Dermatoprotective effects of some plant extracts (genus Ficus) against experimentally induced toxicological insults in rabbits.

AIM: Present study was conducted to evaluate the dermatoprotective effects of plant extracts (Ficus religiosa, Ficus benghalensis, and Ficus racemosa) against known irritants such as sodium dodecyl sulfate (SDS), atrazine, and petrol. METHODS: The study was conducted in adult male rabbits. Ethanol extracts of plants were obtained through Soxhlet. All irritants and Ficus extracts were topically applied to the backs of rabbits daily for 4 days, while pure ethanol served as control. Skin was examined after 24, 48, and 96 h for erythema. Skin biopsies were taken on 5th day for microscopic examination. RESULTS: Erythema produced by irritants reduced significantly with the simultaneous application of Ficus extracts. The mean ± SEM epidermal thickness (micrometer) with SDS was 45.40 ± 1.89, F. religiosa + SDS was 18.60 ± 0.51, F. benghalensis + SDS was 18.40 ± 0.25, F. racemosa + SDS was 18.80 ± 0.37, and mixture of three Ficus species + SDS was 16.80 ± 0.37. Similar findings were revealed after using plant extracts with atrazine and petrol. The mean ± SEM epidermal layer count for SDS was 3.60 ± 0.25, atrazine was 3.40 ± 0.25, petrol was 3.40 ± 0.25, and ethanol (control) was 1.00 ± 0.20. This count reduced to 1.20 ± 0.20 for three Ficus species + SDS, 1.40 ± 0.25 for Ficus species + atrazine, and 1.40 ± 0.25 for Ficus species + petrol. CONCLUSION: Ficus species demonstrated the potential to block the dermatotoxic effects of topical irritants and could be used successfully to prevent skin toxicity.

The kiss of (cell) death: can venom-induced immune response contribute to dermal necrosis following arthropod envenomations?

Introduction: Snakes, insects, arachnids and myriapods have been linked to necrosis following envenomation. However, the pathways involved in arthropod venom-induced necrosis remain a highly controversial topic among toxinologists, clinicians and the public. On the one hand, clinicians report on alleged envenomations based on symptoms and the victims' information. On the other hand, toxinologists and zoologists argue that symptoms are incompatible with the known venom activity of target species. This review draws from the literature on arthropod envenomations, snakebite, and inflammatory processes to suggest that envenomation by a range of organisms might trigger an intense inflammatory cascade that ultimately lead to necrosis. If confirmed, these processes would have important implications for the treatment of venom-induced necrosis. Objectives: To describe two inflammatory pathways of regulated necrosis, tumour necrosis factor (necroptosis) and Neutrophil Extracellular Traps (NETosis); to discuss existing knowledge about snake venom and arachnid-induced necrosis demonstrating the involvement of tumour necrosis factor and neutrophils in the development of tissue necrosis following envenomation and to contribute to the understanding of venom-induced necrosis by arthropods and provide clinicians with an insight into little known inflammatory processes which may occur post envenomation. Methods: ISI Web of Science databases were searched using the terms "spider bite necrosis", "arthropod envenomation necrosis", "venom necrosis", "venom immune response", "loxoscelism", "arachnidism", "necroptosis venom", "necroptosis dermatitis", "tumour necrosis factor TNF venom", "scorpionism", "scolopendrism", "centipede necrosis", "NETosis venom", "NETosis necrosis". Searches produced 1737 non-duplicate citations of which 74 were considered relevant to this manuscript. Non-peer-reviewed sources or absence of voucher material identifying the organism were excluded. What is necrosis? Necrosis is the breakdown of cell membrane integrity followed by inflowing extracellular fluid, organelle swelling and the release of proteolytic enzymes into the cytosol. Necrosis was historically considered an unregulated process; however, recent studies demonstrate that necrosis can also be a programmed event resulting from a controlled immune response (necroptosis). Tumour necrosis factor and the necroptosis pathway: Tumour necrosis factor is a pro-inflammatory cytokine involved in regulating immune response, inflammation and cell death/survival. The pro-inflammatory cytokine TNF-α participates in the development of necrosis after envenomation by vipers. Treatment with TNF-α-antibodies may significantly reduce the manifestation of necrosis. Neutrophil Extracellular Traps and the NETosis pathway: The process by which neutrophils discharge a mesh of DNA strands in the extracellular matrix to entangle ("trap") pathogens, preventing them from disseminating. Neutrophil Extracellular Traps have been recently described as important in venom-induced necrosis. Trapped venom accumulates at the bite site, resulting in significant localized necrosis. Arthropod venom driving necrosis: Insects, myriapods and arachnids can induce necrosis following envenomation. So far, the processes involved have only been investigated in two arachnids: Loxosceles spp. (recluse spiders) and Hemiscorpius lepturus (scorpion). Loxosceles venom contains phospholipases D which hydrolyse sphingomyelin, resulting in lysis of muscle fibers. Subsequently liberated ceramides act as intermediaries that regulate TNF-α and recruit neutrophils. Experiments show that immune-deficient mice injected with Loxosceles venom experience less venom-induced inflammatory response and survive longer than control mice. Necrosis following Hemiscorpius lepturus stings correlates with elevated concentrations of TNF-α. These observations suggest that necrosis may be indirectly triggered or worsened by pathways of regulated necrosis in addition to necrotic venom compounds. Conclusions: Envenomation often induce an intense inflammatory cascade, which under certain circumstances may produce necrotic lesions independently from direct venom activity. This could explain the inconsistent and circumstantial occurrence of necrosis following envenomation by a range of organisms. Future research should focus on identifying pathways to regulated necrosis following envenomation and determining more efficient ways to manage inflammation. We suggest that clinicians should consider the victim's immune response as an integral part of the envenomation syndrome.

