Pseudomonas Aeruginosa Exotoxin A Research Articles

Pseudomonas aeruginosa exotoxin A as a novel allergen induced Non-TH2 inflammation in a murine model of steroid-insensitive asthma

BackgroundDespite the immediate in vivo occurrence of anaphylactic and allergic reactions following treatment with Pseudomonas aeruginosa exotoxin A (PEA)-based immunotoxins, the immunological role of PEA in asthma pathogenesis remains unclear. ObjectiveThis study investigated the allergenic potential of PEA and the specific type of asthma induced. MethodsRecombinant PEA (rPEA) lacking domain Ia (to eliminate non-specific cytotoxicity) was expressed, purified, and employed to detect serum PEA-specific IgE levels in asthmatic patients. Competitive ELISA assays were used to assess rPEA's IgE binding capacity and allergenicity. Additionally, rPEA-challenged C57BL/6 mice were subjected to inflammatory endotyping and therapeutic assays to characterize the allergic nature of PEA. ResultsPEA-specific IgE was identified in 17 (14.2 %) of 120 asthma patients. The rPEA-sensitized and challenged mice had increased PEA-specific immunoglobulins (such as IgE, IgG1 and IgG2a) and developed asthma-like phenotypes with airway hyperresponsiveness, severe airway inflammation, and airway remodeling. Lungs from these mice displayed significant increases in neutrophils and IL-17A+ cells. Innate lymphoid cells (ILCs) produced type 2 cytokines (IL-4, IL-5, and IL-13), whereas Th cells did not. Nonetheless, airway inflammation, rather than hyperresponsiveness, was elicited in non-sensitized mice upon challenge with rPEA. Importantly, rPEA-induced asthmatic mice were unresponsive to dexamethasone treatment. ConclusionPEA is a novel allergen that sensitizes asthmatic patients. Furthermore, mice developed steroid-resistant asthma, characterized by an atypical cytokine profile associated with non-TH2 inflammation, only after being sensitized and challenged with rPEA. These findings suggest a potentially significant role for PEA in asthma development, warranting consideration in clinical diagnosis and treatment strategies.

Characterization and applications of nanobodies against Pseudomonas aeruginosa Exotoxin A selected from single alpaca B cells

We have established a highly efficient 96-well format based strategy to characterize nanobody (Nb) from an expressed alpaca Nb repertoire by combining heavy-chain antibody variable region gene cloning with Nb expression and reactivity profiling at the single cell level. Individual alpaca B lymphocytes were isolated based on B cell surface marker and membrane type antigen specific antibodies expression by fluorescence activated cell sorting (FACS). Single cell reverse transcription and polymerase chain reaction (PCR) amplification of CH1 and CH2 domain enable the identification and exclusion of B cell clones that express regular antibodies. The corresponding heavy-chain antibody variable region gene transcripts, which are CH1-CH2 domain negative, are then amplified from single B cell clones by nested PCR. Then Nbs were expressed in Escherichia coli cells and four Nbs showed high affinity against Pseudomonas aeruginosa Exotoxin A (PEA). Moreover, two PEA Nbs that recognize unique epitopes on PEA have been successfully applied to protect human cells from PEA induced apoptosis. In summary, our FACS and single cell reverse transcription polymerase chain reaction (RT-PCR) based strategy for identifying recombinant Nbs from single alpaca B cells allows highly efficient and unbiased characterization of the expressed Nb repertoires by sequence analysis, expression and parallel antibody reactivity testing.

