Can mesenchymal stem cell lysate reverse aging?

Recent findings regarding uses of adipose-derived mesenchymal stem cell (MSC)-lysate on weight loss and improved glucose tolerance in mice on a high-fat diet suggest an encouraging possibility of using MSC lysate for an anti-aging intervention in humans. However, weight loss and lipopenia during late life can be as life-threatening as hyperglycemia during early adulthood. For this 3-year lifelong experiment, a total of 92 rats were randomized into the vehicle-injected group (F=22; M=24) and the MSC lysate injected group (F=22, M=24). We examined longevity, spontaneous locomotor activity, and body composition in rats maintained on a normal diet and received an intermittent treatment of human adipose-derived MSC lysate (3 times a week, 11 times a month given every second month), starting at 12 months of age until natural death. In substantiating previous knowledge regarding the effects of long-term MSC lysate treatments on fat loss and insulin resistance, the present findings also highlighted a shortened average lifespan, a longer inactive time, and a greater bone loss with a relative increase of lean mass in MSC lysate rats with respect to controls. Conclusion: Our data suggest that MSC lysate treatments stimulate disparity in tissue development and produce a cachexia-like effect to decrease longevity.

Exposure to Anisakis extracts can induce inflammation on in vitro cultured human colonic cells.

Anisakis spp. is a parasitic nematode whose infective third-stage larvae may be found within the flesh of fish species commonly consumed by humans. Thorough cooking or freezing should render the fish safe for consumption; furthermore, marinating solutions containing biocidal agents might have a significant action against Anisakis larvae. Some studies suggest a relationship between some parasitic infections and development of inflammatory bowel disorders, and Anisakis infection might be a risk factor for stomach or colon cancer. The aim of our study was to investigate if crude extracts (CEs) obtained from Anisakis larvae marinated in a solution with added allyl isothiocyanate (ACE-AITC) and frozen, or from frozen only Anisakis larvae (ACE), can induce an inflammatory effect on in vitro differentiated colonic Caco-2 cells exposed or not to LPS. Caco-2 exposure to the two CEs induced a marked COX-2 expression and potentiated LPS-induced COX-2 overexpression, confirming that substances present in Anisakis larvae can induce an inflammatory response in the intestinal epithelium, possibly also exacerbating the effects of other inflammatory stimuli. ACE induced a marked decrease in caspase-3 activation, while AITC-ACE increased its activation. However, LPS-induced caspase-3 activation appeared lower in cells treated with ACE and with the lower concentration of AITC-ACE. Thus, it is evident that Anisakis CEs may affect various cell pathways crucial not only in the inflammatory process but also in cell growth and death. Thus, CEs obtained from nonviable Anisakis larvae retain or are otherwise provided with noxious properties able to induce a strong inflammation response in intestinal epithelial cells. Furthermore, their influence may persist also following pretreatment with the biocidal agent AITC, indicating that the harmful substances contained in crude extracts from Anisakis larvae are resistant to the thermal or biocidal agent treatments.

Cathelicidin related antimicrobial peptide, laminin, Toll-like receptors and chemokines levels in experimental hypersensitivity pneumonitis in mice.

INTRODUCTION: Hypersensitivity pneumonitis (HP) is an interstitial lung disease caused by unresolved inflammation and tissue repair pathologies triggered by repeated organic dust exposure. The aim of the study was to investigate changes in levels of the cathelicidin related antimicrobial peptide (CRAMP), laminin (LAM-A1), selected Toll-like receptors (TLR) and chemokines in experimental HP in mice. MATERIALS AND METHODS: Three and 18-month-old female C57BL/6J mice underwent inhalations of the saline extract of Pantoea agglomerans cells, Gram-negative bacterium common in organic dust and known for its pathogenic impact. The inhalations were repeated daily (28 days). ELISA was used for measuring in lung tissue homogenates concentration of CRAMP, LAM-A1, TLR2, TLR4, TLR8, CXCL9 (chemokine [C-X-C motif] ligand) and CXCL10. RESULTS: Levels of TLR2, TLR4 and CXCL9 were significantly higher in both young and old mice lungs already after 7 days of inhalations, while significant increase of LAM-A1 and CXCL10 was noted after 28 days, compared to untreated samples. TLR8 level was significantly augmented only in young mice. Only CRAMP level significantly declined. Significantly higher TLR8 and CXCL9 concentration in untreated samples were noted in old animals compared to young ones. CONCLUSION: Significant alterations of the examined factors levels indicate their role in HP pathogenesis.