[Biological properties of heat-labile lethal toxin of Yersinia pseudotuberculosis].

The aim of the study was to assess the biological properties of heat-labile lethal protein toxin of Y. pseudotuberculosis. Toxin was extracted from Y. pseudotuberculosis strain 2517 type III serovar pYV-. The toxin killed mice 1-3 days after intraperitoneal administration (LD50=0.3 mcg of the protein). Heating at 56 degrees C during 30 min inactivated lethal activity of the toxin. It had a dose-dependent dermonecrotic effect during intracutaneous administration in rabbits. Hyperimmune rabbit serum to the toxin was obtained. Incubation of the toxin (LD100=1.2 mcg of the protein) with the serum at 37 degrees C during 30 min resulted in neutralization of lethal and dermonecrotic effects. The toxin did not have the cytotoxic effect on HeLa, Hep-2, and SPEV cells, but showed hemolytic activity to human and animal erythrocytes, and weak mitogenic activity to splenic cells of CBA line mice compared with control mitogen (concanavalin A).

Teleocidin, lyngbyatoxin A and their hydrogenated derivatives, possible tumor promoters, induce terminal differentiation in HL-60 cells.

Teleocidin isolated from Streptomyces mediocidicus, its catalytically hydrogenated compound dihydroteleocidin B, and lyngbyatoxin A isolated from marine blue-green alga Lyngbya majuscula as well as its hydrogenated product, tetrahydrolyngbyatoxin A were tested for their ability to induce differentiation of human promyelocytic leukemia cells (HL-60 cells) in culture. All of these indole alkaloids induced differentiation of HL-60 cells, characterized by increased phagocytosis, increased release of lysozyme, and morphological changes resembling macrophages. The concentrations required for the induction were 1-5 ng/ml, showing similarity to those of 12-O-tetradecanoylphorbol-13-acetate (TPA). Teleocidin, lyngbyatoxin A, and tetrahydrolyngbyatoxin A are suggested to be tumor promoters, like TPA and dihydroteleocidin B. The HL-60 cell system might be useful for screening for environmental tumor promoters.

Characterization of the dermal lesions induced by a purified protein from toxigenic Pasteurella multocida.