The immunotoxin activity of exotoxin A is sensitive to domain modifications

Immunotoxins are a class of recombinant proteins which consist of an antibody and a part of a bacterial or herbal toxin. Immunotoxins containing Pseudomonas aeruginosa exotoxin A (PEA) have been found to be very applicable in clinical trials. Many obstacles such as solubility and absorbency reduce their usability in solid tumors. The current study aims to overcome the mentioned barriers by addition and removal of functional and non-functional domains with a structural approach. In the experimental section, we took advantage of molecular dynamics simulations to predict the functionality of candidate immunotoxins which target human HER2 receptors and confirmed our findings with in vitro experiments. We found out when no changes were made to domain II of PEA, addition of solubilizing domains to immunotoxins would not reduce their targeting and anti-tumor activity, while increasing the yield of expression and stability. On the other side, when we replaced domain II with eleven amino acids of furin cleavage site (FCS), the activity of the immunotoxin was mainly affected by the FCS neighboring domains and linkers. A combination of seven beneficial point mutations in domain III was also assessed and reconfirmed that the toxicity of the immunotoxin would be reduced dramatically.The obtained results indicate that the addition or removal of domains cannot depict the activity of immunotoxins and the matter should be assessed structurally in advance.

Differences in Genetic Background Contribute to Pseudomonas Exotoxin A-Induced Hepatotoxicity in Rats

Pseudomonas aeruginosa exotoxin A (PEA) causes severe hepatotoxicity in experimental animals and is useful in investigations of immune-mediated liver injury. However, strain differences in the sensitivity to PEA-induced hepatotoxicity in rats remains be elucidated. In this study, we determined the severity of PEA-induced hepatotoxicity in six genetically different rat strains. Male LE (Long Evans), Wistar, F344, WKY, BN/SsN and LEW rats were administered a single intravenous injection of PEA (20 μg/kg). Significantly elevated serum ALT and AST levels, massive necrosis and hemorrhage, and numerous TUNEL-positive hepatocytes were observed in BN/SsN rats. In contrast, low levels of ALT and AST as well as mild changes in liver histopathology were observed in Wistar and F344 rats. Moderate levels of hepatic injuries were observed in LE, WKY, and LEW rats. Pro-inflammatory cytokines including TNF-α, IL-2 and IL-6 serum levels were markedly increased in BN/SsN rats compared to Wistar and F344 rats. However, the hepatic levels of low density lipoprotein receptor-related protein (LRP), which functions as the PEA receptor, were not significantly different in each strain. Taken together, we suggest that BN/SsN is the most sensitive rat strain, whereas Wistar and F344 were the most resistant rat strains to PEA-induced liver damage. The different genetic background of rat strains plays an important role in the susceptibility to PEA-induced epatotoxicity that may depend on immune-regulation but not LRP receptor levels.

Pseudomonas aeruginosa exotoxin A(PEA)가 사람혀 편평암종세포에서 나타나는 세포자멸사 작용

Objectives : The purpose of the study is to examine the apoptotic effects of Pseudomonas aeruginosa exotoxin A(PEA) in squamous cell carcinoma(SCC) 25 cells. Methods : Cell growth reduction and apoptosis induced by PEA were confirmed by WST-1 assay, Hoechst 33258 staining, flow cytometry analysis, and Western blot assay. Results : The PEA treatment decreased the cell viability in a dose and time dependent manner: control; <TEX>$100{\pm}0^e$</TEX>(p<0.01), 0.1875 nM; <TEX>$87{\pm}4.36^d$</TEX>(p<0.01), 0.375 nM; <TEX>$82{\pm}0.58^d$</TEX>(p<0.01), 0.75 nM; <TEX>$72{\pm}1.67^c$</TEX>(p<0.01), 1.5 nM; <TEX>$51{\pm}1.53^{bc}$</TEX>(p<0.01), 7.5 nM; <TEX>$31{\pm}1.20^{ab}$</TEX>(p<0.01), 15 nM; <TEX>$26{\pm}0.67^a$</TEX>(p<0.01), control; <TEX>$100{\pm}0^a$</TEX>(p<0.05), 24 h; <TEX>$51{\pm}1.53^b$</TEX>(p<0.05), 48 h; <TEX>$16{\pm}0.5^c$</TEX>(p<0.05), 72 h; <TEX>$12{\pm}1.67^d$</TEX>%(p<0.05). The PEA was observed on SCC 25 cells with the half maximal inhibitory concentration(IC50) value of 1.5 nM at 24 hours. The PEA treated SCC 25 cells demonstrated several types of apoptotic indications, such as nuclear condensation, the increase of sub G1, and the cleavage of PARP-1 and DFF 45. Conclusions : PEA showed anti-cancer activity against SCC 25 cells via apoptosis. PEA may potentially contribute to human oral cancer treatment.

