Novel Prodrug-Like Fusion Toxin with Protease-Sensitive Bioorthogonal PEGylation for Tumor Targeting

Highly potent biotoxins like Pseudomonas exotoxin A (ETA) are attractive payloads for tumor targeting. However, despite replacement of the natural cell-binding domain of ETA by tumor-selective antibodies or alternative binding proteins like designed ankyrin repeat proteins (DARPins) the therapeutic window of such fusion toxins is still limited by target-independent cellular uptake, resulting in toxicity in normal tissues. Furthermore, the strong immunogenicity of the bacterial toxin precludes repeated administration in most patients. Site-specific modification to convert ETA into a prodrug-like toxin which is reactivated specifically in the tumor, and at the same time has a longer circulation half-life and is less immunogenic, is therefore appealing. To engineer a prodrug-like fusion toxin consisting of the anti-EpCAM DARPin Ec1 and a domain I-deleted variant of ETA (ETA″), we used strain-promoted azide alkyne cycloaddition for bioorthogonal conjugation of linear or branched polyethylene glycol (PEG) polymers at defined positions within the toxin moiety. Reversibility of the shielding was provided by a designed peptide linker containing the cleavage site for the rhinovirus 3C model protease. We identified two distinct sites, one within the catalytic domain and one close to the C-terminal KDEL sequence of Ec1-ETA″, simultaneous PEGylation of which resulted in up to 1000-fold lower cytotoxicity in EpCAM-positive tumor cells. Importantly, the potency of the fusion toxin was fully restored by proteolytic unveiling. Upon systemic administration in mice, PEGylated Ec1-ETA″ was much better tolerated than Ec1-ETA″; it showed a longer circulation half-life and an almost 10-fold increased area under the curve (AUC). Our strategy of engineering prodrug-like fusion toxins by bioorthogonal veiling opens new possibilities for targeting tumors with more specificity and efficacy.

The protease inhibitor alpha-2-macroglobulin-like-1 is the p170 antigen recognized by paraneoplastic pemphigus autoantibodies in human

Paraneoplastic pemphigus (PNP) is a devastating autoimmune blistering disease, involving mucocutaneous and internal organs, and associated with underlying neoplasms. PNP is characterized by the production of autoantibodies targeting proteins of the plakin and cadherin families involved in maintenance of cell architecture and tissue cohesion. Nevertheless, the identity of an antigen of Mr 170,000 (p170), thought to be critical in PNP pathogenesis, has remained unknown.

Amicrobial pustulosis-like rash in a patient with Crohn's disease under anti-TNF-alpha blocker

Amicrobial pustulosis of the folds (APF) is a recently described entity characterized by relapsing pustular lesions predominantly involving the cutaneous flexures and scalp. This disease typically occurs in association with systemic lupus erythematosus and a variety of other autoimmune diseases. We here describe an APF-like pustular eruption predominantly affecting the scalp, face and trunk, occurring during long-term infliximab treatment for Crohn's disease. Immunohistochemical staining of skin biopsy specimens for myxovirus resistance protein A, a marker for type 1 interferon-inducible proteins, showed increased staining in the epidermis and dermal mononuclear inflammatory infiltrate. Our observation further extends the spectrum of cutaneous adverse reactions potentially related to anti-tumor necrosis factor-α, the clinical context in which APF can occur as well as its clinical presentations.

Characterization of three human sec14p-like proteins: alpha-tocopherol transport activity and expression pattern in tissues