Cytotoxic effects induced in vitro by organic extracts from urban air particulate matter in human leukocytes.

Urban areas represent major pollution sources as a result of anthropogenic activities located in these districts. Among the legislated air pollutants, polycyclic aromatic hydrocarbons (PAHs), which are mostly adsorbed on the surface of dust particles, are known for their adverse health effects. The present study has been carried out to examine the cytotoxic effects induced in vitro on human peripheral monocytes (PBMCs) by extractable organic matter (EOM) from PM10 (characterized for its PAH content) collected at four sites in the urban center of Messina, Italy. Chromatographic analyses showed the presence of PAHs in all EOM. Only EOM from one site induced a marked cell death probably resulting from the highest PAH content in this sample. Conversely, apoptosis activation was evident after PBMC exposure to all the EOM tested. These apoptotic effects do not appear related only to the total PAH content, but are probably influenced by chemical composition. In conclusion, our findings confirm that the cytotoxic potential of organic matter associated to ambient respirable air particles depends predominantly on the quantity and quality of the chemicals contained in it. In particular, the present data strongly evidence that the only evaluation of air concentration of particulate matter and benzo[a]pyrene, as well as the generally used risk models based on additivity, are not sufficient to evaluate air quality and PAH effect on human health because they do not take into account the possible inhibitory or synergic or antagonistic effect of combined exposure and the interference of other organic compounds present in respirable matter.

Effect of oxidative stress induced by Brevibacterium sp. BS01 on a HAB causing species--Alexandrium tamarense.

Harmful algal blooms occur all over the world, destroying aquatic ecosystems and threatening other organisms. The culture supernatant of the marine algicidal actinomycete BS01 was able to lysis dinoflagellate Alexandrium tamarense ATGD98-006. Physiological and biochemical responses to oxidative stress in A. tamarense were investigated to elucidate the mechanism involved in BS01 inhibition of algal growth. Transmission electron microscope analysis revealed that there were some chloroplast abnormalities in response to BS01 supernatant. The decrease in cellular-soluble protein content suggested that cell growth was greatly inhibited at high concentration of BS01 supernatant. The increase in the levels of reactive oxygen species (ROS) and malondialdehyde contents following exposure to BS01 supernatant indicated that algal cells suffered from oxidative damage. The content of pigment was significantly decreased after 12 h treatment, which indicated that the accumulation of ROS destroyed pigment synthesis. Moreover, the decrease of Fv/Fm ratio suggested that in the photosynthetic system, the dominant sites producing ROS were destroyed by the supernatant of the BS01 culture. The activities of the antioxidant enzymes including superoxide dismutase and peroxidase increased in a short time and decreased slightly with increasing exposure time. A real-time PCR assay showed changes in the transcript abundances of two photosynthetic genes, psbA and psbD. The results showed that BS01 supernatant reduced the expression of the psbA gene after 2 h exposure, but the expression of the psbD gene was increased at concentrations of 1.0 and 1.5%. Our results demonstrated that the expression of the psbA gene was inhibited by the BS01 supernatant, which might block the electron transport chain, significantly enhancing ROS level and excess activity of the antioxidant system. The accumulation of ROS destoryed pigment synthesis and membrane integrity, and inhibited or ultimately killed the algal cells.

Ornithodoros brasiliensis (mouro tick) salivary gland homogenates inhibit in vivo wound healing and in vitro endothelial cell proliferation.