Identification of Staphylococcus aureus α‐hemolysin as a protein drug that is secreted by anticancer bacteria and rapidly kills cancer cells

Targeted bacterial delivery of anticancer proteins has the ability to overcome therapeutic resistance in tumors that limits the efficacy of chemotherapeutics. The ability of bacteria to specifically target tumors allows for delivery of aggressive proteins that directly kill cancer cells and cannot be administered systemically. However, few proteins have been tested for this purpose. To identify effective molecules, we systematically sorted proteins that have been shown to cause mammalian cell death. The genes for five proteins were selected and cloned into Escherichia coli and Salmonella. Supernatant from cultures of the transformed bacteria was applied to flasks of MCF-7 mammary carcinoma cells to identify proteins that (1) were expressed, (2) secreted, and (3) rapidly killed cancer cells. Time-lapse images were taken to visualize mammalian cell morphology. Of the investigated proteins, α-hemolysin from Staphylococcus aureus (SAH) was the most promising because it was secreted, caused trauma to cellular membranes, and induced oncosis in 18 min. After exposure for 6 h, SAH decreased cell viability by 90%. In comparison, the positive control, Pseudomonas aeruginosa exotoxin A (PEA), required 11 days to achieve a similar effect, when administered at 3,000 times its LC50 . The maximum death rate induced by SAH was calculated to be a reduction in cell viability of 7.1% per min, which was 200-fold faster than the PEA control. Two proteins, Dermonecrotic Toxin and Phospholipase C were active when extracted from the bacterial cytoplasm but were not secreted. This investigation revealed for the first time SAH as a potent anticancer drug for delivery by bacteria because of its ability to be secreted in a fully functional form and aggressively kill cancer cells.

Pretreatment with lipopolysaccharide ameliorates Pseudomonas exotoxin A-induced hepatotoxicity in rats

Context: Liver injury can be induced by various hepatotoxicants, including Pseudomonas aeruginosa exotoxin A (PEA). Our previous study indicated that PEA-induced rat hepatotoxicity was T cells and Kupffer cells dependent. Several reports have demonstrated that non-toxic doses of bacterial lipopolysaccharide (LPS) can protect liver against the chemicals-induced toxicity such as acetaminophen and concanavalin-A.Objective: This study aimed to investigate the protecting mechanisms of LPS on PEA-induced hepatotoxicity.Results: Rats pretreated with LPS (40 μg/kg, 12 h before PEA admission) significantly decreased animal mortality, serum enzyme (ALT, AST and T-bil) activities, histopathological changes and hepatocytes apoptosis following challenge with PEA. The concentrations of tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ) and interleukin-2 (IL-2) were reduced, but IL-6 and IL-10 were increased in the serum. In addition, prior treatment of these LPS-pretreated rats with gadolinium chloride (GdCl3), a selective Kupffer cell depletion agent, markedly enhanced liver injury after PEA administration. In contrast, the pretreatment of LPS to T-cell deficient athymic nude rats still display significant attenuation of PEA-induced liver injury. This observation further confirmed our hypothesis that LPS ameliorate PEA-hepatotoxicity was through Kupffer cells but not T cells. Moreover, LPS-induced hepatoprotection ability was neutralized by co-treatment with anti-TNF-α antibodies, but not with anti-IFN-γ antibodies. Finally, replacement of LPS with RS-LPS (Rhodobacter sphaeroides LPS), a Toll like receptor-4 (TLR-4) antagonist, resulted in severe hepatotoxicity.Conclusion: These results suggested that Kupffer cells, TNF-α and TLR-4 play central mediator roles during the hepatoprotection against PEA-induced hepatotoxicity conferred by LPS.

