Orthogonal assembly of a designed ankyrin repeat protein-cytotoxin conjugate with a clickable serum albumin module for half-life extension.

The generation of drug conjugates for safe and effective tumor targeting requires binding proteins tolerant to functionalization by rational engineering. Here, we show that Designed Ankyrin Repeat Proteins (DARPins), a novel class of binding proteins not derived from antibodies, can be used as building blocks for facile orthogonal assembly of bioconjugates for tumor targeting with tailored properties. DARPin Ec1, which targets the Epithelial Cell Adhesion Molecule (EpCAM), was genetically modified with a C-terminal cysteine for conjugation of the small molecule cytotoxin monomethylauristatin F (MMAF). In addition, it was N-terminally functionalized by metabolic introduction of the non-natural amino acid azidohomoalanine to enable linkage of site-specifically dibenzocyclooctyne-modified mouse serum albumin (MSA) for half-life extension using Cu(I)-free click chemistry. The conjugate MSA-Ec1-MMAF was assembled to obtain high yields of a pure and stable drug conjugate as confirmed by various analytical methods and in functional assays. The orthogonality of the assembly led to a defined reaction product and preserved the functional properties of all modules, including EpCAM-specific binding and internalization, FcRn binding mediated by MSA, and cytotoxic potency. Linkage of MMAF to the DARPin increased receptor-specific uptake of the drug while decreasing nonspecific uptake, and further coupling of the conjugate to MSA enhanced this effect. In mice, albumin conjugation increased the serum half-life from 11 min to 17.4 h, resulting in a more than 22-fold increase in the area-under-the-curve (AUC). Our data demonstrate the promise of the DARPin format for facile modular assembly of drug conjugates with improved pharmacokinetic performance for tumor targeting.

Neutralization by human serum samples of a transmissible agent isolated from the cerebrospinal fluid of neurological patients.

A transmissible cytotoxic agent thought to be associated with one or more misfolded protein(s) was found in several cerebrospinal fluid (CSF) samples from neurological patients. Since some experiments carried out to identify this unusual infectious factor showed the block of its propagation by rabbit gammaglobulins (IgGs), the search for such an activity by human IgGs was programmed. Neutralizing assays carried out using human sera as IgGs source showed a blocking property displayed by: twenty serum samples from as many patients with a diagnosis of acute infection, two of ten sera from healthy subjects and four serum samples from patients with lupus erythematous (SLE). When neutralizing sera were tested on cell cultures in immunofluorescence assays for the serum ability to label specific protein( s), similar fluorescent pictures resulted in treated and control cells. On the other hand, the SLE serum samples disclosed a granulosity of the nuclear material of cytotoxic cells in accordance with the DNA apoptotic laddering reported in previous papers. Oxidative disorders, as suggested by the immunoblotting analysis of the antioxidant enzymes Mn-superoxide dismutase (SOD2) and heme-oxygenase 1 (HO-1), point to an alteration of the oxidative pathway among the causes of the DNA damage induced by the cytotoxic transmissible agent under study.

Frequency of vacuolating cytotoxin A (VacA)-positive Helicobacter pylori seropositivity and TGF-ß1 decrease in atrial fibrillation.

The study was performed to determine whether there were any associations of VacA positive Helicobacter pylori and TGF-ß1 with atrial fibrillation (AF). The serum levels of antibodies to H. pylori and VacA, and cytokines were assessed using ELISA in 96 subjects. While elevated levels of TNF-α, IL-6 and CRP were associated with AF, TGF-ß(1) was significantly lowered in AF patients (p=0.021). In addition, AF was associated with elevated levels of antibodies to VacA (p=0.023), compared to the control group. Accordingly, the chronic infection of VacA(+)H. pylori may increase the risk for AF by inducing systemic inflammation mediated, partly by suppressed TGF-ß(1) and elevated proinflammatory cytokines.

[Analysis of the etiological structure of sexually transmitted infections and immunological responsiveness in women with papillomavirus infection of the cervix uteri].

