a strong G2‐M arrest, whereas PP2 weakly acted on the G1‐S transition. Furthermore, when compared with PP2, purvalanol A more effectively suppressed the growth of human colon cancer HT29 and SW480 cells, in which Src family kinases and CDKs are activated. These findings demonstrate that the coordinated inhibition of cell cycle progression and tyrosine kinase signaling by the multi‐selective purvalanol A is effective in suppressing cancer progression associated with c‐Src up‐regulation.     

Shiga toxin 2a binds antithrombin and heparin,but does not directly activate platelets

Escherichia coli-induced hemolytic uremic syndrome (eHUS) is a life-threatening complication of infection with Shiga toxin (Stx), in particular Stx2a-producing Escherichia coli. Enhanced coagulation activation with formation of microthrombi seems to be a key event in development of eHUS. Platelet activation has been postulated as a possible, but controversially debated mechanism.The present study investigated the effect of Stx2a on plasmatic coagulation and platelets. Binding studies were initially performed with ELISA and co-immunoprecipitation and supported by quartz crystal microbalance with dissipation monitoring (QCM-D). Antithrombin (AT) activity was measured using the automated BCS XP^® system. ROTEM^® was used for functional coagulation testing. Platelet binding and activation was studied with FACS and light-transmission aggregometry.We found binding of Stx2a to AT, an important inhibitor of blood coagulation, but only a mild albeit significant reduction of AT activity against FXa in the presence of Stx2a. QCM-D analysis also showed binding of Stx2a to heparin and an impaired binding of AT to Stx2a-bound heparin. ROTEM^® using Stx2a-treated platelet-poor plasma revealed a significant, but only moderate shortening of clotting time. Neither binding nor activation of platelets by Stx2a could be demonstrated.In summary, data of this study suggest that Stx2a binds to AT, but does not induce major effects on plasmatic coagulation. In addition, no interaction with platelets occurred. The well-known non-beneficial administration of heparin in eHUS patients could be explained by the interaction of Stx2a with heparin.     

The oxidative stress induced in vivo by Shiga toxin‐2 contributes to the pathogenicity of haemolytic uraemic syndrome

Typical haemolytic uraemic syndrome (HUS) is caused by Shiga toxin (Stx)‐producing Escherichia coli infections and is characterized by thrombotic microangiopathy that leads to haemolytic anaemia, thrombocytopenia and acute renal failure. Renal or neurological sequelae are consequences of irreversible tissue damage during the acute phase. Stx toxicity and the acute inflammatory response raised by the host determine the development of HUS. At present there is no specific therapy to control Stx damage. The pathogenic role of reactive oxygen species (ROS) on endothelial injury has been largely documented. In this study, we investigated the in‐vivo effects of Stx on the oxidative balance and its contribution to the development of HUS in mice. In addition, we analysed the effect of anti‐oxidant agents as therapeutic tools to counteract Stx toxicity. We demonstrated that Stx induced an oxidative imbalance, evidenced by renal glutathione depletion and increased lipid membrane peroxidation. The increased ROS production by neutrophils may be one of the major sources of oxidative stress during Stx intoxication. All these parameters were ameliorated by anti‐oxidants reducing platelet activation, renal damage and increasing survival. To conclude, Stx generates a pro‐oxidative state that contributes to kidney failure, and exogenous anti‐oxidants could be beneficial to counteract this pathogenic pathway.     

IL‐4 blocks M‐CSF‐dependent macrophage proliferation by inducing p21Waf1 in a STAT6‐dependent way