Three closely related human sec14p-like proteins (hTAP1, 2, and 3, or SEC14L2, 3, and 4, respectively) have been described. These proteins may participate in intracellular lipid transport (phospholipids, squalene, tocopherol analogues and derivatives) or influence regulatory lipid-dependent events. Here, we show that the three recombinant hTAP proteins associate with the Golgi apparatus and mitochondria, and enhance the in vitro transport of radioactively labeled alpha-tocopherol to mitochondria in the same order of magnitude as the human alpha-tocopherol transfer protein (alpha-TTP). hTAP1 and hTAP2 are expressed in several cell lines, whereas the expression level of hTAP3 is low. Expression of hTAP1 is induced in human umbilical cord blood-derived mast cells upon differentiation by interleukin 4. In tissues, the three hTAPs are detectable ubiquitously at low level; pronounced and localized expression is found for hTAP2 and hTAP3 in the perinuclear region in cerebellum, lung, liver and adrenal gland. hTAP3 is well expressed in the epithelial duct cells of several glands, in ovary in endothelial cells of small arteries as well as in granulosa and thecal cells, and in testis in Leydig cells. Thus, the three hTAPs may mediate lipid uptake, secretion, presentation, and sub-cellular localization in a tissue-specific manner, possibly using organelle- and enzyme-specific docking sites.

Hypoxia inducible factor-1 alpha induction by tumour necrosis factor-alpha, but not by toll-like receptor agonists, modulates cellular respiration in cultured human hepatocytes

BACKGROUND/AIMS: Genes encoding for some of the mitochondrial proteins are under the control of the transcriptional factor hypoxia inducible factor-1 alpha (HIF-1 alpha), which can accumulate under normoxic conditions in inflammatory states. The aim of this study was to evaluate the effects of cobalt chloride (CoCl(2), a hypoxia mimicking agent), tumour necrosis factor-alpha (TNF-alpha) and toll-like receptor (TLR) -2, -3 and -4 agonists on HIF-1 alpha accumulation, and further on HIF-1 alpha-mediated modulation of mitochondrial respiration in cultured human hepatocytes. METHODS: The human hepatoma cell line HepG2 was used in this study. Cells were treated with CoCl(2), TNF-alpha and TLR-2, -3 and -4 agonists. HIF-1 alpha was determined by Western blotting and mitochondrial respiration in stimulated cells by high-resolution respirometry. RESULTS: CoCl(2), TNF-alpha and TLR agonists induced the expression of HIF-1 alpha in a time-dependent fashion. TNF-alpha and CoCl(2), but not TLR agonists, induced a reduction in complex I-, II- and IV-dependent mitochondrial oxygen consumption. TNF-alpha-associated reduction of cellular oxygen consumption was abolished through inhibition of HIF-1 alpha activity by chetomin (CTM). Pretreatment with cyclosporine A prevented CoCl(2)-induced reduction of complex I- and II-dependent mitochondrial oxygen consumption and TNF-alpha-induced reduction of complex-I-dependent respiration, implicating the involvement of the mitochondrial permeability transition pore openings. TNF-alpha and TLR-2, -3 and -4 agonists induced the expression of vascular endothelial growth factor, which was partially abolished by the blockage of HIF-1 alpha with CTM. CONCLUSIONS: The data suggest that HIF-1 alpha modulates mitochondrial respiration during CoCl(2) and TNF-alpha stimulation, whereas it has no effect when induced with TLR-2, -3 and -4 agonists.

Anti-Alpha-2-Macroglobulin-Like-1 Autoantibodies Are Detected Frequently and May Be Pathogenic in Paraneoplastic Pemphigus

Paraneoplastic pemphigus (PNP) shows autoantibodies mainly to plakin and desmosomal cadherin family proteins. We have recently identified alpha-2-macroglobulin-like-1 (A2ML1), a broad range protease inhibitor, as a unique PNP antigen. In this study, we tested a large number of PNP sera by various methods. Forty (69.0%) of 58 PNP sera recognized A2ML1 recombinant protein expressed in COS7 cells by immunofluorescence (IF) and/or immunoprecipitation (IP)/immunoblotting (IB). IP/IB showed higher sensitivity than IF. In addition, 22 (37.9%) PNP sera reacted with A2ML1 by IB of cultured normal human keratinocytes (NHKs) under non-reducing conditions. Statistical analyses using various clinical and immunological data showed that the presence of anti-A2ML1 autoantibodies was associated with early disease onset and absence of ocular lesions. Next, to investigate the pathogenic role of anti-A2ML1 antibody, we performed additional functional studies. Addition of anti-A2ML1 polyclonal antibody to culture media decreased NHK cell adhesion examined by dissociation assay, and increased plasmin activity detected by casein zymography, suggesting that anti-A2ML1 antibody may decrease NHK cell adhesion through plasmin activation by inhibition of A2ML1. This study demonstrates that autoantibodies to A2ML1 are frequently and specifically detected and may have a pathogenic role in PNP.

