Preparation of Diphtheria and Pseudomonas Exotoxin A Immunotoxins and Evaluation of Their Cytotoxicity Effect on SK-BR-3, BT-474, and MDA-MB-231 Breast Cancer Cell Lines.

Immunotoxin targeted therapy is a promising way of cancer therapy that is made from a toxin attached to an antibody which target a specific protein presented on cancer cells. In this study, we introduce immunotoxins comprising of truncated pseudomonas exotoxin A (PEA) and diphtheria toxin (DT) conjugated to trastuzumab. The effectiveness of 20 and 30 µg/ml immunotoxins and trastuzumab were studied on SK-BR-3 and BT-474 HER2/neu positive breast cancer cell lines by a cell death assay test. The produced immunotoxins have the potential to reduce the therapeutic dose of the trastuzumab and in the same time achieve higher efficiency.

The hidden nature of protein translational control by diphthamide: the secrets under the leather.

The protein translation elongation factor eEF2 undergoes a unique posttranslational modification called diphthamidation. eEF2 is an essential factor in protein translation, and the diphthamide modification has been a famous target of the diphtheria toxin for a long time. On the other hand, the physiological function of this rare modification in vivo remains unknown. Recent studies have suggested that diphthamide has specific functions for the cellular stress response and active proliferation. In this review, we summarize the history and findings of diphthamide obtained to date and discuss the possibility of a specific function for diphthamide in regulating protein translation.

[Whole cell protein profiling characterization of Corynebacterium diphtheriae clinical isolates collected from various infections].

INTRODUCTION: Corynebacterium diphtheriae can cause various infections such as diphtheria, wound infections, septic arthritis, bacteraemia and endocarditis. Different virulence properties of the isolates might be related to different virulence factors expressed by the isolates. The objective of this study was to explore whether whole cell protein profiling might be useful in prediction of pathogenic properties of C. diphtheriae isolates. METHODS: C. disphtheriae isolates collected from diphtheria, invasive and local infections and from asymptomatic carriers in Poland, France, New Caledonia and Canada in 1950-2014 were investigated using whole cell protein profile analysis. RESULTS: All the examined isolates were divided into two clades: A and B with similarity about 47%, but clade B was represented by only one isolate. The clade A was divided in two subclades A.I NS .II with similarity 53,2% and then into four groups: A.Ia, A.Ib, A.Ic and A.Id. The comparative analysis did not distinguish clearly toxigenic and nontoxigenic isolates as well as invasive and noninvasive isolates. CONCLUSIONS: Whole cell protein profile analysis of C. diphtheria exhibits good concordance with other genotyping methods but this method is not able to distinguish clearly invasive from non-invasive isolates.

An alternative method for purifying and detoxifying diphtheria toxin.

Infections caused by Corynebacterium diphtheriae frequently induce situations in which very small doses of antigens injected intradermally can cause strong inflammatory reactions. This bacterium secretes the diphtheria toxin (DT), a virulence factor that can be lethal to the human organism at doses below 0.1 µg/kg of body weight. The present work proposes alternative methods of DT purification using affinity chromatography and of DT detoxification through conjugating with the polymer methoxypolyethylene glycol activated (mPEG). Tests were performed to evaluate: the formation of edemas and the presence of dermonecrotic activity, in vitro cytotoxicity to Vero cells, the neutralizing activity of serum from guinea pigs immunized with the diphtheria toxoid inactivated with mPEG, and the immunogenic activity of the purified and modified toxin. The results indicated that purification with Blue Sepharose was an efficient method, yielding antigen purity equivalent to 2600 Lf/mg of protein nitrogen. The modification of the Purified Toxin with mPEG did not result in the formation of edema or necrosis although it was immunogenic and stimulated the formation of antibodies that could neutralize the Purified Toxin. The toxoid obtained from the purified toxin maintained its immunogenic characteristics, inducing antibodies with neutralizing activity; edema and necrosis were still observed, however.

Interplay between DtxR and nitric oxide reductase activities: a functional genomics approach indicating involvement of homologous protein domains in bacterial pathogenesis.