Macrophages are recruited from the blood stream to the inflammatory loci to carry out their functional activities. In an early phase of the cell cycle, macrophages become activated by Th1‐type cytokines (i.e. IFN‐γ), thereby producing several factors (cytokines, NO, etc.) and developing pro‐inflammatory activities. When bacteria and apoptotic bodies are removed, through the interaction with Th2‐type cytokines (i.e. IL‐4), macrophages become anti‐inflammatory and repair damaged tissues. Incubation of bone‐marrow‐derived macrophages with IFN‐γ or IL‐4 blocked their proliferation. While M‐CSF withdrawal caused cell cycle arrest at the early G₁ phase, treatment of macrophages with IFN‐γ or IL‐4 caused this arrest later, at the G₁/S boundary. Proliferation arrest was not due to an induction of apoptosis. IFN‐γ and IL‐4 induced the expression of the cyclin‐dependent kinase (Cdk) inhibitor p21^Waf1. Using KO mice and iRNA experiments, we found that p21^Waf1is required for IL‐4‐ but not for IFN‐γ‐dependent inhibition of macrophage proliferation. IL‐4 inhibited M‐CSF‐dependent Cdk‐2 and Cdk‐4 activities, which are necessary for entry and passage through the S phase of the cell cycle. The signal transduction used to induce the expression of p21^Waf1after interaction of IL‐4 with the corresponding receptor was mediated by STAT6. Thus, IL‐4 and IFN‐γ blocked M‐CSF‐induced macrophage proliferation through distinct mechanisms.     

Effect of α‐trinositol on secretion induced by Escherichia coli ST‐toxin in rat jejunum

Aim: d ‐myo‐inositol‐1,2,6‐trisphosphate (α‐trinositol, PP56), is a synthetic isomer of the intracellular second messenger, d ‐myo‐inositol‐1,4,5‐trisphospahate. The pharmacological actions of α‐trinositol include potent anti‐inflammatory properties and inhibition of the secretion induced by cholera toxin and obstructive ileus. In the present study, we investigated whether α‐trinositol was able to influence the secretion induced by heat‐stable ST‐toxin from Escherichia coli in the rat jejunum. Methods: A midline abdominal incision was performed in anaesthetized male Sprague–Dawley rats and a 6–7 cm long jejunal segment was isolated with intact vascular supply and placed in a chamber suspended from a force displacement transducer connected to a Grass^® polygraph. Intestinal net fluid transport was continuously monitored gravimetrically. Crystalline ST‐toxin (120 mouse units) was introduced into the intestinal lumen and left there for the rest of the experiment. When a stable secretion was observed, α‐trinositol (60 mg kg^?1 h^?1) or saline were infused during 2 h, followed by a 2‐h control period. Results: α ‐Trinositol induced a significant (P < 0.001) inhibition of ST‐toxin secretion within 30 min, lasting until 2 h after infusion had stopped. The agent also moderately increased (P < 0.05) net fluid absorption in normal jejunum. Mean arterial pressure (P < 0.001) and heart rate (P < 0.001) were reduced by α‐trinositol. Conclusion: The inhibition by α‐trinositol of ST‐toxin induced intestinal secretion is primarily secondary to inhibition of secretory mechanisms and only to lesser extent due to increased absorption. The detailed mechanisms of action have not been clarified but may involve suppression of inflammation possibly by means of cellular signal transduction.     

Autoantibodies against stress‐induced phosphoprotein‐1 as a novel biomarker candidate for ovarian cancer

Detection of autoantibodies against tumor‐associated antigens (TAA) has recently been shown to be a powerful tool for early detection of various cancers. The aim of this study was to investigate the possibility of using autoantibodies against TAA as novel biomarkers by a proteomics‐based approach in patients with ovarian cancer. We used two‐dimensional differential gel electrophoresis analysis of immuno‐precipitated tumor antigens (2D‐DITA) to compare the levels of autoandibodies in pretreatment and posttreatment sera of patients with ovarian cancers. The identified autoantibodies were validated by SYBR Green real‐time polymerase chain reaction (PCR) and immunohistochemistry (IHC). We further evaluated the level of autoantibody in sera of 68 ovarian cancer patients by an enzyme‐linked immunosorbent assay (ELISA). The autoantibody directed against stress‐induced phosphoprotein‐1 (STIP‐1) emerged as a novel biomarker candidate for ovarian cancer. SYBR Green PCR and IHC confirmed that the STIP‐1 mRNA and protein expression levels were significantly up‐regulated in ovarian cancers compared with normal and benign tumors (P = 0.003 and P < 0.001, respectively). A preliminary ELISA study showed that the serum levels of anti‐STIP‐1 autoantibodies were significantly elevated in ovarian cancer patients compared with healthy controls (P = 0.03). The results suggest that 2D‐DITA is a useful tool to detect autoantibodies and that STIP‐1 is a potential biomarker candidate for ovarian cancers. © 2010 Wiley‐Liss, Inc.     