Ornithodoros brasiliensis is a nidicolous tick only found in the southern Brazilian highlands region. O. brasiliensis parasitism is frequently associated with toxicosis syndrome, which can lead to severe reactions, ranging from local pruritus and pain to systemic disturbances both in humans and dogs. One of the most frequent findings associated with an O. brasiliensis bite is a slow healing lesion at the site of tick attachment, which can take several weeks to heal. This work tested the hypothesis that an O. brasiliensis salivary gland homogenate is able to modulate the skin wound-healing process in vivo, using a model of excisional skin lesion in rats, which are divided into two groups: (1) control group and (2) treated group, which topically received salivary gland homogenate equivalent to the protein amount of one whole salivary gland (≈5 µg protein). The hypothesis that O. brasiliensis salivary gland homogenates interfere with endothelial cell proliferation, a key role phenomenon in wound healing, was also tested. O. brasiliensis salivary gland homogenates significantly delay skin wound healing. The time to full healing of skin lesions in control rats was 15 days, contrasting with 24 days in rats topically treated with O. brasiliensis salivary gland homogenates. The calculated HT50 (healing time to recover 50% of the wound area) for control groups was 3.6 days (95% CI, 3.2-3.9) and for salivary gland treated rats was 7.7 days (95% CI, 7.0-8.4). Salivary gland homogenates have a strong cytotoxic activity on cultured endothelial cells (LC50, 13.6 mg/ml). Also, at sublethal concentrations (≤3 mg/ml), salivary gland homogenates have a remarkable anti-proliferative activity (IC50 0.7 mg/ml) on endothelial cells, equivalent to ≈0.03 salivary gland pairs, an activity which seems to be much greater than reported for any other tick species. This is the first report about the biological activities of O. brasiliensis salivary compounds and provides the first in vivo evidence to support the concept of wound-healing modulation by tick salivary secretions. Results shown here contribute to an understanding of O. brasiliensis tick toxicosis syndrome, and also increase our knowledge of tick salivary bioactive compounds.

Tumor promoting effects of cyanobacterial extracts are potentiated by anthropogenic contaminants--evidence from in vitro study.

Inhibition of gap junctional intercellular communication (GJIC) is affiliated with tumor promotion process and it has been employed as an in vitro biomarker for evaluation of tumor promoting effects of chemicals. In the present study we investigated combined effects of anthropogenic environmental contaminants 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) and fluoranthene, cyanotoxins microcystin-LR and cylindrospermopsin, and extracts of laboratory cultures of cyanobacteria Aphanizomenon gracile and Cylindrospermopsis raciborskii, on GJIC in the rat liver epithelial cell line WB-F344. Binary mixtures of PCB 153 with fluoranthene and the mixtures of the two cyanobacterial strains elicited simple additive effects on GJIC after 30 min exposure, whereas microcystin-LR and cylindrospermopsin neither inhibited GJIC nor altered effects of PCB 153 or fluoranthene. However, synergistic effects were observed in the cells exposed to binary mixtures of anthropogenic contaminants (PCB 153 or fluoranthene) and cyanobacterial extracts. The synergistic effects were especially pronounced after prolonged (6-24h) co-exposure to fluoranthene and A. gracile extract, when mixture caused nearly complete GJIC inhibition, while none of the individual components caused any downregulation of GJIC at the same concentration and exposure time. The effects of cyanobacterial extracts were independent of microcystin-LR or cylindrospermopsin, which were not detected in cyanobacterial biomass. It provides further evidence on the presence of unknown tumor promoting metabolites in cyanobacteria. Clear potentiation of the GJIC inhibition observed in the mixtures of two anthropogenic contaminants and cyanobacteria highlight the importance of combined toxic effects of chemicals in complex environmental mixtures.

Pro-inflammatory effects of the aqueous extract of Echinometra lucunter sea urchin spines.

The sea urchin, Echinometra lucunter, can be found along the Western Central Atlantic shores. In Brazil, it is responsible by circa 50% of the accidents caused by marine animals. The symptoms usually surpass trauma and may be pathologically varied and last differently, ranging from spontaneous healing in a few days, to painful consequences lasting for weeks. In this work, we have mimicked the sea urchin accident by administering an aqueous extract of the spine into mice and rats and evaluated the pathophysiological developments. Our data clearly indicate that the sea urchin accident is indeed a pro-inflammatory event, triggered by toxins present in the spine that can cause edema and alteration in the leukocyte-endothelial interaction. Moreover, the spine extract was shown to exhibit a hyperalgesic effect. The extract is rich in proteins, as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but also contains other molecules that can be analyzed by reversed phase high-performance liquid chromatography. Altogether, these effects corroborate that an E. lucunter encounter is an accident and not an incident, as frequently reported by the victims.

Ixodid tick salivary gland products target host wound healing growth factors.