Abstract 5658: Engineered Salmonella effectively produce and preferentially deliver bacterial cytotoxins to solid tumors

Abstract To effectively treat cancer, therapeutics must specifically target tumor tissue and impart minimal toxicity to healthy tissues. Bacterial toxins, such as Pseudomonas aeruginosa Exotoxin A (PEA) and Staphylococcus aureus Alpha-Hemolysin (SAH), are highly cytotoxic and are promising moieties for cancer treatment. Due to their high toxicity and non-specificity towards all cells types, systemic effects limit their effectiveness as a therapeutic. While efforts have been made to impart cancer-cell specificity to these potent molecules, especially through immunotoxin technology, systemic toxicities still prove to be a challenge. Genetically engineered bacteria, such as Salmonella typhimurium, are known to target cancer by preferential accumulation within tumors and thus can be exploited as tumor-specific drug delivery vectors. By using cellular engineering techniques, the genetic machinery of bacteria can be used to produce exogenous bacterial toxins in situ. In addition to the specificity of bacterial accumulation, inducible promoter systems such as the arabinose-inducible system can be used to minimize systemic toxin levels by controlling the timing of protein expression. By coupling these strategies, we have been able to develop an effective bacterial therapy for targeted tumor treatment. PEA and SAH expressed in Salmonella under the PBAD promoter was secreted into the culture medium following arabinose induction. Monolayer cultures of MCF7 human breast carcinoma cells treated with secreted PEA and SAH for three days were depleted by 82% and 99%, respectively. Mechanism of delivery studies in a murine tumor model showed co-localization of salmonella, toxins and cell death within tumors following arabinose induction, while minimizing the localization of toxins to the healthy organs. Survival studies indicate that bacterial delivery of PEA and SAH can minimize systemic toxicities while decreasing tumor growth and increasing survival. Overall, these data indicate that by using bacteria to produce toxins in situ, we were able to overcome the delivery challenges inherent with non-specific toxin moieties and create a highly efficacious drug delivery system for use in the treatment of solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5658. doi:1538-7445.AM2012-5658

Codon optimization enhances secretory expression of Pseudomonas aeruginosa exotoxin A in E. coli

Pseudomonas aeruginosa exotoxin A (PEA) is a number of family of bacterial ADP-ribosylating toxins and possesses strong immunogenicity. The detoxified exotoxin A, as a potent vaccine adjuvant and vaccine carrier protein, has been extensively used in human and animal vaccinations. However, the expression level of PEA gene in Escherichia coli is relative low which is likely due to the presence of rare codon and high levels of GC content. In order to enhance PEA gene expression, we optimized PEA gene using E. coli preferred codons and expressed it in E. coli BL21 (DE3) by using pET-20b(+) secretory expression vector. Our results showed that codon optimization significantly reduced GC content and enhanced PEA gene expression (70% increase compared with that of the wild-type). Moreover, the codon-optimized PEA possessed biological activity and had the similar toxic effects on mouse L292 cells compared with the wild-type PEA gene. Codon optimization will not only improve PEA gene expression but also benefit further modification of PEA gene using nucleotide-mediated site-directed mutagenesis. A large number of purified PEA proteins will provide the necessary conditions for further PEA functional research and application.