Autophagy plays a protective role as an anti‐oxidant system in human T cells and represents a novel strategy for induction of T‐cell apoptosis

Autophagy is an intracellular degradation system that plays an important role in T‐cell survival. However, the precise mechanism linking autophagy and cell death in primary human T cells is unclear because methods for monitoring autophagy in small numbers of primary human cells remain controversial. We established a novel method for assessing autophagy in activated human T cells using a retroviral GFP–LC3 expression system. We found that autophagy was induced after TCR stimulation and that autophagy‐defective naïve CD4⁺ T cells were susceptible to apoptosis through the intrinsic apoptotic pathway. Enhanced apoptosis of autophagy‐defective T cells resulted from accumulation of ROS due to impaired mitophagy. We also demonstrated that effector memory CD4⁺ T cells had lower autophagic activity than naïve CD4⁺ T cells, which contributed to their enhanced apoptosis due to increased ROS. Moreover, blocking autophagy increased intracellular mitochondrial volume and ROS levels in activated T cells. These results suggest a protective role of autophagy as an anti‐oxidant system in activated human T cells. The combination of an autophagy blocker and a mitochondrial electron transport chain inhibitor has a synergistic effect on T‐cell death, which could be a novel strategy for induction of T‐cell apoptosis.     

FCRL3 promotes TLR9‐induced B‐cell activation and suppresses plasma cell differentiation

Fc receptor‐like (FCRL) molecules are preferentially expressed by B lymphocytes and possess tyrosine‐based immunoregulatory function. Although they generally inhibit B‐cell receptor signaling, their influence on other activation pathways remains largely unexplored. In humans, FCRL3 encodes a type I transmembrane protein harboring both cytoplasmic ITAM and ITIM elements that can repress B‐cell receptor activation. Despite this inhibitory property, mounting associations for FCRL3 with autoimmune and lympho‐proliferative disorders imply a role for it in promoting B‐cell pathogenesis. Here, we explore the influence of FCRL3 on B‐cell responses to innate TLR9 stimulation. A detailed survey of blood B‐cell populations found that FCRL3 expression increased as a function of differentiation and was higher among memory subsets with innate‐like features. FCRL3 ligation augmented CpG oligodeoxynucleotide TLR9‐mediated B‐cell proliferation, activation, and survival, but surprisingly, abrogated plasma cell differentiation and antibody production. Although FCRL3 amplified the NF‐κB and mitogen‐activated protein kinase signaling cascades, it halted CpG triggered BLIMP1 induction in an ERK‐dependent fashion. These findings indicate that FCRL3 differentially modulates innate signaling in B cells and provide new insight into the potential of this disease‐associated receptor to counter‐regulate adaptive and innate immunity.     

IL‐34‐ and M‐CSF‐induced macrophages switch memory T cells into Th17 cells via membrane IL‐1α

Macrophages orchestrate the immune response via the polarization of CD4⁺ T helper (Th) cells. Different subsets of macrophages with distinct phenotypes, and sometimes opposite functions, have been described. M‐CSF and IL‐34 induce the differentiation of monocytes into IL‐10^high IL‐12^low immunoregulatory macrophages, which are similar to tumor‐associated macrophages (TAMs) in ovarian cancer. In this study, we evaluated the capacity of human macrophages induced in the presence of M‐CSF (M‐CSF macrophages) or IL‐34 (IL‐34 macrophages) and ovarian cancer TAMs to modulate the phenotype of human CD4⁺ T cells. Taken together, our results show that M‐CSF‐, IL‐34 macrophages, and TAMs switch non‐Th17 committed memory CD4⁺ T cells into conventional CCR4⁺ CCR6⁺ CD161⁺ Th17 cells, expressing or not IFN‐gamma. Contrary, the pro‐inflammatory GM‐CSF macrophages promote Th1 cells. The polarization of memory T cells into Th17 cells is mediated via membrane IL‐1α (mIL‐1α), which is constitutively expressed by M‐CSF‐, IL‐34 macrophages, and TAMs. This study elucidates a new mechanism that allows macrophages to maintain locally restrained and smoldering inflammation, which is required in angiogenesis and metastasis.