The dermonecrotic effect of purified Pasteurella multocida toxin (PMT) was studied sequentially in guinea pigs and rats. The skin reaction was initially an acute inflammatory reaction, with edema and emigration of neutrophils and a few eosinophils and diapedesis of some erythrocytes. Four hours after intracutaneous injection the vessels were congested and thrombocytes were focally attached to the endothelial wall. Twenty-four h after the injection the inflammatory reaction appeared more severe and venules and arterioles were thrombosed. Necrotic changes were seen in hair follicles and in striated muscle fibers. Crude extracts from P. multocida and Clostridium perfringens injected intracutaneously into guinea pigs induced skin lesions qualitatively similar to the lesions induced by the purified PMT, indicating that dermonecrotic bacterial toxins may share similar biochemical properties.

A two-stage mouse skin carcinogenesis study of lyngbyatoxin A.

A strong skin irritant, lyngbyatoxin A, isolated from the marine blue-green alga Lyngbya majuscula is structurally related to teleocidin. Since lyngbyatoxin A satisfied our short-term screening tests for possible tumor promoters, viz. irritation of mouse ear, induction of ornithine decarboxylase (ODC) in mouse skin, and adhesion of human promyelocytic leukemia cells (HL-60), a two-stage carcinogenesis experiment was carried out. Tumor incidences in the groups treated with 7,12-dimethylbenz(a)anthracene (DMBA) plus lyngbyatoxin A and with DMBA plus 12-O-tetradecanoylphorbol-13-acetate (TPA) were 86.7% and 93.3% in week 30, respectively. The average number of tumors per mouse was 3.7 in the former group and 10.5 in the latter group. This paper reports for the first time the potent tumor-promoting activity of lyngbyatoxin A and also the histological examination of tumors.

Prevalence of epidermolytic toxin in clinical isolates of Staphylococcus aureus.

One hundred and sixteen strains of Staphylococcus aureus isolated from exfoliative skin lesions were screened for their ability to produce different serotypes of epidermolytic toxin (ET). Toxin production was assessed by immunodiffusion, analytical isoelectric focussing and examination for the Nikolsky sign in mice. Of 84 strains of phage group II, 72 (85.7%) were toxinogenic as were 10 of 32 (31.3%) non-group-II strains. The ability to produce ET serotypes A and B was not confined to a particular phage group.

Effects of Bordetella bronchiseptica dermonecrotizing toxin on bone formation in calvaria of neonatal rats.

The effects of Bordetella bronchiseptica dermonecrotizing toxin on bone formation were investigated using a purified toxin preparation. Single injection of 4.3 ng of the toxin into the subcutaneous tissue overlying the calvariae of neonatal rats necrotized periosteum of parietal bone and degenerated osteoblasts within two days. Nine days after the injection, the lesion of the bone tissue became severe; the bone matrix became thin and fragmented. These observations indicate that dermonecrotizing toxin without other factors produced by the organisms impairs bone formation.

Dermonecrotic and lethal components of Loxosceles gaucho spider venom.

Loxosceles gaucho spider venom causes a typical dermonecrotic lesion in bitten patients and rarely causes lethal systemic effects. Gel filtration on Sephadex G 100 of L. gaucho spider venom resulted in three fractions: fraction A, containing the higher mol. wt components (approximately 35,000); fraction B, containing lower mol. wt components (approximately 15,000); and fraction C, containing very low mol. wt components (probably small peptides). The dermonecrotic and lethal activities were detected exclusively in fraction A. The venom and fraction A produced large dermonecrotic lesions in rabbits with necrosis spreading by gravity to the skin of the lateral body wall. Analysis by SDS-PAGE showed that the proteins contained in fraction A are approximately 35,000 and 33,000 mol. wt. Immunoblotting analysis showed that the proteins responsible for the dermonecrotic and lethal activity are very immunogenic and the first to be detected by antibodies during the course of immunization.

Characterization of dermonecrotic toxin produced by serotype D strains of Pasteurella multocida.