Pseudomonas aeruginosaExotoxin A Reduces Chemoresistance of Oral Squamous Carcinoma Cell via Inhibition of Heat Shock Proteins 70 (HSP70)

PurposeOral squamous carcinoma (OSCC) cells exhibit resistance to chemotherapeutic agent-mediated apoptosis in the late stage of malignancy. Increased levels of heat shock proteins 70 (HSP70) in cancer cells are known to confer resistance to apoptosis. Since recent advances in the understanding of bacterial toxins have produced new strategies for the treatment of cancers, we investigated the effect of Pseudomonas aeruginosa exotoxin A (PEA) on HSP70 expression and induction of apoptosis in chemoresistant OSCC cell line (YD-9).Materials and MethodsThe apoptotic effect of PEA on chemoresistant YD-9 cells was confirmed by MTT, Hoechst and TUNEL stains, DNA electrophoresis, and Western blot analysis.ResultsWhile YD-9 cells showed high resistance to chemotherapeutic agents such as etoposide and 5-fluorouraci (5-FU), HSP70 antisense oligonucelotides sensitized chemoresistant YD-9 cells to etoposide and 5-FU. On the other hand, PEA significantly decreased the viability of YD-9 cells by deteriorating the HSP70-relating protecting system through inhibition of HSP70 expression and inducing apoptosis in YD-9 cells. Apoptotic manifestations were evidenced by changes in nuclear morphology, generation of DNA fragmentation, and activation of caspases. While p53, p21, and E2F-1 were upregulated, cdk2 and cyclin B were downregulated by PEA treatment, suggesting that PEA caused cell cycle arrest at the G2/M checkpoint.ConclusionTherefore, these results indicate that PEA reduced the chemoresistance through inhibition of HSP70 expression and also induced apoptosis in chemoresistant YD-9 cells.

항암제에 저항성을 가지는 YD-9 human oral squamous carcinoma cell line에서 Pseudomonas aeruginosa exotoxin A의 p53 단백질 누적과 caspase를 활성화 경로를 통해 유도된 세포자멸사

구강편평상피암종은 말기에서 종종 화학치료요법제들이 유도하는 세포자멸사에 저항성을 보인다. 박테리아의 독에 대한 진전된 이해는 암치료에 대한 새로운 치료전략으로 제기되어지고 있다. 본 연구는 Pseudomonas aeruginosa exotoxin A (PEA)가 세포자멸사 기작을 통해 항암제에 저항성을 보이는 YD-9 구강편평상피암종의 생존율을 현격하게 떨어뜨림을 설명하고 있다. 세포자멸사현상은 핵의 형태학적 변화와 DNA 분절 생성을 통해 입증되었다. PEA는 caspase-3, -6, -9 의 분절과 활성화를 일으켰다. 그리고 이러한 반응들은 caspase 의 기질에 해당하는 poly (ADP-ribose) polymerase (PARP), DFF45, 그리고 lamin A 의 단백질 분해를 야기했다. 사립체 막전위 감소, cytochrome c와 Smac/DlABLO의 사립체로부터 세포질로의 유리, 그리고 AIF의 사립체에서 핵으로 이동 등이 관찰되었다. p53, p21 그리고 <TEX>$14-3-3{\gamma}$</TEX>는 증가되는 반면 cyclin B와 cdc2는 감소되었다. 이상의 결과들을 종합해 보면 PEA는 caspase를 활성화시키고, 사립체에 변화를 야기시키고 더 나아가서 세포주기 유전자를 조절함으로써 항암제에 대한 강한 저항성을 보이는 YD-9 세포에서 세포자멸사를 유도한다. Oral squamous carcinoma (OSC) cells present resistance to chemotherapeutic agents-mediated apoptosis in the late stages of malignancy. Advances in the understanding of bacterial toxins have produced new strategies for the treatment of cancers. It was demonstrated here that Pseudomonas aeruginosa exotoxin A (PEA) significantly decreased the viability of chemoresistant YD-9 cells in the apoptosis mechanism. Apoptotic manifestations were evident through changes in nuclear morphology and generation of DNA fragmentation. PEA treatment induced caspase-3, -6 and -9 cleavage, and activation. These events preceded proteolysis of the caspase substrates poly (ADP-ribose) polymerase (PARP), DNA fragmentation factor 45 (DFF45), and lamin A in YD-9 cells. The reduction of mitochondrial membrane potential, release of cytochrome c and SmacjDlABLO from mitochondria to cytosol, andtranslocation of AlF into nucleus were shown. While p53, p21 and <TEX>$14-3-3{\gamma}$</TEX> were upregulated, cyclin Band cdc2 were downregulated by PEA treatment. Taken together, PEA induces apoptosis in chemoresistant YD-9 cells via activation of caspases, mitochondrial events and regulation of cell cycle genes.

