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MHC class I molecules are important components of immune surveillance. There are no available methods to directly visualize and determine the quantity and distribution of MHC/peptide complexes on individual cells or to detect such complexes on antigen-presenting cells in tissues. MHC-restricted recombinant antibodies with the same specificity of T cell receptors (TCR) may become a valuable tool to address these questions. They may also serve as valuable targeting molecules that mimic the specificity of cytotoxic T cells. We isolated by phage display a panel of human recombinant Fab antibodies with peptide-specific, MHC-restricted TCR-like reactivity directed toward HLA-A2-restricted T cell epitopes derived from a novel antigen termed TCRgamma alternative reading frame protein (TARP) which is expressed on prostate and breast cancer cells. We have characterized one of these recombinant antibodies and demonstrated its capacity to directly detect specific HLA-A2/TARP T cell epitopes on antigen-presenting cells that have complexes formed by naturally occurring active intracellular processing of the antigen, as well as on the surface of tumor cells. Moreover, by genetic fusion we armed the TCR-like antibody with a potent toxin and demonstrated that it can serve as a targeting moiety killing tumor cells in a peptide-specific, MHC-restricted manner similar to cytotoxic T lymphocytes.

We have shown previously that high-affinity receptors for interleukin-13 (IL-13Ralpha2) are overexpressed on a variety of solid cancer cells, diseased fibroblasts, and other cells, and a chimeric fusion protein composed of human IL-13 and mutated Pseudomonas exotoxin (IL-13-PE38) is highly and specifically cytotoxic to these cells in vitro and in vivo. To improve the specificity for the target, we isolated specific antibodies against IL-13Ralpha2 from human single-chain Fv (scFv) antibody phage library and developed immunotoxin by selecting two high-affinity clones of scFv and fused to PE. The fusion chimeric gene was expressed in Escherichia coli, and highly purified IL-13R-specific immunotoxin, termed anti-IL-13Ralpha2(scFv)-PE38, was tested for its cytotoxicity. This molecule was highly cytotoxic to U251 glioma and PM-RCC renal cell carcinoma cell lines in vitro. The cytotoxic activity was neutralized by purified extracellular domain of IL-13Ralpha2 but not by IL-13, indicating that cytotoxic activity is specific. Anti-IL-13Ralpha2(scFv)-PE38 showed significant antitumor activity in immunodeficient mice with s.c. glioma tumors. Both i.p. and i.t. routes of administration showed antitumor activity in a dose-dependent manner. The maximum tolerated dose of anti-IL-13Ralpha2(scFv)-PE38 was 200 microg/kg i.p. twice daily for 5 days. These results indicate that anti-IL-13Ralpha2(scFv)-PE38 is a highly selective therapeutic agent for cancer therapy and should be further tested in animal models of human cancer.

Aim: To evaluate the long-term effects of gonadotropinreleasing hormone (GnRH)-based vaccine on levels of GnRH antibody and testosterone, and vaccine-induced immunocastration on sexual behavior of male rats., Methods: The rats were treated with GnRH-PE40 intraperitoneally every other day for 12 wk. GnRH antibody and testosterone level in rat blood were determined by ELISA and radioimmunoassay, respectively. Morphological changes in testes and sexual behavior of rats were evaluated., Results: GnRH-PE40 induced a high production in GnRH antibody, decreased the serum testosterone level, testis atrophy and sexual function in rats., Conclusion: Intraperitoneal administration of GnRHPE40 produces structural and functional castration of male rat reproductive system by inducing anti-GnRH antibody.

Our previous work suggested that streptococcal pyrogenic exotoxin (SPE) B-induced apoptosis is mediated through a receptor-like mechanism. In this study, we have identified alpha(v)beta(3) and Fas as the SPE B receptors for this function. The SPE B fragment without the RGD motif and G308S, a SPE B mutant with the RSD motif, induced less apoptosis than did native SPE B, suggesting that the RGD motif is critical for SPE B-induced apoptosis. Fluorescein isothiocyanate-SPE B binding assays and immunoprecipitation analysis showed that SPE B specifically interacted with alpha(v)beta(3). Anti-alpha(v)beta(3) antibody partially inhibited SPE B-induced apoptosis but had no effect on G308S-induced apoptosis. In addition, Fas binding to SPE B was verified in an affinity column and an immunoprecipitation analysis. Anti-Fas antibody inhibited SPE B- and G308S-induced apoptosis in a dose-dependent manner, suggesting that Fas-mediated SPE B-induced apoptosis also occurs RGD independently. Both anti-alpha(v)beta(3) and anti-Fas antibodies synergistically inhibited SPE B-induced apoptosis. The apoptotic cascades were activated by SPE B and G308S, with a little delay by the latter. After SPE B binding, the cell surface level of alpha(v)beta(3), but not of Fas, was decreased. The decreased alpha(v)beta(3) level was restored by treatment with the proteasome inhibitor MG132, suggesting a SPE B-mediated endocytosis of integrin alpha(v)beta(3) via the ubiquitin-proteasome system. Taken together, our results demonstrate that SPE B-induced apoptosis is mediated through alpha(v)beta(3) integrin and Fas in a synergistic manner.

Purpose: We investigated the expression of interleukin-13 receptor alpha2 (IL-13R alpha 2), EphA2, and Fos-related antigen 1 (Fra-1) in astrocytomas and normal brain. We sought to document whether the expression of the three factors changed with progression to higher grade malignancy and whether two or three targets in combination might be sufficient to target all patients with high-grade astrocytomas., Experimental Design: Immunohistochemistry was done for IL-13R alpha 2, EphA2, and Fra-1 using human brain tumor tissue microarrays containing 30 specimens of WHO grades II and III astrocytomas, 46 glioblastoma multiformes (GBM), and 9 normal brain samples. Sections were scored based on frequency and intensity of expression. Western blotting was done for all three markers using GBM tumor specimens and xenograft cell lines. Two cytotoxins, IL-13.E13K.PE38QQR and ephrinA1-PE38QQR, which target IL-13R alpha 2 or EphA2, respectively, were tested for cytotoxicity against human GBM primary explant cells and established cells., Results: Expression of all three proteins was significantly higher in GBM compared with normal brain, low-grade, and anaplastic astrocytomas. Greater than 95% of GBM overexpressed at least two of the three markers. Importantly, every GBM overexpressed at least one marker. Human GBM primary explant cells and cell lines were potently killed by IL-13.E13K.PE38QQR and ephrinA1-PE38QQR, in accordance with their level of expression of IL-13R alpha 2 and EphA2, respectively., Conclusions: IL-13R alpha 2, EphA2, and Fra-1 are attractive therapeutic targets representing molecular denominators of high-grade astrocytomas. One hundred percent of GBM tumors overexpress at least one of these proteins, providing the basis for rational combinatorial targeted therapies/diagnostics suitable for all patients with this disease.

Background: Nosocomial infections often lead to sepsis and multisystem organ failure in critically injured patients, including burn and trauma patients. A better understanding of the bacterial response to the host immune system is essential to develop better antimicrobials against pathogens. Pseudomonas aeruginosa combats host-initiated oxidant stress through expression of the transactivating factor, OxyR. Here we have tested the premise that OxyR regulates Pseudomonal cytotoxicity through secreted exotoxin production., Materials and Methods: Wild-type P. aeruginosa (PAO1) and a deletion mutant lacking the oxyR gene (Delta oxyR) were grown for 18 h in Luria broth and the supernatant containing the secreted products was removed using centrifugation. Secreted proteins were isolated using ammonium sulfate precipitation. ER-MP20(+) myeloid progenitor cells were harvested from the bone marrow of C57Blk/6J mice. These cells were differentiated into dendritic cells and macrophages. Various concentrations (0-20 microg/100 microL) of the bacterial proteins were added to the medium and cells were allowed to differentiate for 7 days. Cellular viability was then assayed using a proliferation assay. These studies were repeated on two other macrophage cell lines, human U937 and murine P388D1., Results: At a protein concentration of 5 microg/100 microL PAO1 supernatant protein, cellular proliferation was significantly reduced to 4.2 +/- 2.8% compared to untreated controls, while the DeltaoxyR supernatant protein remained at 103.3 +/- 4.0% of untreated controls (P < 0.05). Similar significant results were seen in the U937-, P388D1-, and ER-MP20(+)-derived macrophage cells., Conclusions: Taken together, our data indicate that OxyR regulates the secretion of potent cytotoxic factors by P. aeruginosa.