Dermonecrotic toxin (DNT) produced by serotype D strains of Pasteurella multocida, isolated from pigs, was characterized and compared with DNT produced by Bordetella bronchiseptica. The DNT prepared by sonication from P multocida or B bronchiseptica had dermonecrotic activity and lethal toxicity for guinea pigs and mice, and also induced marked atrophy of spleens in the mice. Toxicity of P multocida or B bronchiseptica DNT was completely inactivated by heating at 70 C for 30 minutes, and was reduced by treatment with trypsin, formalin, or glutaraldehyde, indicating that the DNT may be a protein. Although biologic and toxic properties of P multocida DNT were similar to those of B bronchiseptica DNT, cross-neutralization tests between P multocida and B bronchiseptica indicated that DNT from the 2 bacterial species were serologically distinct.

Intracellular locations of dermonecrotic toxins in Pasteurella multocida and in Bordetella bronchiseptica.

Location of dermonecrotic toxin (DNT) in the cells of Pasteurella multocida or Bordetella bronchiseptica was investigated. After cell lysis by various procedures, various fractions prepared from bacterial cells grown in liquid culture media were assayed for dermonecrotic activity by skin testing of guinea pigs. During the death phase of the growth tested for the 2 bacterial species, little cell-free DNT was detected in the culture supernatants. Throughout the log and stationary phases of the growth, DNT activity was cell associated, but was not seen in the culture supernatants, which indicated that DNT was not secreted by actively growing P multocida or B bronchiseptica cells. Little DNT was released by subjecting whole cells to osmotic shock, a common procedure that releases proteins from the periplasmic space of many gram-negative bacteria. After sonication and centrifugation of whole cells, a substantial amount of DNT was released; results were similar when spheroplasts were used instead of whole cells. Treatment of whole cells with trypsin did not decrease the DNT activity, but trypsin treatment of sonicated cells resulted in a significant decrease in the DNT activity (P less than 0.01). The results indicated an intracellular location of the DNT of P multocida or B bronchiseptica. The DNT of P multocida or of B bronchiseptica is probably located in the cytoplasmic space.

Induction of nasal turbinate atrophy in germ-free pigs, using Pasteurella multocida as well as bacterium-free crude and purified dermonecrotic toxin of P multocida.

To establish the role of the dermonecrotic toxin (DNT) of Pasteurella multocida in the cause and pathogenesis of atrophic rhinitis, germ-free pigs were inoculated with several strains of P multocida, crude DNT, or purified DNT. In some experiments, the aforementioned inocula were combined with Bordetella bronchiseptica. All DNT-producing P multocida strains induced severe turbinate atrophy. Histologic examination of the remnants of the nasal turbinates revealed intact, but undulated, ciliated epithelium and numerous osteoclasts. Inflammation was minimal or absent. A DNT-producing B bronchiseptica strain induced only mild turbinate atrophy. The lesions were characterized histologically by loss of cilia and ciliated cells and by an infiltration of predominantly mononuclear cells. Bone formation seemed impaired. Turbinate lesions were most severe in pigs infected with a combination of B bronchiseptica and a DNT-producing P multocida strain. Intranasal administration of sterile DNT-containing culture filtrate of P multocida or purified DNT of P multocida did not result in turbinate atrophy. In contrast, turbinate atrophy developed when these preparations were injected IM or when intranasal administration of DNT was preceded by inoculation of B bronchiseptica.

Histomorphologic features of the nasal cavity of pigs exposed to Pasteurella multocida type-D dermonecrotic toxin.