Co-exposure of lipopolysaccharide and pseudomonas aeruginosa exotoxin A-induced multiple organ injury in rats

Pseudomonas aeruginosa Exotoxin A (PEA) induces hepatotoxicity in experimental animals. Lipopolysaccharide (LPS) interacts synergistically with xenotoxics to induce severe organ injury. We examined the combination of non-injurious doses of LPS and sub-hepatotoxic PEA in the induction of multiple organ injury (MOI). Rats treated with 20 or 40 μg/kg LPS plus 10 μg/kg PEA developed severe liver, kidney, and lung injury; elevation of TNF-α, IFN-γ, and IL-2; and high mortality. Depletion of Kupffer cells or T-cells by pretreatment with Gadolinium Chloride or FK506, respectively, attenuated MOI. Thus LPS + PEA acted synergistically on Kupffer and T-cells to induce proinflammatory cytokines contributing to MOI.

Pseudomonas aeruginosa Exotoxin A-Induced Hepatotoxicity: An Animal Model in Rats

Pseudomonas aeruginosa exotoxin A (PEA) has been generally used to induce liver injury in mice for experimental study. No PEA-induced hepatotoxicity study has ever been conducted in rats, although rats are the most common rodents used in toxicologic bioassay and pharmacological evaluation. The present study was conducted in male Wistar rats that were injected (i.v.) with PEA at doses of 0, 10, 20, 30 or 40 microg/kg body weight and evaluated at 12, 24, 36, 48 and 60 hr post-exposure (HPE). Rats exposed to PEA at 40 microg/kg died before 36 HPE, and the mortality was dose and time dependent. Liver injury was noted as increases in serum enzymes, along with alterations of liver histology in the 40 microg/kg group at 12 HPE. TUNEL-positive staining indicative of hepatocyte apoptosis was observed in the 20 microg/kg group at 12 HPE. Significant levels of DNA fragmentation ladder were observed in the 30 microg/kg group starting at 24 HPE. Serum levels of TNF-alpha was increased in the 30 and 40 microg/kg groups at 48 and 24 HPE, respectively. Other cytokines, such as IL-2, IL-6, and IL-10 were also increased at various doses and times. Furthermore, the elevated serum hepatic index levels decreased significantly by dexamethasone pretreatment. In contrast, these markers were exacerbated by co-administration of a non-toxic dose LPS. In overall evaluation, the PEA-induced liver injury can be used as a model for study of hyperimmune-mediated hepatotoxicity.

The furin inhibitor hexa-D-arginine blocks the activation of Pseudomonas aeruginosa exotoxin A in vivo.

The Pseudomonas aeruginosa exotoxin A (PEA) protein requires furin-mediated cleavage for manifestation of toxicity. We show here that the small stable furin inhibitor hexa-D-arginine amide effectively blocks PEA-induced cell lysis and is itself noncytotoxic. Administration of hexa-D-arginine to PEA-treated mice significantly improves their survival rate and also decreases circulating levels of tumor necrosis factor alpha.

The effect of Pseudomonas exotoxin A on cytokine production in whole blood exposed to Pseudomonas aeruginosa

To determine the effect of Pseudomonas aeruginosa exotoxin A (P-ExA) on cytokine production, we studied cytokine release induced by heat-killed P. aeruginosa (HKPA) in human whole blood in the presence or absence of P-ExA. P-ExA (0.01–1 μg ml −1) caused a dose-dependent decrease in HKPA-induced production of tumor necrosis factor α (TNF), interleukin (IL-) 10, IL-6 and IL-8 (all P<0.05). P-ExA-induced inhibition of IL-10, IL-6 and IL-8 release was not dependent on reduced TNF concentrations, since the relative attenuation of the production of these cytokines was similar in the presence or absence of a neutralizing anti-TNF antibody. The effect of P-ExA on cytokine production may offer a disadvantage to the host with respect to clearance of the infection.