Aggregatibacter actinomycetemcomitans leukotoxin (Ltx) is a repeats-in-toxin (RTX) cytolysin that kills human leukocyte function-associated antigen-1 (LFA-1; alpha(L)/beta(2))-bearing cells. In order to determine whether the alpha(L) portion of the heterodimer is involved in Ltx recognition, we transfected human, mouse and bovine alpha(L) cDNAs into J-beta(2).7, an alpha(L)-deficient cell line, and looked for restoration of Ltx susceptibility. Cells expressing either bovine or human alpha(L) in conjunction with human beta(2) were efficiently killed by Ltx, an indication that bovine alpha(L) could substitute for its human counterpart in critical regions used by Ltx for attachment to LFA-1. On the other hand, cells expressing murine alpha(L) and human beta(2) were not susceptible to the lethal effects of Ltx indicating that the toxin recognition sites are not present in the corresponding mouse sequence. To further identify the region(s) of alpha(L) recognized by Ltx, we constructed and evaluated a panel of chimeric human/murine alpha(L) genes in J-beta(2).7 cells. Analysis of the alpha(L) mutant panel showed that the presence of human N-terminal 128 amino acids on a mouse CD11a background, a region that includes beta-sheets 1 and 2 of the beta-propeller of the human alpha(L) chain, was sufficient for Ltx cytolysis.

Previous reports have shown the presence of streptococcal erythrogenic exotoxin type B (ETB), leukocyte infiltration, interleukin-8 (IL-8), transforming growth factor-beta (TGF-beta) and glomerular proliferation in renal biopsies from patients with acute post-streptococcal glomerulonephritis (APSGN). In addition, increased levels of plasma interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFalpha), and urinary IL-6, have also been reported in this disease. To determine the effect of streptococcal proteins on leukocyte proliferation and leukocyte production of IL-6, TNFalpha, IL-8 and TGF-beta1, we cultured human mononuclear leukocytes with ETB or ETB precursor (ETBP). After 24 h, 48 h and 96 h, culture supernatants were assessed for cytokines by enzyme-linked immunosorbent assay (ELISA), and for leukocyte proliferation by a monoclonal antibody anti-proliferating cellular nuclear antigen (PCNA). A significant increase in all cytokines was found in ETB- or ETBP-treated cultures when compared with controls. A polyclonal anti-ETB antibody diminished the cytokine stimulatory effect of ETB. An increased number of PCNA-positive cells was observed in ETB or ETBP treated cultures at 48 h and 96 h. Cytokine production and proliferation were not correlated. The stimulatory effect of streptococcal exotoxin B on leukocyte cytokine production may be relevant in renal tissue during the course of APSGN.

Life-threatening Clostridium sordellii infections have recently been reported in women undergoing therapeutic abortion, during natural childbirth, and in injection drug users. Shock, diffuse capillary leak, and a leukemoid reaction (LR) are cardinal features of these infections. The magnitude of the LR is highly correlated with mortality. We have isolated a 42-kDa extractable protein from C. sordellii culture supernatant that stimulates proliferation of promyelocytic HL-60 cells in vitro. Using mass spectrometry, we have identified this protein as the C. sordellii neuraminidase, NanS. Recombinant NanS (rNanS) dose dependently stimulated HL-60 cell proliferation. Increased proliferation was observed when HL-60 cells were cocultured with both rNanS and granulocyte-macrophage colony stimulating factor. In addition, NanS also modified vascular cell adhesion molecule 1, which orchestrates the release of mature and immature granulocytes from bone marrow stromal cells. Thus, neuraminidase likely plays an important role in the characteristic LR in C. sordellii infection.

We generated a mouse model with a conditional deletion of TGF-beta signaling in the neurons by crossing TGF-beta receptor I (TbetaRI) floxed mice with neurofilament-H (NF-H) Cre mice. 35% of F1 conditional knockout (COKO) mice developed spontaneous squamous cell carcinomas (SCCs) in periorbital and/or perianal regions. Transplantation of these tumors into athymic nude mice resulted in 62% tumorigenicity. To determine whether evasion of the immune response plays any role in this tumorigenesis, we analyzed the expression levels of receptors for interleukin-13 (mIL-13R), a key negative regulator of tumor immunosurveillance, and found that 33% of COKO tumors expressed the IL-13R alpha2 chain. Primary cultures of the SCCs expressing IL-13R alpha2 were sensitive to the cytotoxic effect of IL-13R-directed cytotoxin treatment. This is the first demonstration that loss of TbetaRI can lead to spontaneous tumor formation. These mice can serve as a unique mouse model of SCC to evaluate the tumorigenicity and effect of anti-cancer therapeutics.

Programs that direct cellular differentiation are dependent on the strict temporal expression of regulatory factors that can be provided by Rho GTPases. Ciliogenesis is a complex sequence of events involving the generation and docking of basal bodies at the apical membrane, followed by ciliary axoneme generation. Although a cilia proteome has been assembled, programs that direct ciliated cell differentiation are not well established, particularly in mammalian systems. Using mouse primary culture airway epithelial cells, we identified a critical stage of ciliogenesis requiring the temporal establishment of an apical web-like structure of actin for basal body docking and subsequent axoneme growth. Apical web formation and basal body docking were prevented by interruption of actin remodeling and were dependent on RhoA activation. Additional evidence for this program was provided by analysis of Foxj1-null mice that failed to dock basal bodies and lacked apical actin. Foxj1 expression coincided with actin web formation, activated RhoA and RhoB, and persisted despite RhoA inhibition, suggesting that Foxj1 promoted RhoA during ciliogenesis. Apical ezrin localization was also dependent on Foxj1, actin remodeling, and RhoA, but was not critical for ciliogenesis. Thus, temporal Foxj1 and RhoA activity are essential regulatory events for cytoskeletal remodeling during mammalian ciliogenesis.

Diverse pathogenic bacteria have developed similar mechanisms to subvert host cell responses. In this Progress article, we focus on bacterial virulence factors with different enzymatic activities that can increase the expression of the Kruppel-like factor (KLF) family of mammalian transcriptional regulators through their ability to modify the activity of a common host-cell target - the Rho protein family. By using a common virulence strategy, both Gram-negative and Gram-positive pathogens exploit the KLF regulatory cascade to modulate nuclear factor kappaB activation, pro-inflammatory cytokine expression, actin cytoskeletal dynamics and phagocytosis.

Targeted therapy encompasses a wide variety of different strategies, which can be divided into direct or indirect approaches. Direct approaches target tumor-associated antigens by monoclonal antibodies (mAbs) binding to the relevant antigens or by small-molecule drugs that interfere with these proteins. Indirect approaches rely on tumor-associated antigens expressed on the cell surface with antibody-drug conjugates or antibody-based fusion proteins containing different kinds of effector molecules. To deliver a lethal cargo into tumor cells, the targeting antibodies should efficiently internalize into the cells. Similarly, to qualify as targets for such drugs newly-discovered cell-surface molecules should facilitate the internalization of antibodies that bind to them. Internalization can be studied be several biochemical and microscopy approaches. An undisputed proof of internalization can be provided by the ability of an antibody to specifically deliver a drug into the target cells and kill it. We present a novel IgG binding toxin fusion, ZZ-PE38, in which the Fc-binding ZZ domain, derived from Streptococcal protein A, is linked to a truncated Pseudomonas exotoxin A, the preparation of complexes between ZZ-PE38 and IgGs that bind tumor cells and the specific cytotoxicity of such immunocomplexes is reported. Our results suggest that ZZ-PE38 could prove to be an invaluable tool for the evaluation of the suitability potential of antibodies and their cognate cell-surface antigens to be targeted by immunotherapeutics based on armed antibodies that require internalization.