Two sexually transmitted infections or more were more frequently encountered in persistent papillomavirus infection (PVI) than those in transient PVI. The found immunological parameters in PVI arrested further infection progression, suppressed the persistence of human papillomavirus infection types 16 and 18, and prevented related cancer. This might eliminate the virus from the body.

Serologic evidence for effective production of cytolysin A in Salmonella enterica serovars typhi and paratyphi A during human infection.

ClyASTy and ClyASPaA are closely related pore-forming cytolysins of Salmonella enterica serovars Typhi and Paratyphi A whose expression is strongly repressed under standard in vitro growth conditions. We show here that human infections by these pathogens cause a specific antibody response to ClyA, indicating effective toxin production during infection.

Antimicrobial and cytolytic peptides of venomous arthropods.

As a response to invading microorganisms, the innate immune system of arthropods has evolved a complex arrangement of constitutive and inducible antimicrobial peptides that immediately destroy a large variety of pathogens. At the same time, venomous arthropods have developed an additional offensive system in their venom glands to subdue their prey items. In this complex venom system, several enzymes, low-molecular-mass compounds, neurotoxins, antimicrobial and cytolytic peptides interact together, resulting in extremely rapid immobilization and/or killing of prey or aggressors. This review provides an overview of antimicrobial peptides identified in the hemolymph of venomous arthropods, and especially of cytolytic peptides in their venom. For these peptides a dual role is proposed: acting as antimicrobials as well as increasing the potency of the venom by influencing excitable cells.

[Cytotoxic activity of natural killer cells in children with chronic type C hepatitis]. / Aktywnosc cytotoksyczna komórek NK u dzieci chorych na przewlekle zapalenie watroby typu C.

UNLABELLED: The aim of this study was to evaluate the activity of NK cells in peripheral blood in children with chronic type C hepatitis, and to determine the correlation between the activity of NK cells and histopathological changes in the liver. The study included 25 children with chronic type C hepatitis. The control group consisted of 10 children without liver diseases in past medical history and normal activity of amino-transferases. In all children the activity of NK cells was evaluated in relation to leukaemic cells of erytroleukaemia K-562 without stimulation and after stimulation with IL-2 (in vitro), and the percentage and absolute count of NK cells in peripheral blood were determined by flow cytometry. RESULTS: In the studied group of children NK cells accounted for 11.68 +/- 6.73% of peripheral white blood cells, and their count was 241.08 +/- 128.56/ml. The cytotoxic activity of NK cells was 91.8 +/- 1.07% in solution E:T 25:1; and 6.72 +/- 3.68% in solution E:T 12.5:1. After stimulation with IL-2 it was 92.8 +/- 1.01% and 7.58 +/- 3.95%, respectively. The count of NK cells in serum and cytotoxic activity of NK cells in the studied group were not different from those of the control group. CONCLUSIONS: Key parameters of NK cells activity are not changed in children with chronic type C hepatitis.

Endotoxin and cytokine removal in sepsis.

Sepsis, the leading cause of mortality in intensive care units, is a complex series of interrelated effects caused by the overproduction of multiple mediators and their unrestrained biological activity. Both proinflammatory and antiinflammatory mediators participate in the high complexity of sepsis and explain the failure of specific therapies to improve survival. Continuous extracorporeal therapies have been proposed as therapeutic options and as tools for blood purification in sepsis. Along these lines and in order to achieve higher clearances and mass removal rates, we studied the effects of plasmafiltration coupled with adsorption and provided in vitro and in vivo evidence that adsoprtion of multiple cytokines, activated complement components, and lipid mediators such as the platelet-activating factor occurs. We also showed that such treatment may lead to improved survival in a rabbit model of sepsis and to improved hemodynamics, reduced norepinephrine dose, and restoration of near-to-normal responsiveness of blood leukocytes to endotoxin in humans. It is anticipated that treatment of plasma, as a modular device to conventional hemofiltration, may pave the way to innovative approaches in the extracorporeal treatment of septic patients.