For successful blood-feeding, ticks must confront the host immune system comprising many cells and signaling molecules, mainly cytokines and growth factors. These factors bind to specific receptors on the cell membranes, thereby initiating a signaling cascade that leads to distinct cellular activities. Ticks are able to manipulate host immune responses via molecules secreted from their salivary glands. Saliva of ixodid ticks contains factors binding important cytokines and their subgroup, chemokines. Here we demonstrate that constituents of tick salivary gland extract (SGE) also appear to bind growth factors: transforming growth factor beta (TGF-ß1), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF-2), and hepatocyte growth factor (HGF), depending on tick species. SGE derived from Amblyommavariegatum reacted with TGF-ß1, PDGF, FGF-2 and HGF; Dermacentorreticulatus and Rhipicephalusappendiculatus with TGF-ß1, FGF-2 and HGF; and Ixodes ricinus and Ixodesscapularis with PDGF. SGE from the species targeting PDGF (A. variegatum and I. ricinus) also inhibited cell proliferation in vitro and induced a change in morphology of different cell lines. These effects correlated with disruption of the actin cytoskeleton. Such effects were not observed with SGE of the two species that did not target PDGF. Targeting of wound healing growth factors appears to be yet another strategy ixodid ticks adopt for suppression of inflammation and successful haematophagy.

In vitro antiviral activity against herpes simplex virus in the abalone Haliotis laevigata.

As viruses are extremely abundant in oceans, marine organisms may have evolved novel metabolites to protect themselves from viral infection. This research examined a well-known commercial gastropod, abalone (Haliotidae), which in Australia have recently experienced disease due to a neurotropic infection, abalone viral ganglioneuritis, caused by an abalone herpesvirus (AbHV). Due to the lack of molluscan cell lines for culturing AbHV, the antiviral activity of the abalone Haliotis laevigata was assessed against another neurotropic herpesvirus, herpes simplex virus type 1 (HSV-1), using a plaque assay. The concentration range at which abalone extract was used for antiviral testing caused minimal (<10 %) mortality in Vero cells. Haemolymph (20 %, v/v) and lipophilic extract of the digestive gland (3000 µg ml(-1)) both substantially decreased the number and size of plaques. By adding haemolymph or lipophilic extract at different times during the plaque assay, it was shown that haemolymph inhibited viral infection at an early stage. In contrast, the antiviral effect of the lipophilic extract was greatest when added 1 h after infection, suggesting that it may act at an intracellular stage of infection. These results suggest that abalone have at least two antiviral compounds with different modes of action against viral infection, and provide a novel lead for marine antiviral drug discovery.

Evaluation of the potential biological toxicities of aqueous extracts from red tide phytoplankton cultures in in vitro and in vivo systems.

The biological toxic potentials of aqueous extracts from the dinophycean flagellates Gymnodinium impudicum and Alexandrium affine and the raphidophycean flagellate Chattonella ovata were examined in both in vitro and in vivo systems. Interestingly, the extract from A. affine was the only one that showed potent cytotoxicities towards HeLa, Vero, and Neuro-2a cells in a concentration-dependent manner. Mice given intraperitoneal injections of the extracts revealed that none of the extracts exhibited serious toxicities in mice. However, temporal body weight loss was observed in the mice injected with the extract from A. affine during the early stage, and the dramatic enlargement of spleens was also observed in the mice on the 7th day after injection. Since A. affine extract showed potent hemolytic activity in vitro towards mouse erythrocytes, hemolytic anemia may be a possible mechanism responsible for the splenomegaly in the mice injected with A. affine extract. Similar marginal effects were observed in the mice injected with the extract from C. ovata; however, no significant toxic or detrimental effects were detected in the mice injected with the extract from G. impudicum. These results suggest that the extract from G. impudicum may not be contaminated with detectable levels of biologically hazardous compounds and may be relatively safe compared with the other two extracts.

Differential uptake and oxidative stress response in zebrafish fed a single dose of the principal copper and zinc enriched sub-cellular fractions of Gammarus pulex.