The novel recombinant anthrax toxin, PrAgU2/FP59, composed of the urokinase-activated protective antigen and a fusion protein of Pseudomonas exotoxin and lethal factor was tested for anti-lung cancer efficacy in an in vivo human tumor model. Male athymic nude mice (age 4-6 weeks) were inoculated s.c. with 10 million H1299 non-small cell lung cancer (NSCLC) cells in the left flank. When tumor volumes reached 200 mm(3) (6-8 days), i.p. injection of 100 muL saline or different ratios and doses of PrAgU2/FP59 in 100 muL saline were given every 3 days for four doses and an additional dose at day 29. Animals were monitored twice daily and tumor measurements were made by calipers. The maximum tolerated doses of PrAgU2/FP59 differed dependent on the ratios of PrAgU2 to FP59 over the range of 3:1 to 25:1, respectively. At tolerated doses, tumor regressions were seen in all animals. Complete histologic remission lasting 60 days occurred in 30% of animals. PrAgU2/FP59 showed dramatic anti-NSCLC efficacy and warrants further clinical development for therapy of patients with advanced NSCLC.

MUC1 is a mucin family protein, overexpressed in more than 90% of breast cancers in an underglycosylated form, exposing the core peptides of the extracellular domain that act as a potential target for antibody-mediated therapy. We have developed an anti-MUC1 scFv antibody from a phage library of mice immunized with synthetic peptide MUC1-variable number of tandem repeats. MUC1 binding phages were affinity selected through biopanning using a biotin-streptavidin pull-down method. The selected phage clones showed target-specific binding to MUC1-expressing cells. Fusion of truncated Pseudomonas aeruginosa exotoxin A (ETA) to a high binder, phage-derived scFv clone and bacterial expression and purification of recombinant scFv(MUC1)-ETA immunotoxin were done with good yield and purity. In vitro target-specific cytotoxic activity and target-specific binding of immunotoxin were shown on MUC1-expressing cells and primary breast tumor samples. A truncated ETA fusion protein expressed from the same vector but lacking scFv did not show cytotoxic effects, confirming target specificity. Our results suggest that the scFv(MUC1)-ETA immunotoxin has therapeutic potential and deserves further development and characterization for MUC1-specific breast cancers treatment.

At fertilization, the sea urchin egg undergoes an internal pH (pHi) increase mediated by a Na+ -H+ exchanger. We used antibodies against the mammalian antiporters NHE1 and NHE3 to characterize this exchanger. In unfertilized eggs, only anti-NHE3 cross-reacted specifically with a protein of 81-kDa, which localized to the plasma membrane and cortical granules. Cytochalasin D, C3 exotoxin (blocker of RhoGTPase function), and Y-27632 (inhibitor of Rho-kinase) prevented the pHi change in fertilized eggs. These inhibitors blocked the first cleavage division of the embryo, but not the cortical granule exocytosis. Thus, the sea urchin egg has an epithelial NHE3-like Na+ -H+ exchanger which can be responsible for the pHi change at fertilization. Determinants of this pHi change can be: (i) the increase of exchangers in the plasma membrane (via cortical granule exocytosis) and (ii) Rho, Rho-kinase, and optimal organization of the actin cytoskeleton as regulators, among others, of the intrinsic activity of the exchanger.

Actinobacillus actinomycetemcomitans produces a leukotoxin (Ltx) that kills leukocyte function-associated antigen-1 (LFA-1)-bearing cells from man, the Great Apes and Old World monkeys. The unique specificity of Ltx for the beta2 integrin, LFA-1, suggests it is capable of providing insight into the pathogenic mechanisms of Ltx and other RTX toxins. Using the Jurkat T cell line and an LFA-1-deficient Jurkat mutant (Jbeta2.7) as models, we found the initial effect of Ltx is to elevate cytosolic Ca2+ [Ca2+]c, an event that is independent of the Ltx/LFA-1 interaction. [Ca2+]c increases initiate a series of events that involve the activation of calpain, talin cleavage, mobilization to, and subsequent clustering of, LFA-1 in cholesterol and sphingolipid-rich regions of the plasma membrane known as lipid rafts. The association of Ltx and LFA-1 within lipid rafts is essential for cell lysis. Jbeta2.7 cells fail to accumulate Ltx in their raft fractions and are not killed, while cholesterol depletion experiments demonstrate the necessity of raft integrity for Ltx function. We propose that toxin-induced Ca2+ fluxes mobilize LFA-1 to lipid rafts where it associates with Ltx. These findings suggest that Ltx utilizes the raft to stimulate an integrin signalling pathway that leads to apoptosis of target cells.

Recombinant exotoxin A (rEPA) from Pseudomonas aeruginosa conjugated to Vibrio cholerae O1 serotype-specific polysaccharides (mono-, di- and hexasaccharide) were immunogenic in mice. Monosaccharide conjugates boosted the humoral responses to the hexasaccharide conjugates. Prior exposure to purified Ogawa lipopolysaccharide (LPS) enabled contra-serotype hexasaccharide conjugates to boost the vibriocidal response, but Inaba LPS did not prime for an enhanced vibriocidal response by a contra-serotype conjugate. Prior exposure to the carrier, and priming B cells with the LPS of either serotype, resulted in enhanced vibriocidal titers if the Ogawa hexasaccharides were used, but a diminished response to the Inaba LPS. These studies demonstrate that the 'functional' B cell epitopes on the LPS differ from those of the neoglycoconjugates and that the order of immunization and the serotype of the boosting conjugate can influence the epitope specificity and function of the antisera.

Highly active antiretroviral therapy (HAART) against human immunodeficiency virus type 1 (HIV-1) infection dramatically suppresses viral load, leading to marked reductions in HIV-1 associated morbidity and mortality. However, infected cell reservoirs and low-level replication persist in the face of suppressive HAART, leading invariably to viral rebound upon cessation of treatment. Toxins engineered to target the Env glycoprotein on the surface of productively infected cells represent a complementary strategy to deplete these reservoirs. We described previously highly selective killing of Env-expressing cell lines by CD4(178)-PE40 and 3B3(Fv)-PE38, recombinant derivatives of Pseudomonas aeruginosa exotoxin A containing distinct targeting moieties against gp120. In the present report, we compare the in vitro potency and breadth of these chimeric toxins against multiple clinical HIV-1 isolates, replicating in biologically relevant primary human target cell types. In PBMCs, 3B3(Fv)-PE38 blocked spreading infection by all isolates examined, with greater potency than CD4(178)-PE40. 3B3(Fv)-PE38 also potently inhibited spreading HIV-1 infection in primary macrophages. Control experiments demonstrated that in both target cell types, most of the 3B3(Fv)-PE38 activity was due to selective killing of infected cells, and not merely to neutralization by the antibody moiety of the chimeric toxin. High-dose treatment of rhesus macaques with 3B3(Fv)-PE38 did not induce liver toxicity, whereas equivalent dosage of CD4(178)-PE40 induced mild hepatotoxicity. These findings highlight the potential use of 3B3(Fv)-PE38 for depleting HIV-infected cell reservoirs persisting in the face of HAART.