'Pergularain e I'--a plant cysteine protease with thrombin-like activity from Pergularia extensa latex

Pergularain e I, a cysteine protease with thrombin-like activity, was purified by ion exchange chromatography from the latex of Pergularia extensa. Its homogeneity was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), native PAGE and reverse-phase high-performance liquid chromatography (RP-HPLC). The molecular mass of pergularain e I by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) was found to be 23.356 kDa and the N-terminal sequence is L-P-H-D-V-E. Pergularain e I is a glycoprotein containing approximately 20% of carbohydrate. Pergularain e I constituted 6.7% of the total protein with a specific activity of 9.5 units/mg/min with a 2.11-fold increased purity. Proteolytic activity of the pergularain e I was completely inhibited by iodoacetic acid (IAA). Pergularain e I exhibited procoagulant activity with citrated plasma and fibrinogen similar to thrombin. Pergularain e I increases the absorbance of fibrinogen solution in concentration-dependent and time-dependent manner. At 10 microg concentration, an absorbance of 0.48 was reached within 10 min of incubation time. Similar absorbance was observed when 0.2 NIH units of thrombin were used. Thrombin-like activity of pergularain e I is because of the selective hydrolysis of A alpha and B beta chains of fibrinogen and gamma-chain was observed to be insusceptible to hydrolysis. Molecular masses of the two peptide fragments released from fibrinogen due to the hydrolysis by pergularain e I at 5-min incubation time were found to be 1537.21 and 1553.29 and were in close agreement with the molecular masses of 16 amino acid sequence of fibrinopeptide A and 14 amino acid sequence of fibrinopeptide B, respectively. Prolonged fibrinogen-pergularain e I incubation releases additional peptides and their sequence comparison of molecular masses of the released peptides suggested that pergularain e I hydrolyzes specifically after arginine residues.

Lethal toxin of Clostridium sordellii is associated with fatal equine atypical myopathy

The lethal toxin of Clostridium sordellii (TcsL) evokes severe, mostly fatal disease patterns like toxic shock syndrome in humans and animals. Since this large clostridial toxin-induced severe muscle damaging when injected intramuscularly into mice, we hypothesized that TcsL is also associated with equine atypical myopathy (EAM), a fatal myodystrophy of hitherto unknown etiology. Transmission electron microscopy revealed skeletal and heart muscles of EAM-affected horses to undergo degeneration ultrastructurally similar to the damage found in TcsL-treated mice. Performing immunohistochemistry, myofibers of EAM-affected horses specifically reacted with sera derived from horses with EAM as well as an antibody specific for the N-terminal part of TcsL, while both antibodies failed to bind to the myofibers of either healthy horses or those with other myopathies. The presence of TcsL in myofibers of horses with EAM suggests that it plays a role as trigger or even as lethal factor in this disease.

Innate-like control of human iNKT cell autoreactivity via the hypervariable CDR3beta loop