The sub-cellular compartmentalisation of trace metals and its effect on trophic transfer and toxicity in the aquatic food chain has been a subject of growing interest. In the present study, the crustacean Gammarus pulex was exposed to either 11 microg Cu l(-1), added solely as the enriched stable isotope 65Cu, or 660 microg Zn l(-1), radiolabeled with 2MBq (65)Zn, for 16 days. Post-exposure the heat stable cytosol containing metallothionein-like proteins (MTLP) and a combined granular and exoskeletal (MRG+exo) fractions were isolated by differential centrifugation, incorporated into gelatin and fed to zebrafish as a single meal. Assimilation efficiency (AE) and intestinal lipid peroxidation, as malondialdehyde (MDA) were measured. There was a significant difference (p<0.05) between the retention of the MTLP-Zn (39.0+/-6.4%) and MRG+exo-Zn (17.2+/-3.7%) and of this zinc retained by the zebrafish a significantly greater proportion of the MTLP-Zn feed had been transported away from the site of uptake. For 65Cu, although the results pointed towards greater bioavailability of the MTLP fraction compared to MRG+exo during the slow elimination phase (24-72 h) these results were not significant (p=0.155). Neither zinc feed provoked a lipid peroxidation response in the intestinal tissue of zebrafish compared to control fish (gelatin fed), but both 65Cu labeled feeds did. The greater effect was exerted by the MRG+exo (2.96+/-0.29 nmol MDA mg protein(-1)) feed which three-fold greater than control (p<0.01) and almost twice the MDA concentration of the MTLP feed (1.76+/-0.21 nmol MDA mg protein(-1), p<0.05). The oxidative stress response produced by Zn and Cu is in keeping with their respective redox potentials; Zn being oxidatively inert and Cu being redox active. These results are similar, in terms of bioavailability and stress response of each feed, to those in our previous study in which 109Cd labeled G. pulex fractions were fed to zebrafish. Thus it appears that when a metal (Cu or Cd) has the potential to cause cytotoxicity via lipid peroxidation, a feed consisting of a largely unavailable fraction (MRG+exo) causes a greater intestinal stress response than the more bioavailable (MTLP) feed.

Identification, mycotoxin risk and pathogenicity of Fusarium species associated with fig endosepsis in Apulia, Italy.

In a survey carried out on 87 rotted fig fruits samples collected in the Apulia region of Italy, the authors isolated 126 Fusarium strains identified as F. ramigenum (69 strains), F. solani (49), F. proliferatum (five) and three not identified. Investigation on the fertility of the strains belonging to F. proliferatum and F. ramigenum revealed that only strains of F. proliferatum were fertile. The identity of F. ramigenum strains was confirmed by sequencing a portion of the translation elongation factor-1alpha gene. When Fusarium species were analysed for their toxigenicity, 37/69 strains of F. ramigenum produced fusaric acid (FA) up to 525 mg kg(-1); 30 strains produced beauvericin (BEA) up to 190 mg kg(-1); 60 strains produced fumonisin B(1) (FB(1)) and fumonisin B(2) (FB(2)) up to 1575 mg kg(-1) of total FBs; and two strains produced fusaproliferin (FUP) up to 345 mg kg(-1); all five strains of F. proliferatum produced FA at low levels; two strains produced BEA up to 205 mg kg(-1); one strain produced FB(1) and FB(2), 1100 and 470 mg kg(-1), respectively; and one strain produced FUP, 820 mg kg(-1); F. solani (30 strains) produced FA, 13 strains up to 215 mg kg(-1). Few fungal extracts showed high toxicity toward brine shrimp larvae and in some cases in relation to BEA and FA content. A pathogenic assay on fig fruits showed that all three species were pathogenic, with higher virulence of F. ramigenum. These data report for the first time the production of BEA and FB(1)/FB(2) by F. ramigenum and show that it is a main agent of fig endosepsis in Apulia and can contribute to fumonisin contamination of fresh and dried figs.

The effects of staphylococcal bacteriophage lysates on cancer cells in vitro.