Background: Cases of pyomyositis and myositis have been increasing in frequency at Texas Children's Hospital (Houston) since 2000. The increase appears to correlate with the emergence of community-acquired methicillin-resistant Staphylococcus aureus (MRSA)., Methods: The medical records of patients with pyomyositis and myositis hospitalized at Texas Children's Hospital during the period from January 2000 through December 2005 were reviewed. Available S. aureus isolates were obtained for susceptibility testing, to determine the presence of pvl (lukS-PV and lukF-PV), and for pulsed-field gel electrophoresis analysis., Results: Forty-five previously healthy children with bacterial pyomyositis or myositis were analyzed. The causes were S. aureus (in 57.8% of children) and Streptococcus pyogenes (in 2.2%); 40.0% of children had negative culture results. The number of cases increased between 2000 and 2005, primarily as a result of an increase in the prevalence of community-acquired MRSA. The mean patient age was 5.5 years (range, 0.06-15 years). The thigh (40.0% of children) and pelvis (28.9%) were the most commonly affected sites. The mean abscess diameter was 3.5 cm. Eighteen children required at least 1 muscle drainage procedure. Of the 24 available S. aureus isolates (15 community-acquired MRSA isolates and 9 community-acquired, methicillin-susceptible S. aureus [MSSA] isolates), 16 were found to be USA300 by pulsed-field gel electrophoresis, and 17 carried pvl. Patients with community-acquired MRSA, USA300, and/or pvl-positive strains required more drainage procedures than did those with community-acquired MSSA, non-USA300, and/or pvl-negative strains (81% vs. 40% [P=.05], 82% vs. 29% [P=.02], and 81% vs. 38% [P=.07], respectively)., Conclusions: Community-acquired MRSA is an increasing cause of pyomyositis and myositis in children. Community-acquired MRSA, USA300, pvl-positive S. aureus isolates caused more severe disease than did community-acquired MSSA, non-USA300, and pvl-negative isolates, respectively.

Crosspresentation of exogenous antigens (Ags) to CD8(+) T cells by dendritic cells generally requires their entry into the cytosol. Here we show that both soluble and phagocytosed extracellular Ags accessed the cytosol via molecular components required for endoplasmic reticulum (ER)-associated degradation (ERAD). Exogenous Pseudomonas aeruginosa Exotoxin A, which inhibits protein translocation from the ER to the cytosol, abrogated crosspresentation. Exotoxin A also prevented the transporter associated with antigen processing (TAP) inhibitor, ICP47, from entering the cytosol and blocking TAP-mediated peptide transport. In an in vitro model of retrotranslocation, the AAA ATPase p97, an enzyme critical for ERAD, was the only cytosolic cofactor required for protein export from isolated phagosomes. Functional p97 was also required for crosspresentation but not conventional presentation. Thus, crosspresentation appears to result from an adaptation of the retrotranslocation mechanisms involved in the degradation of misfolded ER proteins.

A novel single-chain immunotoxin was constructed by combining a CD33-specific single chain Fv (scFv) antibody fragment with an engineered variant of Pseudomonas exotoxin A (ETA). The variant toxin carries the KDEL peptide at its C-terminus, a cellular peptide mediating improved retrograde transport to the endoplasmic reticulum. The purified recombinant fusion protein induced potent apoptosis of the human myeloid cell lines U937, HL-60 and THP-1. Up to 98% of U937 cells were eliminated after treatment for 72 h with a single dose of 500 ng/ml (c. 7 nmol/l). Killing was antigen-specific and occurred by apoptosis. A control protein, consisting of a CD19-specific scFv antibody fragment fused to the ETA-KDEL toxin, failed to induce death of the CD19-negative cell lines U937, HL-60 and THP-1. The CD33-ETA toxin also mediated apoptosis of fresh patient-derived acute myeloid leukaemia cells from bone marrow and peripheral blood. The pronounced antigen-restricted cytotoxicity of the novel fusion protein makes it a candidate for further evaluation of its therapeutic potential.

Diphtheria toxin and exotoxin A are well-characterized members of the ADP-ribosyltransferase toxin family that function as virulence factors in the pathogenic bacteria Corynebacterium diphtheriae and Pseudomonas aeruginosa. Recent high-resolution structural data of the Michaelis (enzyme-substrate) complex of the P. aeruginosa toxin with an NAD(+) analog and eukaryotic elongation factor 2 (eEF2) have provided insights into the mechanism of inactivation of protein synthesis caused by these protein factors. In addition, rigorous steady-state and stopped-flow kinetic analyses of the toxin-catalyzed reaction, in combination with inhibitor studies, have resulted in a quantum leap in our understanding of the mechanistic details of this deadly enzyme mechanism. It is now apparent that these toxins use stealth and molecular mimicry in unleashing their toxic strategy in the infected host eukaryotic cell.

Objective: The aim of this study was to evaluate the effects of Staphylococcus aureus exotoxin B (SE-B) on proinflammatory cytokine/chemokine releases in primary nasal epithelial cell cultures (NECC) of subjects with and without chronic rhinosinusitis (CRS)., Study Design and Setting: NECC (CRS: n = 14;, Controls: n = 11) were stimulated with SE-B. Protein concentrations of interleukin-(IL)-1beta, IL-6, and IL-8 levels were measured in NECC supernatants by ELISA before (T0) and after 24 hr stimulation with SE-B (T1)., Results: T0: supernatants of the NECC of CRS patients contained significant lower levels of IL-8 (2.1 ng/ml) compared to CONTROLS (IL-8: 6.2 ng/ml; P < 0.01). T1: SE-B induced a significant increase of IL-6 in NECC (P < 0.001). IL-1beta was not detectable., Conclusions: This is the first study evaluating the effects of exotoxins on NECC. SE-B showed proinflammatory effects on NECC., Significance: Our data suggest that resident NECC are involved in immunological responses to Staphylococcus aureus toxins, supplementing the so-called "superantigen hypothesis" in CRS.

Background/aims: Previous reports have shown the presence of streptococcal erythrogenic toxin type B (ETB), IL-8, transforming growth factor-beta (TGF-beta) and glomerular proliferation in renal biopsies from patients with acute poststreptococcal glomerulonephritis (APSGN). In addition, increased levels of plasma IL-6 and tumor necrosis factor-alpha (TNFalpha) and urinary IL-6 have also been reported in this disease. To determine the effect of ETB in mesangial cell cytokine production and proliferation, the concentration of several cytokines (IL-6, IL-1beta, TNFalpha, IL-10, IL-4, RANTES), soluble TNF receptor I (STNFR-I), soluble TNF receptor II (STNFR-II) and proliferation were measured in rat mesangial cells cultures after treatment with ETB or its precursor (ETBP)., Methods: To analyze the levels of cytokines and production of soluble receptors as well as proliferation, rat mesangial cells were cultured with ETB or ETBP (50 microg/ml). After 24, 48 and 96 h of incubation, culture supernatants were assessed for cytokines and receptors by ELISA and for proliferation by incorporation of radioactive thymidine., Results: A significant increase in IL-6 levels was found in mesangial cell cultures treated with either ETBP or ETB when compared with controls. Streptococcal proteins treated mesangial cells also showed elevated levels of proliferation at 96 h. Increased production of IL-6 was not correlated with proliferation. A polyclonal anti-ETB antibody abolished the IL-6 stimulatory effect of ETB on mesangial cells. ETB/ETBP failed to increase the levels of other cytokines and cytokine soluble receptors., Conclusion: Streptococcal ETB/ETBP is capable of inducing increased production of IL-6 and proliferation on mesangial cells. These findings could be relevant in a possible early interaction of streptococcal proteins with mesangial cells and during the course of APSGN., (Copyright 2006 S. Karger AG, Basel)

Bacillus cereus is mainly known as a human food-borne opportunistic pathogen. Here, we used biological assays and HPLC to investigate the ability of B. cereus to produce insecticidal exotoxins during the stationary growth phase. None of the 575 B. cereus strains screened produced detectable levels of beta-exotoxin I, a small, heat-stable insecticidal nucleotide analogue. However, six out of a subset of 270 B. cereus strains produced several small, nonproteinaceous insecticidal exotoxins different from beta-exotoxin I. Thus, B. cereus can secrete a large array of proteinaceous and nonproteinaceous toxins acting on insects and mammals.