Corynebacterium diphtheriae pathogenesis depends on the production of toxin (Dtx), which in turn depends on a micromolar concentration of nitric oxide (NO)-mediated deactivation of DtxR (an iron-dependent regulator). Inside a host, the pathogen often encounters excess of NO that acts as an oxidative toxicant. Therefore a critical level of NO needs to be maintained by the pathogen. This necessitates reduction of excess NO by the presence of a reductase, namely nitric oxide reductase (NOR). Similar to the expression of toxin, the expression of NOR is possibly regulated by another regulator NorR, as has been found in other gram positive and gram-negative bacteria. Therefore, a correlation between concentration of NO on the deactivation of DtxR and transactivation of NorR becomes apparent. However, unlike many other pathogens the presence of NOR and NorR in C. diphtheriae has not been established. We applied a combination of bioinformatics and comparative genomics approach on C. diphtheriae genome using Escherichia coli as a model organism to find some structural and functional homologoues for the two genes in question. The various domain characteristics for the two proteins (NOR and NorR) have been taken into account in this analysis. Through extensive genome and proteome search we have been able to identify key regulatory genes, which are possibly involved in coordination and control of NO stress in C. diphtheriae. Our finding will progress the understanding of the complete regulatory mechanism for evasion and maintenance of pathogenesis by this and other pathogenic organisms.

[Expression of NADPH-diaphorase in the peripheral nerve and its changes at different stages of diphtheritic polyneuropathy]. / Ekspressiia NADPH-diaforazy v perifericheskom nerve i ee izmenenie na raznykh stadiiakh difteriinoi polineiropatii.

Distribution and intensity of NADPH-d reactivity, a marker for enzyme of the nitric oxide synthesis, in nervus suralis biopsies in severe DP were studied at light and electron microscopic levels. The study of control specimens has shown that NADPH-d reactivity was permanently present in Schwann cells (SC) and was distributed in all parts of their cytoplasm. Axon and myelin were devoid of NADPH-d reactivity. A decrease of enzyme reactivity in SC cytoplasm of the damaged nerve fibers and rising enzyme reactivity in the cytoplasm of activated SC were observed in DP. High reactivity in SC of small fibers was found at earlier stages and that of thick fibers at later stages. This distinction reflected, apparently, sequence of entering at first thin, then thick fibres in the reparative process. Under the electron microscope, the reaction product was deposited on membranes of endoplasmic reticulum, nuclear membrane and Golgi complex. The enzyme was also located in nucleus of activated SC. Ultrastructural location and the fact that the highest intensity of reaction is present in SC of nervous fibres with morphological signs of remyelination suggest link of this enzyme with the reparative process. This study provides the first evidence of NADPH-d reactivity in SC and shows that NADPH-d histochemistry is a useful tool for peripheral nerve biopsies study.

The protective effect of carnitine in human diphtheric myocarditis.

Carnitine, an important cofactor in the transport of fatty acids to the interior of cell mitochondria, is depleted in myocardial tissue of guinea pigs submitted to diphtheric toxin administration. Mortality rates were reduced in these animals by supplying exogenous amounts of carnitine. The accumulation of fatty acids in the cytoplasm of human heart cells reported in cases of diphtheria suggests that carnitine might possibly be depleted in human myocardium as well. For the purpose of studying the effect of carnitine administration, 132 diphtheric patients were randomly divided into two groups, one of them (carnitine-treated group, n = 73) receiving DL-carnitine, 100 mg/kg/day during 4 days after admission, in addition to routine treatment, which was prescribed for this and the control group (n = 59). The presence of myocardial damage was evaluated by clinical, electrocardiographic, radiological, and enzymatic criteria. Carnitine administration resulted in decreased incidence of heart failure (P = 0.0475), of pacemaker implants (P = 0.0256), and of lethality indexes due to myocarditis (P = 0.013). We suggest that carnitine can play an important role in the treatment of diphtheric patients.

[Synthesis and breakdown of pyridine nucleotide coenzymes in the cell nuclei of guinea pig kidneys in diphtheria intoxication]. / Sintez i raspad piridinnukleotidnykh kofermentov v iadrakh kletok pochek morskoi svinki pri difteriinoi intoksikatsii.