Invariant Natural Killer T cells (iNKT) are a versatile lymphocyte subset with important roles in both host defense and immunological tolerance. They express a highly conserved TCR which mediates recognition of the non-polymorphic, lipid-binding molecule CD1d. The structure of human iNKT TCRs is unique in that only one of the six complementarity determining region (CDR) loops, CDR3beta, is hypervariable. The role of this loop for iNKT biology has been controversial, and it is unresolved whether it contributes to iNKT TCR:CD1d binding or antigen selectivity. On the one hand, the CDR3beta loop is dispensable for iNKT TCR binding to CD1d molecules presenting the xenobiotic alpha-galactosylceramide ligand KRN7000, which elicits a strong functional response from mouse and human iNKT cells. However, a role for CDR3beta in the recognition of CD1d molecules presenting less potent ligands, such as self-lipids, is suggested by the clonal distribution of iNKT autoreactivity. We demonstrate that the human iNKT repertoire comprises subsets of greatly differing TCR affinity to CD1d, and that these differences relate to their autoreactive functions. These functionally different iNKT subsets segregate in their ability to bind CD1d-tetramers loaded with the partial agonist alpha-linked glycolipid antigen OCH and structurally different endogenous beta-glycosylceramides. Using surface plasmon resonance with recombinant iNKT TCRs and different ligand-CD1d complexes, we demonstrate that the CDR3beta sequence strongly impacts on the iNKT TCR affinity to CD1d, independent of the loaded CD1d ligand. Collectively our data reveal a crucial role for CDR3beta for the function of human iNKT cells by tuning the overall affinity of the iNKT TCR to CD1d. This mechanism is relatively independent of the bound CD1d ligand and thus forms the basis of an inherent, CDR3beta dependent functional hierarchy of human iNKT cells.

Role of Toll interleukin-1 receptor (IL-1R) 8, a negative regulator of IL-1R/Toll-like receptor signaling, in resistance to acute Pseudomonas aeruginosa lung infection

Toll interleukin-1 receptor (IL-1R) 8 (TIR8), also known as single Ig IL-1 receptor (IL-R)-related molecule, or SIGIRR, is a member of the IL-1R-like family, primarily expressed by epithelial cells. Current evidence suggests that TIR8 plays a nonredundant role as a negative regulator in vivo under different inflammatory conditions that are dependent on IL-R and Toll-like receptor (TLR) activation. In the present study, we examined the role of TIR8 in innate resistance to acute lung infections caused by Pseudomonas aeruginosa, a Gram-negative pathogen responsible for life-threatening infections in immunocompromised individuals and cystic fibrosis patients. We show that Tir8 deficiency in mice was associated with increased susceptibility to acute P. aeruginosa infection, in terms of mortality and bacterial load, and to exacerbated local and systemic production of proinflammatory cytokines (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], IL-1β, and IL-6) and chemokines (CXCL1, CXCL2, and CCL2). It has been reported that host defense against P. aeruginosa acute lung infection can be improved by blocking IL-1 since exaggerated IL-1β production may be harmful for the host in this infection. In agreement with these data, IL-1RI deficiency rescues the phenotype observed in Tir8-deficient mice: in Tir8-/- IL-1RI-/- double knockout mice we observed higher survival rates, enhanced bacterial clearance, and reduced levels of local and systemic cytokine and chemokine levels than in Tir8-deficient mice. These results suggest that TIR8 has a nonredundant effect in modulating the inflammation caused by P. aeruginosa, in particular, by negatively regulating IL-1RI signaling, which plays a major role in the pathogenesis of this infectious disease.

A novel steroid-like compound F90927 exerting positive-inotropic effects in cardiac muscle

Here we report a novel steroid-like compound F90363, exhibiting positive inotropy in vivo and in vitro in various cardiac muscle preparations. F90363 is a racemic mixture composed of the stereoisomers (-)-F90926 and (+)-F90927. Only F90927 exerted positive inotropy, while F90926 induced a weak negative inotropy, but only at concentrations 10(3) times higher than F90927 and most likely resulting from an unspecific interaction. The rapid time course of the action of F90927 suggested a direct interaction with a cellular target rather than a genomic alteration. We could identify the L-type Ca2+ current I(Ca(L)) as a main target of F90927, while excluding other components of cardiac Ca2+ signalling as potential contributors. In addition, several other signaling pathways known to lead to positive inotropy (e.g. alpha- and beta-adrenergic stimulation, cAMP pathways) could be excluded as targets of F90927. However, vessel contraction and stiffening of the cardiac muscle at high doses (>30 microM, 0.36 mg kg(-1), respectively) prevent the use of F90927 as a candidate for drug development. Since the compound may still find valuable applications in research, the aim of the present study was to identify the cellular target and the mechanism of inotropy of F90927.