Because the most characteristic property of ovarian cancer is i.p. spread, the majority of patients are diagnosed at an advanced stage, leading to limited availability of options for curative therapies. With an intent to identify targeted therapeutic approaches, we have observed that approximately 60% of 21 ovarian cancer tissue samples express a high density of interleukin-4 receptor (IL-4R), whereas normal ovarian tissues tested (n = 7) expressed no or low levels of IL-4R. To target IL-4R, we have developed IL-4 cytotoxin, in which circular-permuted IL-4 is fused to a mutated form of Pseudomonas exotoxin. This cytotoxin is specifically and highly cytotoxic to PA-1, IGROV-1, and SK-OV3 ovarian carcinoma cell lines in vitro. In addition, it shows remarkable antitumor activities against established s.c. ovarian tumors in immunodeficient animals. i.p. administration of IL-4 cytotoxin in mice with orthotopically implanted ovarian tumors caused regression of established tumors and prevented these animals from tumor metastasis. Continuous i.p. infusion of IL-4 cytotoxin prolonged survival of tumor-bearing mice even with bulky disease. These results indicate that IL-4R-targeted cytotoxin may be a useful agent for the management of patients with ovarian cancer, and further studies need to be done to evaluate its safety, tolerability, and efficacy.

We have determined that CLL B cells consistently express type 3 membrane receptors for the Th2-derived cytokine IL-4 (IL-4R). Furthermore, when added to CLL B cells, IL-4 induces increased apoptosis resistance, increased protein synthesis in CLL B cells and rapid onset activation of STAT1, STAT5 and STAT6. Since the IL-4-IL-4R pathway is intact in CLL B cells and is related to apoptosis resistance, we considered whether we could target this pathway. A recombinant IL-4 Pseudomonas exotoxin fusion protein (IL-4 PE), known to bind to IL-4R, was incubated with CLL B cells. IL-4 PE (10 ng/ml) cultured with CLL B cells resulted in an increase of apoptosis/death from mean levels of 46.6+/-7.0 of non-exposed cells to 69+/-8.6 (n=6). By measuring in vitro protein synthesis, two predominant patterns of sensitivity were observed. In one, CLL B cell clones (n=4) were found to be extremely sensitive to IL-4 PE (IC50's range=6-25 ng/ml). In the second, low concentrations of IL-4 PE induced agonist activity while increasing concentrations induced cytotoxicity in 6 of 21 patient-derived cells. These studies suggest that the IL-4R, on B-CLL cells, can serve as a unique molecular target for directing cytotoxic agents in the therapy of B-CLL.

NeoPharm Inc, under license from the National Institutes of Health and the FDA, and in collaboration with the Japanese licensee Nippon Kayaku Co Ltd, is developing cintredekin besudotox, a chimeric human IL-13 conjugated to a genetically engineered Pseudomonas exotoxin molecule, as a potential antitumor agent. This agent is currently undergoing phase III clinical trials.

The staphylococcal superantigen-like proteins (SSLs) are close relatives of the superantigens but are coded for by a separate gene cluster within a 19-kb region of the pathogenicity island SaPIn2. rSSL7 (formally known as SET1) bound with high affinity (K(D), 1.1 nM) to the monomeric form of human IgA1 and IgA2 plus serum IgA from primate, pig, rat, and horse. SSL7 also bound the secretory form of IgA found in milk from human, cow, and sheep, and inhibited IgA binding to cell surface FcalphaRI (CD89) and to a soluble form of the FcalphaRI protein. In addition to IgA, SSL7 bound complement factor C5 from human (K(D), 18 nM), primate, sheep, pig, and rabbit serum, and inhibited complement-mediated hemolysis and serum killing of a Gram-negative organism Escherichia coli. SSL7 is a superantigen-like protein secreted from Staphylococcus aureus that blocks IgA-FcR interactions and inhibits complement, leading to increased survival of a sensitive bacterium in blood.

IL-13 cytotoxin comprised of IL-13 and a mutated form of Pseudomonas exotoxin (fusion protein termed IL-13-PE38QQR) has been shown to inhibit protein synthesis leading to necrotic and apoptotic cell death in glioblastoma cells that express high levels of interleukin-13 receptors (IL-13R). To identify target genes of cell death and other cellular genes with IL-13 receptors in glioblastoma cells, we utilized the cDNA microarrays to analyze global gene expression profiles after IL-13 cytotoxin and IL-13 treatment. IL-13 cytotoxin mediated cytotoxicity to U251 cells in a dose-dependent manner. Hierarchical cluster analysis of differentially expressed genes in U251 glioma cells at different time points after IL-13 cytotoxin treatment showed three major groups, each representing a specific expression pattern. Randomly selected differentially expressed genes from each group were confirmed by RT-PCR analysis. Most down-regulated genes belong to cell adhesion, motility, angiogenesis, DNA repair, and metabolic pathways. While up-regulated genes belong to cell cycle arrest, apoptosis, signaling and various metabolic pathways. Unexpectedly, at early time points, both IL-13 and IL-13 cytotoxin induced several genes belonging to different pathways most notably IL-8, DIO2, END1, and ALDH1A3 indicating that these genes are early response genes and their products may be associated with IL-13R. In addition, IL-13 cytotoxin induced IL-13Ralpha2 mRNA expression during the treatment in glioma cells. Our results indicate that novel cellular genes are involved with IL-13 receptors and that IL-13 cytotoxin induced cell death involves various target genes in human glioblastoma cells. On going studies will determine the role of associated genes and their products in the IL-13R functions in glioma cells.

Actinobacillus actinomycetemcomitans produces a leukotoxin that selectively kills human leukocytes. Recently, we reported that macrophages are highly sensitive to leukotoxin and that their lysis involves activation of caspase 1. In this study, we show that leukotoxin also induces the production and release of proinflammatory cytokines from human macrophages. The macrophages were challenged with leukotoxin or lipopolysaccharide (LPS) from A. actinomycetemcomitans or LPS from Escherichia coli, and the production and secretion of interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor alpha (TNF-alpha) were determined at the mRNA and protein levels by reverse transcription-PCR and enzyme-linked immunosorbent assay, respectively. Leukotoxin (1 to 30 ng/ml) induced abundant production and secretion of IL-1beta, while the effects on IL-6 and TNF-alpha production were limited. Leukotoxin (1 ng/ml) caused a 10-times-higher release of IL-1beta than did LPS (100 ng/ml). The secreted IL-1beta was mainly the bioactive 17-kDa protein. At higher concentrations (>30 ng/ml), leukotoxin caused secretion of mainly inactive cytokine, the 31-kDa pro-IL-1beta. The presence of specific antibodies to IL-1beta or of a caspase 1 inhibitor blocked the secretion and production of the cytokine. Supernatants of leukotoxin-challenged macrophages stimulated bone resorption when tested in a mouse calvarial model. The activity could be blocked by an IL-1 receptor antagonist or specific antibodies to IL-1beta. We concluded that A. actinomycetemcomitans leukotoxin can trigger abundant production and secretion of bioactive IL-1beta by human macrophages, which is mediated by activation of caspase 1.