The effect of pseudomonas exotoxin A on cytokine production in whole blood exposed to Pseudomonas aeruginosa.

To determine the effect of Pseudomonas aeruginosa exotoxin A (P-ExA) on cytokine production, we studied cytokine release induced by heat-killed P. aeruginosa (HKPA) in human whole blood in the presence or absence of P-ExA. P-ExA (0.01-1 microgram ml(-1)) caused a dose-dependent decrease in HKPA-induced production of tumor necrosis factor alpha (TNF), interleukin (IL-) 10, IL-6 and IL-8 (all P<0.05). P-ExA-induced inhibition of IL-10, IL-6 and IL-8 release was not dependent on reduced TNF concentrations, since the relative attenuation of the production of these cytokines was similar in the presence or absence of a neutralizing anti-TNF antibody. The effect of P-ExA on cytokine production may offer a disadvantage to the host with respect to clearance of the infection.

Effect of Pseudomonas aeruginosa exotoxin A on IFN-γ synthesis: expression of costimulatory molecules on monocytes and activity of NK cells

The aim of the study was (1) to evaluate the effect of Pseudomonas aeruginosa Exotoxin A (P-ExA) on the production of IFN-γ in anti-CD3 induced human peripheral blood mononuclear cells (PBMC) and (2) to establish the effect of P-ExA on the IFN-γ dependent cellular activities such as the expression of costimulatory molecules on monocytes and cytotoxicity of NK cells. The toxin in a high dose (100 ng/ml) inhibited IFN-γ synthesis. Inhibitory effect of P-ExA was abolished by IL-1α which in a combination with P-ExA exerted a strong synergistic effect on IFN-γ synthesis. Other monokines such as IL-1β, IL-6, TNF-α neither reversed the inhibitory effect of P-ExA nor induced production of IFN-γ. P-ExA also inhibited IFN-γ-induced cellular events: (1) expression of costimulatory molecules on monocytes (CD80, CD86, ICAM-1, HLA-DR); (2) cytotoxic activity of NK cells. Inhibition of NK cells activity by P-ExA was not reversed by cytokines such as IL-2, IFN-α and TNF-α, which are known to enhance effector functions of NK cells. From these results we conclude that: (1) inhibition of IFN-γ synthesis, as well as IFN-γ-induced expression of costimulatory molecules and NK-cell effector functions may lead to suppression of specific and non-specific defense mechanisms, respectively, which are necessary for elimination of PA bacteria; (2) enhancement of IFN-γ synthesis induced by P-ExA in a combination with IL-1α may cause harmful, Th1 cells dependent, inflammatory reactions of the host (septic shock, tissue damage) during infection with Pseudomonas aeruginosa.

Effect of Pseudomonas aeruginosa exotoxin A on CD3-induced human T-cell activation