Helicobacter pylori infection, the cytotoxin gene A strain, and carotid artery intima-media thickness.

BACKGROUND: Chronic Helicobacter pylori infection has been implicated as a risk factor for stroke and other cardiovascular disease. It has been suggested that it acts by promoting atherogenesis. A particular strain of H. pylori, the cytotoxin gene A strain (CagA strain), which has been associated with increased inflammation, has been reported to account for much of this relationship. An early estimate of atherosclerosis can be obtained by ultrasonic imaging of the carotid artery to determine intima-media thickness (IMT). We determined whether H. pylori infection, and particularly the CagA strain, is associated with increased IMT. METHODS: In 983 normal population individuals we measured common carotid artery (CCA) IMT. From serum samples we determined H. pylori and CagA status. RESULTS: There was no significant association between H. pylori seropositivity and CCA IMT after controlling for age and gender. There were significant relationships between H. pylori seropositivity and a number of conventional cardiovascular risk factors (age, systolic and diastolic blood pressure, body mass index, smoking) and after controlling for these the magnitude of the regression coefficient was reduced by a further half. Amongst H. pylori seropositives the CagA strain was associated with increased IMT after controlling for age and gender (B = 0.0256, 95% CI 0.001-0.050, P = 0.038). The relationship was no longer significant after controlling for other cardiovascular risk factors. (B = 0.0194, 95% CI -0.004-0.042, P = 0.098). CONCLUSIONS: H. pylori and the CagA strain are not major risk factors for early arteriosclerosis as assessed by carotid artery intima-media thickness.

[CagA and VacA cytotoxin antibodies and risk for peptic ulcer disease in patients with Helicobacter pylori infection]. / Anticuerpos frente a las citotoxinas CagA y VacA y riesgo de úlcera péptica en pacientes con infección por Helicobacter pylori.

BACKGROUND: The presence of cagA antibodies constitutes a serum marker of infection caused by virulent strains of Helicobacter pylori. The objective of this study was to determine the risk of peptic ulcer in patients with H. pylori infection, in relation to the detection of CagA and VacA antibodies. PATIENTS AND METHOD: Prospective case-control study including 104 peptic ulcer patients with active H. pylori infection (positive urease test and/or histology, or positive urea breath test) and 104 age- and sex-matched controls, without peptic ulcer history, with active H. pylori infection (positive urea breath test). Serum CagA and VacA antibodies were determined by Western blot. Non-steroidal antiinflamatory drugs (NSAID) use was determined by structured data collection. A multivariate analysis (logistic regression) was carried out to determine the odds ratio (OR). RESULTS: Presence of serum antibodies against CagA was higher in peptic ulcer patients (74%) than in controls (46.2%) (OR = 5.7; 95% CI = 2.1-15.6). However, presence of serum antibodies against VacA in patients (46.2%) was similar to that in controls (36.5%). NSAID use was also more frequent in patients (51.9%) than in controls (21,2%) (OR = 6.5; 95% CI = 2.2-19,5). CONCLUSIONS: Serum antibodies against CagA and use of NSAID are the most important risk factors for peptic ulcer disease.

Virulence properties of motile aeromonads isolated from farmed frogs Rana tigerina and R. rugulosa.

Virulence factors were compared in Aeromonas species isolated from clinically normal and septicaemic farmed frogs from Thailand. Haemolysin activities against frog erythrocytes were significantly different within the collection of aeromonads. Groups of high haemolytic activity (unspeciated Aeromonas, Au), moderate haemolytic activity (A. hydrophila), and low haemolytic activity (A. veronii biovar sobria, A. veronii biovar veronii, A. caviae, A. schubertii) were noted. DNA colony hybridisation studies revealed that Au isolates possessed a haemolysin gene (ASH1) which was not present in any of the other Thai aeromonads or type strains tested. Elastinolytic activity was demonstrated in 90% of the Au isolates, 60% of the A. hydrophila isolates and in none of the other motile aeromonads. The cytotoxic activity of the Aeromonas isolates varied according to the source of cells used in the assays. Cells from rainbow trout were extremely sensitive to Au toxins but less so to toxins produced by other species. In contrast mammalian cells showed very little sensitivity to Au toxins but were more sensitive to toxins produced by A. hydrophila. Selection of suitable assay substrates is therefore important.