Actinobacillus actinomycetemcomitans is associated with localized aggressive periodontitis, a disease characterized by rapid loss of the alveolar bone surrounding the teeth. Receptor activator of NF-kappaB Ligand (RANKL) and osteoprotegerin (OPG) are two molecules that regulate osteoclast formation and bone resorption. RANKL induces osteoclast differentiation and activation, whereas OPG blocks this process by acting as a decoy receptor for RANKL. The purpose of this study was to investigate the effect of A. actinomycetemcomitans on the expression of RANKL and OPG in human gingival fibroblasts and periodontal ligament cells. RANKL mRNA expression was induced in both cell types challenged by A. actinomycetemcomitans extract, whereas OPG mRNA expression remained unaffected. Cell surface RANKL protein was also induced by A. actinomycetemcomitans, whereas there was no change in OPG protein secretion. A cytolethal distending toxin (Cdt) gene-knockout strain of A. actinomycetemcomitans did not induce RANKL expression, in contrast to its wild-type strain. Purified Cdt from Haemophilus ducreyi alone, or in combination with extract from the A. actinomycetemcomitans cdt mutant strain, induced RANKL expression. Pretreatment of A. actinomycetemcomitans wild-type extract with Cdt antiserum abolished RANKL expression. In conclusion, A. actinomycetemcomitans induces RANKL expression in periodontal connective tissue cells. Cdt is crucial for this induction and may therefore be involved in the pathological bone resorption during the process of localized aggressive periodontitis.

Objective: To study inhibitory effect of recombinant transforming growth factor alpha-Pseudomonas exotoxin fusion protein (TP40) on proliferation of the human bladder cancer T24 cells., Methods: Expression of epidermal growth factor receptor (EGFR) in cultured T24 cells was analyzed with Western blot assay. Human bladder cancer T24 cells were exposed to TP40 at 5 - 1 000 microg/L. Methyl thiazolyl tetrazolium assay was applied to evaluate the cell proliferation by measuring the absorbance (A) at 570 nm with a microplate reader. Tritium labeled thymine deoxyriboside ([(3)H]-TdR) uptake was measured to observe DNA synthesis. Competition assays were performed by the EGF at 1 - 7 500 microg/L., Results: Expression of EGFR was high in human bladder cancer T24 cells. Cell growth was suppressed by 10%, 19%, 27%, 41%, 47%, 53% and 61% after 96 h treatment with TP40 at 5, 50, 100, 250, 500, 750 and 1 000 microg/L, respectively. [(3)H]-TdR incorporation was 80%, 69%, 48% and 51% after 24 h, 48 h, 72 h, 96 h treatment with TP40 at 750 microg/L, respectively. When the concentration was 1 - 7 500 microg/L, EGF could block the inhibitory effect of TP40 to some extent., Conclusions: Human bladder cancer T24 cells express EGFR at a high level. TP40 could inhibit the growth of T24 cells effectively in a dose- and time-dependent manner. The cytotoxic effects of TP40 were specifically mediated by EGFR.

Objective: To establish a protocol for the targeting gene therapy against cancer with rich epidermal growth factor receptor (EGFR)., Methods: A recombinant pcDNA3.1-PE III mut was constructed and combined with a non-viral vector, a fusion protein histone H1, epidermal growth factor C-loop previously expressed by us, to be a protein-DNA complex in vitro. Using the complex to treat BT-325 and Hela cancer cells with EGFR and JK cells without EGFR. The killing rates of the cells was calculated after 48 h of incubation at 37 degrees C., Results: To BT-325 and Hela cells, the killing rates were 46.03% and 48.12% respectively. To JK cells, the complex had no killing function., Conclusion: The protocol for targeting gene therapy against cancer with EGFR has been established successfully.

The objective was to identify some biochemical and physical properties for fusion protein IL6D24-PE40KDEL. Edman degradation, SDS-PAGE, peptide mass fingerprinting, Western blot and MTT were used for identification of the protein. The results showed that the sequence of N-terminus is Met-Ile-Asp-Lys-Gln-Ile, Met was added because of prokaryotic expression system; Western blot revealed that the purified protein could react with IL6 and PEA antibody. The purified protein IL6D24-PE40KDEL could kill the multiple myeloma cell lines U266 expressing high affinity IL6R, but it could not kill the cell lines CEM which not expressed IL6R; The molecular weight was 58.7 kD measuring by SDS-PAGE; peptide mass fingerprinting (PMF) confirmed that the construction of IL6D24-PE40KDEL was correct. A novel protein by Peptident database in EXPASY web site was identified. In conclusion, IL6D24-PE40KDEL is a new targeting protein with bioactivity of specific killing effect.

Pseudomonas exotoxin (PE)-containing immunotoxins (ITs) act by arresting protein synthesis and promoting apoptosis, but the mechanisms of the induced apoptosis and the relationship to protein synthesis inhibition is not well elucidated. We studied these effects in MA-11 human breast cancer cells treated with 425.3PE, an unmodified PE covalently linked to the 425.3 antibody, which targets the EGF receptor. This IT induced efficient inhibition of protein synthesis with simultaneous induction of apoptosis. Thus, treatment of cells with 10 ng/ml of IT for 5 hr caused 85% inhibition of protein synthesis in parallel with caspase-3, -8 and -9 activation and PARP inactivation. Even after 72 hr of IT treatment, preincubation with the broad-spectrum caspase inhibitor z-VAD-FMK caused a significant increase in cell survival without affecting IT-induced protein synthesis inhibition. Interestingly, a combination of z-VAD-FMK and the cathepsin B/L inhibitor z-FA-FMK prevented completely IT-induced cell death in MA-11 cells after 24 hr, indicating that cathepsin activation may be important for optimal induction of IT-induced cell death. IT treatment caused after 2.5 hr a significant decrease in the level of the antiapoptotic protein Mcl-1 but not of Bcl-2 and Bcl-XL. Furthermore, Mcl-1 expression was not sensitive to caspase inhibitors but was totally prevented by the lactacystin proteasome inhibitor, suggesting that IT-induced apoptosis may be triggered by a reduction in the Mcl-1 level. Mitochondrial membrane potential (DeltaPsi mito) decreased concurrently with caspase activation, showing the involvement of DeltaPsi mito as a regulator of IT-induced apoptosis. Our results demonstrate that 425.3PE-mediated cell death involves simultaneous induction of apoptosis and protein synthesis inhibition in MA-11 cells, thus contributing to an understanding of the mechanisms involved in IT-induced apoptosis.

Purpose: This study was undertaken to determine whether low intratumoral doses of the epidermal growth factor receptor ligand-transforming growth factor alpha (TGF-alpha) fused to Pseudomonas exotoxin (TGF-alpha-PE38)-abrogated head and neck squamous cell carcinoma (HNSCC) tumor growth in vitro and in vivo., Experimental Design: In vitro cytotoxicity assays were carried out to determine the sensitivity of HNSCC cells to TGF-alpha-PE38. TGF-alpha-PE38-treated HNSCC cells were examined by immunoblotting for cleaved poly(ADP-ribose) polymerase to evaluate apoptosis. Nude mice bearing established HNSCC xenografts were treated with several doses of TGF-alpha-PE38 to evaluate the antitumor efficacy in vivo. Tumor sections were stained with terminal deoxynucleotidyl transferase-mediated nick end labeling for apoptosis. To determine the effect of oral administration of TGF-alpha-PE38, gavage injections of TGF-alpha-PE38 were administered, and the esophagus and surrounding soft tissue were then stained for apoptotic cells., Results: HNSCC cell lines examined were sensitive to low doses of TGF-alpha-PE38 (EC(50) in the range of 1.6 to 10 ng/mL). HNSCC cells treated with TGF-alpha-PE38 undergo apoptosis. Antitumor effects were observed using 0.1 and 0.03 microg of TGF-alpha-PE38 administered intratumorally. At these doses, the treatment was well tolerated. Tumors treated with the toxin had a higher number of apoptotic cells compared with the control tumors. No apoptotic cells were observed in the pharyngoesophageal tissues of the mice after gavage administration of the toxin suggesting that the toxin could be orally administered without toxicity., Conclusions: These results indicate that topical or intratumoral administration of low doses of TGF-alpha-PE38 may demonstrate antitumor effects in HNSCC without associated systemic toxicity.