Bacteriophages constitute a serious alternative to antibiotic therapy of bacterial infections. They are also extremely numerous entities: phages can be found in almost all places on Earth and are constantly present in human and animal bodies. Observations of the effect of therapeutic staphylococcal phages and their bacterial hosts on melanoma migration in vitro are reported in this article. Together with bacteriophage preparations, disrupted Staphylococci (host strains) were investigated to compare the effects of bacteria with those of bacteriophages. Migration was decreased by all the investigated preparations in various ways and this was rather due to the activity of the bacterial components. Importantly, none of the investigated bacteriophage or bacterial preparations induced an increase in the migration activity of melanoma cells, which is important from the perspective of the therapeutic use of phage lysates. The possible presence of staphylococcal enterotoxins in the therapeutic bacteriophage preparations was also verified. All the studied therapeutic bacteriophage preparations were negative for the Staphylococcal enterotoxins A, B, C, D, and E (i.e., the enterotoxin content was less than 0.2-0.5 ng/ml).

Mechanisms of action of two different natural mixtures of persistent organic pollutants (POPs) in ovarian follicles.

The present work investigated the effects of two different natural mixtures on aryl hydrocarbon receptor (AhR) and oestrogen receptor (ER)beta protein levels, as well as on the activity of cytochrome P450 (CYP) 1A1 and CYP2B. Consequently, the authors observed the effects of these mixtures on gonadotropine-stimulated steroid secretion by ovarian follicles. The natural mixtures that were studied were 'Mjosa' extracted from burbot liver, which contains a high level of PBDEs, and 'Marine mix', extracted from Atlantic cod liver, which contains a high level of polychlorinated biphenyls (PCBs). Follicular cells were exposed in vitro to 'Marine mix' and 'Mjosa mix' at doses of 3.6 and 1.4 microg ml(-1), respectively. Media were collected and used for steroid analysis and cell viability assays. Cells were used to estimate aromatase activity (CYP19), AhR and ER protein levels, and CYP1A1 and CYP2B1 activity. Western blot analysis indicated down-regulation of AhR by 'Marine mix' and down-regulation of ERbeta by Mjosa mix. Up-regulation of CYP1A1 expression and activity were seen following treatment with Marine mix, but not Mjosa mix. Increased CYP2B1 activity was noted after treatment with both 'Marine mix' and Mjosa mix. Both mixtures increased luteinizing hormone (LH)-stimulated progesterone and testosterone secretion, follicle-stimulating hormone (FSH)-stimulated oestradiol secretion, and CYP19 activity. These results suggest that: (1) 'Marine mix' is a mixed-type CYP inducer; (2) 'Mjosa mix' is an inducer of ERbeta and CYP2B; and (3) both 'Marine mix' and 'Mjosa mix' stimulate aromatase activity as a consequence of oestradiol secretion through activation of CYP19.

Effects of cyanobacterial extracts containing anatoxin-a and of pure anatoxin-a on early developmental stages of carp.

This study compares the effects of pure anatoxin-a and cyanobacterial extracts of an anatoxin-a producing strain on early stages of development of carp. Carp eggs were exposed from 2:30 h to 4 days post-fertilization to different ecologically relevant concentrations of anatoxin-a, provided as pure toxin or contained in the cyanobacterial extracts. Data on time to mortality, mortality rate, time to hatching, hatching rate, skeletal malformations rate, and larval standard length were registered until 8 days post-fertilization. At any tested concentration of anatoxin-a, the pure toxin was almost harmless to carp early stages of development, contrarily to cell extracts that were highly toxic. Only an adverse effect on the larval length was found at the highest concentration of pure toxin, while increasing concentrations of cell extracts caused increasing adverse effects in all the analyzed parameters. Anatoxin-a producing cyanobacteria should be regarded as putative modulators of aquatic ecosystems communities.

Assessment of the genotoxicity of S9-generated metabolites using the GreenScreen HC GADD45a-GFP assay.

Genotoxicity can be assessed by monitoring expression of a GADD45a-GFP reporter in the human lymphoblastoid cell line TK6. A flow cytometric method has been developed to effectively distinguish GFP fluorescence from coloured and fluorescent test samples as well from the S9 liver extracts used to generate metabolites from pro-genotoxins. The method includes the use of propidium iodide exclusion for the determination of cellular viability. This paper describes the method development, the derivation of decision thresholds for the identification of genotoxins using the method, and presents data from a 56-compound validation study of the method. The results illustrate that the method permitted the detection of the majority of pro-genotoxins tested and, importantly, the high specificity of the GADD45a-GFP assay was maintained.

In vitro haemolytic and cytotoxic activity of soluble extract antigen of T. vaginalis isolates from symptomatic and asymptomatic women.