Cellular immunity in healthy volunteers treated with an octavalent conjugate Pseudomonas aeruginosa vaccine

Humoral immunity in response to an octavalent O-polysaccharide-toxin A conjugate Pseudomonas aeruginosa vaccine is well studied, and a phase III clinical study in cystic fibrosis (CF) patients is currently ongoing. In contrast, little is known about cellular immunity induced by this vaccine. Fifteen healthy volunteers were immunized on days 1 and 60. Parameters of cellular immunity were studied before vaccination on day 1, and on day 74. Analyses included flow cytometry of whole blood and antigen-induced proliferation of and cytokine production by lymphocyte cultures. The effects of immunization on the composition of peripheral blood lymphocytes as determined by flow cytometry were minor. In contrast, after immunization a highly significant increase of proliferation in response to stimulation with detoxified toxin A was noted: the stimulation index rose from 1.4 on day 1 to 42.2 on day 74 (restimulation with 0.4 microg/ml; P = 0.003). Immunization led to significant production of interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha by antigen-stimulated lymphocytes. In contrast, no significant induction of interleukin (IL)-4 or IL-10 was observed. In conclusion, immunization of healthy volunteers led to activation of cellular immunity including strong antigen-specific proliferation and cytokine production. In CF patients priming of the cellular immune system towards a Th1-like pattern would be of potential advantage. Therefore, confirmatory analyses in immunized CF patients with and without chronic infection with P. aeruginosa are foreseen.

CopY-like copper inducible repressors are putative 'winged helix' proteins

CopY of Enterococcus hirae is a well characterized copper-responsive repressor involved in copper homeostasis. In the absence of copper, it binds to the promoter. In high copper, the CopZ copper chaperone donates copper to CopY, thereby releasing it from the promoter and allowing transcription of the downstream copper homeostatic genes of the cop operon. We here show that the CopY-like repressors from E. hirae, Lactococcus lactis, and Streptococcus mutans have similar affinities not only for their native promoters, but also for heterologous cop promoters. CopZ of L. lactis accelerated the release of CopY from the promoter, suggesting that CopZ of L. lactis acts as copper chaperone, similar to CopZ in E. hirae. The consensus binding motif of the CopY-like repressors was shown to be TACAxxTGTA. The same binding motif is present in promoters controlled by BlaI of Bacillus licheniformis, MecI of Staphylococcus aureus and related repressors. BlaI and MecI have known structures and belong to the family of 'winged helix' proteins. In the N- terminal domain, they share significant sequence similarity with CopY of E. hirae. Moreover, they bind to the same TACAxxTGTA motif. NMR analysis of the N-terminal DNA binding domain of CopY of L. lactis showed that it contained the same alpha-helical content like the same regions of BlaI and MecI. These findings suggest that the DNA binding domains of CopY-like repressors are also of the 'winged helix' type.

Opioids trigger alpha 5 beta 1 integrin-mediated monocyte adhesion

Inflammatory reactions involve a network of chemical and molecular signals that initiate and maintain host response. In inflamed tissue, immune system cells generate opioid peptides that contribute to potent analgesia by acting on specific peripheral sensory neurons. In this study, we show that opioids also modulate immune cell function in vitro and in vivo. By binding to its specific receptor, the opioid receptor-specific ligand DPDPE triggers monocyte adhesion. Integrins have a key role in this process, as adhesion is abrogated in cells treated with specific neutralizing anti-alpha5beta1 integrin mAb. We found that DPDPE-triggered monocyte adhesion requires PI3Kgamma activation and involves Src kinases, the guanine nucleotide exchange factor Vav-1, and the small GTPase Rac1. DPDPE also induces adhesion of pertussis toxin-treated cells, indicating involvement of G proteins other than Gi. These data show that opioids have important implications in regulating leukocyte trafficking, adding a new function to their known effects as immune response modulators.