Aim: To explore the ability of recombinant toxin luteinizing hormone releasing hormone-Pseudomonas aeruginosa exotoxin 40 (LHRH-PE40) and LHRH binding to LHRH receptor (LHRHR) on the membrane surface of human liver cancer HEPG cells., Methods: LHRH was labeled by using (125)I with enzymatic reaction. The affinity and receptor volume of LHRH-PE40 and LHRH binding to LHRHR on the membrane surface of human liver cancer cells were measured with radioligand receptor assay., Results: The specific activity of LHRH labeled with (125)I was 2.7 x 10(4) kBq/microL, and its radiochemical purity reached to 99.2-99.7%. The binding of (125)I to LHRH was maximal for 240 min in the warm cultivation, and this binding was stabilized. The inhibiting rates of (125)I-LHRH and LHRH on the proliferation of human liver cancer HEPG cells were not significantly different. On the basis of the saturation curve of (125)I-LHRH binding to the membrane LHRHR of HEPG cells, (125)I-LHRH of 1 x 10(5) cpm was selected for radioligand receptor assay. The affinity constants (Kd) of LHRH-PE40 and LHRH binding to the membrane LHRHR of HEPG cells were 0.43+/-0.12 nmol/L and 4.86+/-1.47 nmol/L, respectively, and their receptor volumes were 0.37+/-0.15 micromol/g and 0.42+/-0.13 micromol/g, respectively. The binding of LHRH-PE40 to the membrane protein of normal liver cells was not observed., Conclusion: The recombinant toxin LHRH-PE40 binding to the membrane surface of LHRHR of human liver cancer HEPG cells was very strong, while the specific binding of it to normal liver cells was not observed. The results provide an important experimental basis for the clinical application of LHRH-PE.

We have shown previously that a chimeric fusion protein composed of human interleukin-13 (IL-13) and Pseudomonas exotoxin (PE), termed IL-13 cytotoxin (IL13-PE38), is specifically cytotoxic to various cancer cell lines and primary cell cultures derived from a variety of solid cancers. In addition, we have shown that IL-13 mutant IL-13E13K, in which glutamic acid (E) residue at position 13 of IL-13 molecule was substituted by a lysine (K), is a powerful antagonist of IL-13 and binds to IL-13 receptor with a higher affinity compared with wild-type IL-13. In this study, we have generated an IL-13 cytotoxin IL13E13K-PE38, in which IL-13 antagonist is fused to PE to determine whether this molecule has improved cytotoxicity to tumor cells compared with wild type (wt)IL13-PE38. Highly purified IL13E13K-PE38 was tested in various tumor cell lines including seven glioblastoma multiforme cell lines to compare its binding to the cells, in vitro cytotoxicity, in vivo antitumor activity, and safety in mouse model with wtIL13-PE38. IL13E13K-PE38 bound to U251MG and IL-13Ralpha2 chain-transfected tumor cell lines with 3 to 10 times higher affinity compared with wtIL13-PE38. However, IL13E13K-PE38 did not show higher cytotoxicity compared with wtIL13-PE38 in glioblastoma multiforme or any other cell lines tested. The antitumor activity of IL13E13K-PE38, when administered intraperitoneally to nude mice bearing U251 tumors, was also similar to wtIL13-PE38. Some improvement in antitumor activity was observed when lower doses of IL13E13K-PE38 were injected intratumorally in subcutaneous tumors. These results indicate that in general, IL13E13K-PE38 mediates similar cytotoxicity and antitumor activity to wtIL13-PE38 despite its improved binding affinity to IL-13 receptors.

Mast cells play an important role in both allergy and innate immunity. Recently, we demonstrated an active interaction between human mast cells and Pseudomonas aeruginosa leading to the production of multiple cytokines. Here, we show that both primary cultured human cord blood-derived mast cells and the human mast cell line HMC-1 undergo apoptosis as determined by single-stranded DNA (ssDNA) formation after stimulation with P. aeruginosa exotoxin A (ETA), a major toxin produced by this bacterium. ETA-induced ssDNA formation was completely inhibited by Z-VAD (where Z is benzyloxycarbonyl), which blocks multiple caspases, suggesting a role for caspases in this process. Active caspase-3 formation in mast cells after an ETA challenge was detected by both Western blotting and flow cytometry analysis. ETA-induced caspase-3 activity in human mast cells was demonstrated by the detection of a characteristic 23 kDa product of D4-GDI (where GDI is guanine nucleotide dissociation inhibitor), an endogenous caspase-3 substrate. Interestingly, a specific caspase-8 inhibitor, Z-IETD-fmk (where fmk is fluoromethyl ketone), blocked ETA-induced cleavage of D4-GDI, but a caspase-9 inhibitor (Z-LEHD-fmk) did not. Treatment of mast cells with caspase-3 inhibitor Z-DEVD-fmk or caspase-8 inhibitor Z-IETD-fmk reduced the generation of ssDNA induced by ETA, suggesting a role for caspase-8 and -3 in ETA-induced mast cell apoptosis. Furthermore, treatment of mast cells with ETA induced decreases of the short form and a long form (p43) of Fas-associated death domain protein (FADD)-like interleukin-1beta-converting enzyme (FLICE) (caspase-8)-inhibitory proteins (FLIPs), which are endogenous caspase-8 inhibitors. Taken together, these results suggest that ETA-induced mast cell apoptosis involves down-regulation of antiapoptotic proteins, FLIPs, and activation of caspase-8 and -3 pathways.

gamma-Aminobutyric acid (GABA)(B) receptor-mediated modulation of spontaneous GABA release onto Purkinje cells was investigated in cerebellar slices from 3- to 5-week-old mice. The GABA(B) receptor agonists baclofen and CGP 44533 each reduced the frequency of miniature inhibitory postsynaptic currents (mIPSCs), with no significant effect on mIPSC amplitude; together, consistent with a presynaptic site of action. The GABA(B) receptor antagonist CGP 55845 blocked baclofen-induced inhibition. The sulphydryl alkylating agent N-ethylmaleimide occluded baclofen effects, implicating G(i/o) subunits in mediating a GABA(B) G protein-coupled receptor pathway. Baclofen-induced inhibition persisted in the presence of Ba(2+), a blocker of K(+) channels, and Cd(2+), a blocker of Ca(2+) channel-mediated GABA release. Application of nominally Ca(2+)-free extracellular solutions reduced mIPSC frequency and amplitude; however, baclofen produced a significant inhibition in mIPSC frequency, further suggesting that this pathway was independent of Ca(2+) influx. Spontaneous GABA release was increased by the adenylate cyclase activator, forskolin, and the phorbol ester, phorbol 12,13-dibutyrate. However, baclofen-induced inhibition was not significantly changed in either condition. Baclofen action was also not affected by the adenylate cyclase inhibitor SQ 22536 or the protein kinase C inhibitor chelerythrine chloride. Baclofen still reduced mIPSC frequency in the presence of the polyvalent cation ruthenium red, which acts as a secretagogue here; however, baclofen-induced inhibition was reduced significantly. Furthermore, baclofen produced no clear inhibition during high-frequency mIPSCs bursts induced by the potent secretagogue alpha-Latrotoxin. Together, these results suggest that GABA(B) inhibition occurs downstream of Ca(2+) influx and may be mediated, in part, by an inhibition of the vesicular release mechanism.