The effect of diphteria toxin on the synthesis and degradation of NAD+ and the hydrolysis of NADP+ in the nuclei of guinea pig kidney were studied. Treatment of animals with diphteria toxin (DT) results in considerable reduction of NADpyrophosphorylase activity, which starts 12 hours after incubation and is minimal (50% of that of the control animals) 18 hours after it. During this time interval DT does not affect the activity of NADase and decreases that of NADPase in the nuclei. Thus under diphterial intoxication a decrease of NADPase activity, the synthesis of NAD+ being reduced, probably provides for kidney cells more favourable conditions for maintenance of their synthetic reactions and for restoration of their normal metabolism. It was found that the non-ionic detergent Triton X-100 promotes the NADpyrophosphorylase activity in cell nuclei of intact animals by 40% (on the average) and does not affect the NAD+ synthesis in DT injected animals. Therefore DT not only reduces the nuclear NADpyrophosphorylase activity but also affects the nuclear envelope. The alteration of nuclei under DT treatment is suggested by the decrease of their histone content (30% on the average as compared with the control). The decrease of NADpyrophosphorylase activity under DT intoxication can be considered as an adaptive reaction limiting the availability of NAD+ as the substrate of the EF2 mono (ADP-ribosilation) which results in its unability to promote translation.

[Content of serotonin and histamine in rat submandibular gland under conditions of botulism and toxic diphtheria]. / Soderzhanie gistamina i serotonina v podcheliustnoi sliunnoi zheleze pri botulizme i toksicheskoi difterii u krys.

Content of histamine and serotonin was estimated spectrofluorimetrically in rat submandibular salivary glands under conditions of experimental botulinic and diphtheric intoxication. Decrease in secretion of saliva in botulism was due not only to parasympathetic denervation of the salivary glands but also to alterations in serotonin and, especially, histamine metabolism in the gland tissue. Content of these biogenic amines was almost unaltered after increase in secretion of saliva caused by blocking of the sympathetic nervous impulsation by diphtheria toxin.

[Morphologic reflection of protein biosynthesis disorders in the myocardial cells of rabbits with acute diphtheritic intoxication]. / Morfologicheskoe otrazhenie narusheniia biosinteza belkov v miokardial'nykh kletkakh krolikov pri ostroi difteriinoi intoksikatsii

The lethal dose of diphtheria toxin (IDLM per 1 kg of body weight) was injected to Chinchilla line adult rabbits. Histological and electron-microscopy investigations were carried out in time periods from 3 to 24 hours following the administration of the toxin. Degenerative and necrotic changes in the myocardial cells under conditions of the given experiment were not observed. In many muscular cells dilatation of the perinuclear spaces due to the thinning of the myofibrila layer moved to the periphery of the cell was noted. Electron-microscopy investigation of the perinuclear spaces showed a decrease in the number of mitochondria, reduction of the elements of sarcoplasmatic reticulum and disappearance of ribosomes. Cytochemically, in the opened cytoplasmatic matrix there were revealed numerous beta-granules of glycogen. In nuclei fragmentation of nucleoli with reduction of their granular component was observed. The changes described justify the conclusion that acute diphtheria intoxication at early stages of exposure to the toxin brings about impairment of the protein synthesis not only at the level of translation on cytoplasmatic ribosomes, as was suggested before, but at the level of ribosome formation in the nucleus as well.

Free fatty acid oxidation and carnitine levels in diphtheritic guinea pig myocardium.

Previous studies from this laboratory and by Wittels and Bressler have suggested that myocardial carnitine depletion and an accompanying decrease in fatty acid oxidation may contribute to the myocardial disease associated with diphtheria. In addition, administration of carnitine was found to prolong survival of diphtheritic guinea pigs and improve ventricular function in diphtheritic dogs. The present studies document the hypothesis that the defect in myocardial carnitine, directly assayed, could be repleted in diphtheritic guinea pigs to whom carnitine was administered intraperitoneally. The decreased fatty acid oxidation previously reported only for homogenates was confirmed in an isolated perfused diphtheritic guinea pig heart preparation. The addition of L-carnitine to this perfusate augmented fatty acid oxidation to normal levels. Taken together, these and previous studies would support a pathogenetic role for carnitine depletion in diphtheritic myocarditis and suggest the possibility of experimental therapy with L-carnitine.