Lysine 77 is a key residue in aggregation of equinatoxin II, a pore-forming toxin from sea anemone Actinia equina.

Among eighteen point mutants of equinatoxin II produced in E. coli, containing a single cystein substitution at variable position, EqtIIK77C was chosen for its peculiar properties. It was almost 100 times less hemolytic than the wild-type, but its hemolytic activity could be restored by chemical modification of the thiol group, provided that a positive charge was reintroduced. This indicates that a positive charge at this position is necessary for toxin activity. The mutant formed larger pores as compared to the wild type, but displayed the same cation selectivity. The pores reverted to normal size upon reintroduction of the positive charge. The conformation of EqtIIK77C and its binding to lipid membranes, either vesicles or red blood cells, was almost normal. However the kinetics of calcein release from lipid vesicles was substantially slower than that of the wild-type. Taken together with the different size of the pore formed, this is an indication that mutation of Lys77 --> Cys influences the normal development of the aggregate which is required for assembling the functional pore.

Serum cytotoxin and oxidant stress markers in N-acetylcysteine treated thioacetamide hepatotoxicity of rats.

N-acetylcysteine (NAC) is a glutathione precursor used to treat several clinical conditions where intracellular oxidant-antioxidant balance is disturbed, among which, acetaminophen induced hepatotoxicity may be counted. In this study, administering thioacetamide (TAA) as a hepatotoxic agent, a rat model of hepatotoxicity has been established, to investigate some of the immune mediated basic oxidant-antioxidant homeostatic mechanisms involved, and potential serum markers for follow-up of disease and treatment. To do this, four experimental groups receiving saline/saline, saline/NAC, saline/TAA and NAC/TAA as intraperitoneal injections, have been formed. Rat serum tumor necrosis factor-alpha (TNF-alpha), Interleukin1-beta (IL1-beta), malondialdehyde (MDA) as a measure of final oxidant damage and the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) have been assayed. Hepatocellular damage has been measured via the biochemical estimates ALT, AST and LDH as well as histopathological grading. It was found that both TNF-alpha and IL1-beta were significantly elevated in saline/TAA receivers (P<0.01) when compared to NAC/TAA receivers. Serum MDA was also increased in TAA receivers in addition to SOD (P<0.05) and GSH-Px (P<0.05). Serum nitrite levels have also been assayed to give an estimate of nitric oxide that is suggested as a counter-balancer of oxidant stress. NAC/saline receivers had the highest levels of nitrites in the serum (P<0.05). Our results indicate that part of the hepatocellular injury to rat liver, induced by TAA is mediated by oxidative stress caused by the action of cytokines imparted by the enzymatic SOD and GSH-Px and non-enzymatic gaseous nitric oxide mechanisms causing an alleviation on administration of NAC. In addition, TNF-alpha, IL1-beta, MDA, SOD, GSH-Px and nitrites are potential candidates of serum indicators for monitorization of pathophysiological stage of liver disease.

Capability of serum to convert streptomycin to cytotoxin in patients with aminoglycoside-induced hearing loss.