Streptococcus pyogenes thiol proteinase, also known as streptococcal pyrogenic exotoxin B (SpeB), has been suggested to be a major virulence factor in S. pyogenes infection. SpeB was reported to induce apoptosis of host cells, but its mechanism of action is not yet fully understood. In this study, we examined the involvement of matrix metalloproteinases (MMPs) in SpeB-induced apoptosis. We first developed a large-scale preparation of recombinant SpeB and precursors of human MMP-9 and -2 (proMMPs) by using Escherichia coli Rosetta (DE3)pLysS and baculovirus-insect cell expression systems, respectively. Treatment with SpeB induced effective proteolytic activation of both proMMP-9 and -2. When RAW264 murine macrophages were incubated with SpeB-activated proMMP-9, the level of tumor necrosis factor alpha (TNF-alpha) in conditioned medium (CM), assessed by an enzyme immunoassay, was elevated. This increase was completely inhibited by addition of the MMP inhibitor SI-27 to the cell culture. The CM also produced marked induction of apoptosis of U937 human monocytic cells. Similarly, soluble Fas ligand (sFasL) was detected in CM of cultures of SW480 cells expressing FasL after treatment with SpeB-activated proMMPs; this CM also induced apoptosis in U937 cells. SpeB had a direct effect as well and caused the release of TNF-alpha and sFasL from the cells. SpeB-dependent production of MMP-9 and -2 and proapoptotic molecules (TNF-alpha and sFasL) was evident in a murine model of severe invasive S. pyogenes infection. These results suggest that SpeB or SpeB-activated MMPs contribute to tissue damage and streptococcal invasion in the host via extracellular release of TNF-alpha and sFasL.

Translocating protein and translocating peptides have therapeutic potential against tumors by exposing the cytotoxic domains of toxic proteins to the cell cytosol. The aim of this study is to investigate the effect of N-terminally fused PE translocating peptides on granzyme B (GrBa) activity. PE II-GrBa fusion protein genes were constructed by replacing N-terminal signal and acidic dipeptide sequence of human granzyme B gene with two truncated translocating sequences of Pseudomonas exotoxin A (PE II aa 280-364/358) by recombinant PCR, and then cloned into pIND inducible expression vector. The resulting pIND-PE II-GrBa expression vectors were co-transfected with assistant plasmid pVgRXR into HeLa cells through lipofectamine, followed by selection on G418 and zeocin. The resistant cells were collected and induced with ponasterone A. Western blot analysis demonstrated that ponasterone A induction caused the expression of PE II-GrBa fusion proteins, and indirect immunofluorescence detected giant sized multinucleated cells, suggesting cytoskeletal and mitotic abnormalities as reported in our previous studies. Western blot, enzymatic activity assay and cell counting analysis indicated that two types of PE II-GrBa fusion proteins were capable of cleaving both endogenous and exogenous substrates of granzyme B, and inhibiting the growth of cells. The PE II (aa 280-358)-GrBa was shown to have higher serine protease activity and stronger growth inhibitory effect. Such inhibition was presumably associated with G2 arrest as determined by cell cycle analysis. These data prove that PE II-GrBa fusion proteins have cell inhibitory effect similar to GrBa, and that the shorter PE-derived peptide exerts less influence on GrBa activity. This study helps to optimize the construction of recombinant protein comprising translocating peptides and cytotoxic molecules for tumor cell killing.

A recombinant immunotoxin named CEA/PE38/KDEL was constructed, which was composed of anti-CEA single-chain Fv and the truncated and modified form of Pseudomonas exotoxin (PE38/KDEL). The CEA/PE38/KDEL immunotoxin was expressed in the E. coli strain BL21 (DE3)-star as inclusion bodies. The denatured inclusion bodies were purified with Ni-NTA chelate agarose, then the constant gradient dialysis was used to perform the refolding of the CEA/PE38/KDEL immunotoxin. Results of FACS and MTT assay indicate that the refolded immunotoxins keep potent and specific cytotoxicity to tumor cells bearing CEA antigens.

Phaseolotoxin (PT) is a non-host specific phytotoxin produced by the plant pathogenic bacterium Pseudomonas syringae (P.s.) pv. phaseolicola. In the present study, the inhibitory effect of PT on the proliferation of leukemia cells was studied. After 4 days of treatment, PT decreased cell growth of leukemia cell lines HL-60, K-562 and L1210 in a dose-dependent manner. In addition, PT also reduced cell growth of the insulinoma pancreatic cell line RIN-m5F. IC50 values were 2.1 +/- 1.0 microM (HL-60), 13.3 +/- 3.7 microM (K-562), 2.5 +/- 0.4 microM (L1210) and 5.5 +/- 0.3 microM (RIN-m5F). Although the exact mechanism by which PT inhibits cell growth in these cells is currently not known, we present first evidence that PT may in part be active via inhibition of ornithine decarboxylase (ODC). Based on our findings, PT presents a lead compound with potential for further development into a new anti-cancer agent.

Although several immunotoxins that selectively kill HIV-1-infected cells have been described, their clinical utility is limited by low potency against spreading viral infection. We show here that changing the carboxyterminal sequence of an anti-HIV-1 envelope immunotoxin to the consensus endoplasmic reticulum retention sequence KDEL substantially improves its ability to block infection of peripheral blood mononuclear cells by primary HIV-1 isolates without increasing nonspecific toxicity. Polychromatic flow cytometry of peripheral blood mononuclear cells (PBMC) infected with an HIV-1-GFP reporter virus demonstrated that the improved immunotoxin is active against a variety of primary cell types including memory T cells, NK-T cells, and monocyte/macrophages. The subnanomolar potency of this agent suggests that it could be clinically useful either as an adjuvant to highly active antiretroviral therapy (HAART) in drug-resistant patients or to reduce the reservoir of latently infected cells that is implicated in HIV-1 persistence.

Pseudomonas syringae pv. phaseolicola synthesizes a non-host-specific toxin, phaseolotoxin, and also synthesizes a phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) to protect itself from its own toxin. ROCT is encoded by argK, which is expressed coordinately with phaseolotoxin synthesis at 18 degrees C. To investigate the regulatory mechanisms of this system, null mutants were constructed for argK, argF (encoding the phaseolotoxin-sensitive OCTase [SOCT]), and amtA (encoding an amidinotransferase involved in phaseolotoxin synthesis). The argF mutant did not exhibit arginine auxotrophy when grown in M9 medium at 28 degrees C, because under this condition SOCT was replaced by ROCT. This loss of thermoregulation of argK was apparently caused by accumulation of carbamoylphosphate, one of the substrates of SOCT. Carbamoylphosphate, which has a structure similar to that of the inorganic moiety of phaseolotoxin, was used in induction assays with wild-type P. syringae pv. phaseolicola and was shown to be able to induce argK expression in M9 medium at 28 degrees C. These results indicate that argK expression is independent of temperature and is regulated directly by a compound resembling the inorganic moiety of phaseolotoxin.

Purpose: A group B-streptococcus exotoxin (CM101) was administered following optic nerve injury in adult rats in order to analyze putative effects on macrophages, glial scar formation and regrowth of axons in the lesioned optic nerve., Methods: After a standardized intraorbital optic nerve crush, animals were randomized to treatment with CM101 (30 microm/kg body weight, iv, repeated every other day) or vehicle alone. Morphology (semithin sections) and immunohistochemistry directed towards macrophages (ED1), neurofilament (NF), astrocytes (GFAP) and regenerative sprouts (GAP43) were employed at different time-points up to 28 dpi., Results: A significant increase of ED1-positive macrophages (p < 0.05) was observed at 7, 14 and 28 dpi in treated animals compared to untreated, indicative of macrophage stimulation. Less degenerative structures were found in sections distal to the injury in treated animals, seemingly due to a pro-phagocytic effect. Reactive gliosis was significantly (p < 0.05) less pronounced in CM101-treated animals. The presence of GAP43-positive sprouts and neurofilament-positive neurites distal to the lesion in treated animals indicate regrowth of axons crossing the glial scar., Conclusion: Treatment with group B-streptococcus exotoxin leads to macrophage stimulation, increased phagocytosis of inhibitory debris, and a less dense reactive gliosis, which in turn allows for regrowth of axons through the glial scar.