Microscopic examination of the nasal mucosa of clinically normal specific-pathogen-free pigs and of toxicogenic type-D Pasteurella multocida toxin challenge-exposed specific-pathogen-free pigs indicated that the surface epithelium in pigs of both groups was microscopically normal; erosions or appreciable inflammatory changes were not evident. In pigs of both groups and in all 3 regions of the nasal cavity, the endothelial lining of all blood vessels appeared normal without detectable changes to the walls at postinoculation day 10. Vascular injury in the cartilage or the bone was not discernible in control or challenge-exposed pigs. There were marked differences in the osseous structures of the conchae when the 2 groups were compared. In control pigs, active bone formation and remodeling were observed, and the septal cartilage was normal. In toxin challenge-exposed pigs, there likewise was normal bone formation and remodeling in the vestibular region, and the septal cartilage was normal. In marked contrast, conspicuous changes were observed in the osseous core of the conchae of the respiratory and, sometimes, the olfactory regions. These changes consisted of bone necrosis and resorption by large numbers of osteoclasts with variable replacement by dense mesenchymal stroma, which resulted in conchal atrophy. In the absence of any discernible damage or injury (angiopathy) to the nasal vessels, it appears that the action of the dermonecrotoxin of P multocida serotype D is on the most active osteoblasts and the associated organic matrix of the bone, with subsequent disruption of normal bone formation and remodeling of the nasal conchae.

[Isolation and certain properties of the dermonecrotic toxin from Pasteurella multocida]. / Vydelenie i nekotorye svoistva dermonekroticheskogo toksina Pasteurella multocida.

The procedure for isolation and purification of Pasteurella multocida serovariant D toxin has been described. It includes the three steps of protein precipitation from cultural filtrates by 70% ammonium sulfate, chromatography of the concentrated material on Ultragel AcA44 gel-filtration on Sephracryl S-200. The proposed technique permits one the 155-fold purification of the preparation with 32.6% yield estimated by biological activity. The obtained purified preparation is homogeneous in polyacrylamide gel electrophoresis. The immunological methods also confirm the homogeneity of the preparation. The minimal dermonecrotic dose for guinea pigs of the purified 120 kDa toxin is 78 ng and LD50 for mice is 280 ng. Pasteurella multocida toxin is found to be a thermolabile protein sensitive to trypsin, glutaraldehyde and formaldehyde treatments.

[The expression of the pathogenic properties of the Vibrio cholerae O139 serogroup in vitro]. / Ekspressiia patogennykh svoistv kholernykh vibrionov O139 serogruppy in vitro.

Cholera toxin, hemolysin, dermonecrotic and proteolytic factors have been detected and identified in V. cholerae O139. The production of these substances has been found to depend on the conditions of the cultivation of vibrios, and the types of proteases have been determined.

[The isolation of the Vibrio cholerae dermotoxin and the characterization of its biological properties]. / Vydelenie dermotoksina Vibrio cholerae i kharakteristika ego biologicheskikh svoistv.

The dermonecrotic factor (dermotoxin) inducing skin necrosis in rabbits has been isolated from V. cholerae strain B-53-2-38 and partially purified. Dermotoxin has a molecular weight of about 110 kD and possesses pronounced cytotoxic and general toxic action, differing from that of enterotoxin. The introduction of this factor into the blood and peritoneum of laboratory animals causes their death.

Spatial and thematic distribution of research on cyanotoxins.

Cyanobacteria in surface water are well known for their ability to form toxic blooms responsible for animal mortality and human poisoning. Accompanying major progress in science and technology, the state of knowledge of cyanotoxins has dramatically increased over the last two decades. The bibliometric approach applied in this study shows the evolution of research and identifies major gaps to be filled by future work. Although the publication rate has gradually increased from one hundred to three hundred articles per year since the 1990s, half of the literature available focuses on microcystins and another quarter on saxitoxins. Other cyanotoxins such as beta-N-methylamino-l-alanine or cylindrospermopsin remain vastly disregarded. Moreover, most of the publications deal with toxicity and ecology while other research areas, such as environmental and public health, require additional investigation. The analysis of the literature highlights the main journals for the communication of knowledge on cyanotoxins but also reveals that 90% of the research is originated from only ten countries. These countries are also those with the highest H-index and average number of citation per article. Nonetheless, the ranking of these countries is significantly altered when the amount of publications is normalized based on the population, the number of universities, the national gross domestic product or the government revenue. However, the lower amount of publications from Eastern Europe, Africa and South America could also reflect the lack of monitoring campaigns in these regions. This lack could potentially lead to the underestimation of the prevalence of toxic cyanobacterial blooms and the diversity of toxins worldwide.