Individual variations in sensitivity to the ototoxic effects of aminoglycoside antibiotics are well documented. Our research demonstrates that there is an apparent difference in serum from patients who are resistant or susceptible to aminoglycoside ototoxicity. In the first study, the cytotoxicity of sera from patients with and without hearing loss after various time periods following the discontinuation of aminoglycoside treatment was assayed using the isolated outer hair cell toxicity assay. The results indicate that sera from patients with hearing loss were significantly more toxic than sera from patients with normal hearing or minimal hearing loss. This toxicity may persist for up to 1 year after discontinuation of aminoglycoside therapy. In a second study, sera were obtained from patients who had received aminoglycoside therapy several years previously. None of these sera was toxic to isolated outer hair cells in vitro. Streptomycin was then incubated with the sera or a protein fraction isolated from sera, and the incubation mixtures were tested for toxicity. The percentage of damaged outer hair cells was significantly higher when streptomycin had been treated with sera or a serum protein fraction from patients with hearing loss (58+/-10% and 68+/-9%, respectively) than with sera or a serum protein fraction from a control group (10+/-5% and 17+/-4%, respectively). In addition, several incubation mixtures were analyzed using high performance liquid chromatography. A new chromatographic peak was only found in the incubations of streptomycin with serum protein from patients with hearing loss. The results suggest that sera from individuals sensitive to aminoglycoside antibiotics may metabolize these drugs to cytotoxins.

Cytokines and anticytokines in the pathogenesis of sepsis.

Clinical trials with anti-inflammatory agents in patients with sepsis are based on the assumption that excessive proinflammatory activity of the cytokine network negatively influences the outcome of severe bacterial infections. The failure of these trials to show clinical benefit, in conjunction with recent experimental data, raises doubt about the validity of this assumption. This article reevaluates the role of cytokines in the pathogenesis of sepsis and severe bacterial infections. The cytokine network is discussed as consisting of proinflammatory cytokines, antiinflammatory cytokines, and soluble inhibitors of proinflammatory cytokines.

Cytotoxic factor induced in murine serum after intravenous administration of a dehydrogenation polymer of p-coumaric acid (a synthetic lignin).

A cytotoxic factor (CF) toward cultured murine leukemia L1210 cells was induced in mouse serum by intravenous injection of a dehydrogenation polymer of p-coumaric acid (DHP-pCA). When the serum from the treated mice was diluted with ethanol, CF was preserved in its supernatant (EtOH-sup). An EtOH-sup prepared from untreated control mice also showed cytotoxicity, although at much higher concentrations. The CF activity of EtOH-sups from both treated and untreated mice was completely eliminated by acid treatment at pH 2 at 90 degrees C for 30 min but kept intact by alkali treatment. In addition, the CF activity of both EtOH-sups was not affected by digestion with chymotrypsin. CF was recovered in a neutral MeOH-eluate from a DEAE-cellulofine column but not in HCI-MeOH eluate, in which lignified materials including DHP-pCA should have been recovered. These findings strongly suggest that CF is not a metabolite of DHP-pCA but an endogenous component of the normal serum which is augmented by DHP-pCA administration.

Dietary Fusarium moniliforme culture material induces in vitro tumor necrosis factor-alpha like activity in the sera of swine.

Sera obtained from a group of pigs (n = 5) fed a diet amended with fumonisin containing Fusarium moniliforme culture material was used to determine the levels of Tumor Necrosis Factor-Alpha (TNF) activity by a functional bioassay utilizing the TNF sensitive WEHI 140 mouse fibrosarcoma cell line. Two pigs developed signs consistent with pulmonary edema which was confirmed by pathologic examination in only one pig. Significant, time dependent increases in TNF-like activity were observed in all pigs during the five days of the trial. Another group of pigs (n = 5) was given a defined daily dose of the same culture material by gastric intubation. Two pigs developed fulminant pulmonary edema and sharp increases in TNF activity were observed during the 3 days of the trial in all pigs. In both cases the activity was not abrogated by addition of a neutralizing anti-human TNF monoclonal antibody suggesting that other factors may have been responsible for these effects, possibly the increased levels of sphingoid bases in the serum. Since the pig has become an important model in the study of TNF mediated endotoxic shock, these studies illustrate the relevance of certifying the absence of this important mycotoxin from corn based animal diets, specially if functional assays are used to monitor the activity of TNF in serum.

Cytotoxic protein from human platelets.

The 14 kDa protein was purified from human platelets. It displays high cytotoxic activity to the human ACL cells at 10 M concentration (21.8+/-7.1%). Its N-terminal sequence is YAPQXQFGP-, being highly homologous to region 241-249 residues of the human Cls complement component.