The effect of Pseudomonas aeruginosa (PA) exotoxin A (P-ExA) on CD3-induced T-cell activation was studied on the level of T-cells (proliferation, synthesis of interleukin (IL)-2, expression of IL-2R complex, ICAM-1,2 and LFA-1 molecules), and on the level of monocytes (expression of ICAM-1,2, LFA-1 molecules, as well as FcRI and CD14 receptors). We found that: (1) P-ExA blocked T-cell proliferation and this effect was totally reversed by intact monocytes, and partially by IL-2 or TPA but not by costimulatory cytokines (IL-1 α, IL-1 β, TNF- α or IL-6); (2) P-ExA transiently, in short-term cultures (48 h), inhibited synthesis of IL-2; (3) prolonged stimulation (96 h) of peripheral blood mononuclear cells (PBMC) or CD4+ T-cells with P-ExA in high or low doses (100 and 10 ng/ml, respectively), enhanced the level of IL-2 in the cultures; (4) P-ExA at low dose, combined with IL-1 β, TNF- α or IL-6, up-regulated synthesis of IL-2; and (5) stimulation of T-cells with anti-CD3 monoclonal antibody (mAb) and P-ExA at high dose diminished the expression of the p55 chain but not of the p75 chain of IL-2R complex and slightly affected the expression of CD3 complex, ICAM-1,2 and LFA-1 molecules. Hence, P-ExA can regulate the level of IL-2 in cultures of CD3-induced T-cells either by inhibition of IL-2 consumption (when P-ExA is applied in high dose), or by induction of IL-2 production (a costimulatory effect exerted by P-ExA in low dose in combination with monokines). Action of P-ExA on monocytes resulted in: (1) inhibition of the expression of ICAM-1,2 molecules and their ligand LFA-1 molecule; (2) low expression of FcRI receptor (a ligand for Fc part of CD3 mAb); and (3) inhibition (over 90%) of the expression of CD14 molecule. In conclusion, P-ExA-induced anergy of T-cells depends on: (a) decrease in the affinity of IL-2R complex on activated T-cells; and (b) inhibition of the accessory activities of monocytes.

RICIN-MEDIATED CELL-LYSIS AND APOPTOSIS OF DRUG SENSITIVE AND RESISTANT TUMOR-CELLS

Ricin is a potent plant toxin that inhibits protein synthesis of eukaryotic cells resulting eventually in cell death. The mechanism of ricin-mediated cytotoxicity on human tumor cell lines was examined in view of the recent findings that bacterial toxins like diphtheria toxin (DTX) and Pseudomonas aeruginosa exotoxin A (PEA) cause programmed cell death or apoptosis in addition to their activity of inhibition of protein synthesis. Ricin is shown to be cytolytic in a short term assay (less-than-or-equal-to 24 h) and cytostatic in a long term assay (greater-than-or-equal-to 72 h) as determined by the MTT assay. The cytostatic activity of ricin paralleled its protein synthesis inhibitory activity whereas ricin-mediated cytolytic activity did not correlate with protein synthesis inhibition. Ricin is shown to mediate programmed cell death or apoptosis against several drug sensitive and drug resistant cell lines. DNA fragmentation is initially detected following 4 h of treatment. Although cytotoxicity by ricin is energy dependent, it is not reduced by the addition of a lysosomotropic agent NH4Cl. We have recently demonstrated that the bacterial toxins DTX and PEA synergize with tumor necrosis factor-alpha (TNF-alpha) (J Immunol 147: 2609-2616, 1991 and J Immunol 149: 2089-2094, 1992). However, unlike DTX or PEA, ricin did not synergize with TNF-alpha, suggesting that ricin and DTX/PEA may mediate their cytolytic activity by different mechanisms. These findings demonstrate that ricin can mediate both protein synthesis inhibition and programmed cell death or apoptosis against a variety of drug sensitive and resistant human tumor cell lines. The significance of these findings in cancer therapy is discussed.

A human-human hybridoma secreting anti-Pseudomonas aeruginosa exotoxin-A monoclonal antibody with highly potent neutralizing activity.

A hybridoma secreting human monoclonal antibody (MAB) against Pseudomonas aeruginosa exotoxin A (PEA) was constructed by fusing Epstein-Barr virus-transformed peripheral blood lymphocytes with human B lymphoblastoid cell line TAW-925. The human-human hybridoma stably produced human IgG2 MAB at the rate of 0.4-0.5 microgram/ml per 10(6) cells per day for more than six months, and the MAB was capable of neutralizing the in vitro cytotoxic and in vivo lethal effects of PEA with approximately 100- and 70-fold, respectively, higher activity than serum polyclonal antibody preparations.