Human Trichomonas vaginalis infection ranges from asymptomatic to mild, moderate or severe clinical manifestations. The reasons for diverse symptomatology have been found to vary in several attributes and both parasite and host factors appear to play a role in the pathogenesis. The present study reports the in vitro haemolytic and cytotoxic activity of crude soluble extract (CSE) antigen of T. vaginalis isolates from 15 symptomatic and 15 asymptomatic women. The haemolytic activity following the interaction of CSE antigen with human erythrocytes and cytotoxic activity by adding CSE antigen to normal human vaginal epithelial cells was significantly higher with the use of CSE antigen of isolates from symptomatic women as compared to those from asymptomatic women. Furthermore, cytotoxic effect was found to be pH dependent. The study demonstrates, for the first time, the significant effect of parasite antigen of isolates from symptomatic women as compared to those from asymptomatic women in inducing haemolytic and cytotoxic effect and supports the earlier report that contact-independent mechanism(s) may be playing a role in establishing symptomatic trichomoniasis.

Distribution of cytotoxic and DNA ADP-ribosylating activity in crude extracts from butterflies among the family Pieridae.

Cabbage butterflies, Pieris rapae and Pieris brassicae, contain strong cytotoxic proteins, designated as pierisin-1 and -2, against cancer cell lines. These proteins exhibit DNA ADP-ribosylating activity. To determine the distribution of substances with cytotoxicity and DNA ADP-ribosylating activity among other species, crude extracts from 20 species of the family Pieridae were examined for cytotoxicity in HeLa cells and DNA ADP-ribosylating activity. Both activities were detected in extracts from 13 species: subtribes Pierina (Pieris rapae, Pieris canidia, Pieris napi, Pieris melete, Pieris brassicae, Pontia daplidice, and Talbotia naganum), Aporiina (Aporia gigantea, Aporia crataegi, Aporia hippia, and Delias pasithoe), and Appiadina (Appias nero and Appias paulina). All of these extracts contained substances recognized by anti-pierisin-1 antibodies, with a molecular mass of approximately 100 kDa established earlier for pierisin-1. Moreover, sequences containing NAD-binding sites, conserved in ADP-ribosyltransferases, were amplified from genomic DNA from 13 species of butterflies with cytotoxicity and DNA ADP-ribosylating activity by PCR. Extracts from seven species, Appias lyncida, Leptosia nina, Anthocharis scolymus, Eurema hecabe, Catopsilia pomona, Catopsilia scylla, and Colias erate, showed neither cytotoxicity nor DNA ADP-ribosylating activity, and did not contain substances recognized by anti-pierisin-1 antibodies. Sequences containing NAD-binding sites were not amplified from genomic DNA from these seven species. Thus, pierisin-like proteins, showing cytotoxicity and DNA ADP-ribosylating activity, are suggested to be present in the extracts from butterflies not only among the subtribe Pierina, but also among the subtribes Aporiina and Appiadina. These findings offer insight to understanding the nature of DNA ADP-ribosylating activity in the butterfly.

The influence of culture conditions on vasculitis and anaphylactoid shock induced by fungal pathogen Candida albicans cell wall extract in mice.

To explore whether Candida cell wall mannan is responsible for induction of vasculitis similar to Kawasaki syndrome and anaphylactoid shock in mice, we examined the biological effects of various mannan structures from Candida cell wall extracts prepared using various culture conditions. Intraperitoneal injection of 3 of 4 Candida cell wall extracts dramatically induced coronary arteritis and acute anaphylactoid shock in mice; only the cell wall extract derived from YPD medium culture at 27 degrees C had no toxic effect. It is of note that these biological effects depended on culture conditions around the cells such as culture temperature and media. These conditions lead to the structural rearrangement of cell wall mannan as confirmed by reactivity against antisera and NMR spectroscopy. Since the expression of beta-1,2-linked mannan varies dramatically between biologically active and inactive mannan, beta-1,2-linked mannan might negatively affect Candida cell wall extract-induced coronary arteritis and acute anaphylactoid shock in mice. Our findings indicate that Candida cell wall mannan might contribute to coronary arteritis and acute shock, and that an alteration of mannan structure could be responsible for Candida pathogenicity.