Reduction Of Tetrazolium Salt Research Articles

Ta’ ta’, Huayani: Perception of leishmaniasis and evaluation of medicinal plants used by the Chayahuita in Peru. Part II

Aim of the study A knowledge attitude and practice study centred on leishmaniasis and its treatment was performed among the Chayahuita, an Amazonian Peruvian ethnic group living in an endemic area. This study documents traditional Chayahuita plant's use and disease concepts. Also, activty of some medicinal plants used by the Chayahuita is highlighted and discussed. Materials and methods Ninety-three Chayahuita people were interviewed, following a semi-structured questionnaire focussed on disease knowledge and perception, personal attitude and healing practices. Simultaneously, a collection of plants was performed in different ecotopes, in order to make an extensive inventory of the pharmacopoeia. Results For the Chayahuita, cutaneous (CL) and muco-cutaneous leishmaniasis (MCL) are considered as diseases of their own, with specific names, aetiologies, mode of transmission. Regarding CL, Chayahuita people consider that the humid characteristic of the skin ulcer is a discriminative fact orienting the diagnostic for Ta’ ta’ (leishmaniasis). Forty-six different species were designated useful against LC and /or MCL (29 species by means of the questionnaire and 27 species when collecting in different ecotopes). Thirty-seven extracts corresponding to 31 species used medicinally were screened in vitro against Leishmania amazonensis axenic amastigotes, assessing their viability by the reduction of tetrazolium salt (MTT). Six species displayed a good activity (10 μg/ml < IC50 < 20 μg/ml): an undetermined hemi-epiphytic Clusiaceae, Cybianthus anthuriophyllus Pipoly (Myrsinaceae), two Piper, Piper sanguineispicum Trel., and Piper loretoanum Trel. (Piperaceae), Desmodium axillare Sw. DC. (Fabaceae), and Clibadium sylvestre (Aubl.) Baill. (Asteraceae). Conclusion Perception of leishmaniasis, attitude, treatments and diet prohibitions still largely reflects traditional Chayahuita cosmovision, even if some tentative of bio-medical re-interpretation is arising.

Addition of β-Malic Acid-Containing Poly(ethylene glycol) Dimethacrylate To Form Biodegradable and Biocompatible Hydrogels

Poly(malic acid) is water-soluble, functionalizable, and biodegradable, making it attractive as a precursor of hydrogels for biomedical applications. However, homopoly(malic acid), with pK(1/2) of 4.3, is too acidic for biocompatibility. To overcome the acidity, we have synthesized beta-malic acid-containing poly(ethylene glycol) dimethacrylate (PEGMAc) with pK(a) of 5.02. Solutions of methacrylated O-carboxymethylchitosan (OCMCS), PEGMAc, and poly(ethylene glycol) diacrylate (PEGDA; 7:7:86 and 6:20:74 (w/w/w)) in water (80%) have near neutral pHs (6.8-6.9). These solutions form firm hydrogels when photopolymerized. These are referred to as O7-PEGMAc7-B86 and O6-PEGMAc20-B74 (where the numerals refer to the weight content of each component, O is OCMCS and B is PEGDA added to make blend). The carboxyl groups in PEGMAc permit the surface grafting of hydrogels with Arg-Gly-Asp (RGD). The cytocompatibilities of smooth muscle cells (SMCs) on RGD-grafted hydrogels were studied. From the tetrazolium salt reduction assay, O6-PEGMAc20-B74 was found to have significantly better 10th day cytocompatibility compared to hydrogels containing lower or no PEGMAc. These gels degrade upon hydrolysis releasing malic acid, PEG and OCMCS. The increased cell compatibility of O6-PEGMAc20-B74 is possibly due to increased surface RGD content and near neutral pH even during biodegradation. Our novel PEGMAc-modified blends are a promising functionalizable biodegradable hydrogel precursor providing improved cell proliferation.

Colorimetric microbial viability assay based on reduction of water-soluble tetrazolium salts for antimicrobial susceptibility testing and screening of antimicrobial substances

The applicability of a colorimetric microbial viability assay based on reduction of a tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2 H-tetrazolium, monosodium salt [WST-8]} via 2-methyl-1,4-naphthoquinone (2-methyl-1,4-NQ) as an electron mediator for determining the susceptibility of various bacteria to antibiotics and screening antimicrobial substances was investigated. The measurement conditions, which include the effects of the concentration of 2-methyl-1,4-NQ, were optimized for proliferation assays of gram-negative bacteria, gram-positive bacteria, and pathogenic yeast. In antimicrobial susceptibility testing, there was excellent agreement between the minimum inhibitory concentrations determined after 8 h using the WST-8 colorimetric method and those obtained after 22 h using conventional methods. The results suggest that the WST-8 colorimetric assay is a useful method for rapid determination of the susceptibility of various bacteria to antibiotics. In addition, the current method was applied to the screening of bacteriocin-producing lactic acid bacteria and its efficiency was demonstrated.

The influence of light-curing time on the bacterial colonization of resin composite surfaces

Objectives Bacterial colonization of composite surfaces represents the main factor in the etiology of secondary caries around adhesive restorations. The authors’ aim was to evaluate the influence of light-curing time on mutans streptococci colonization (MS) of a resin composite material. Methods Specimens obtained from a dental resin composite were divided into 12 groups and light-cured with the same light source respectively for 10, 20, 30, 40, 60 or 80 s using two different curing-power levels: 400 and 800 mW/cm 2. A wild strain of MS was isolated and a 24-h-monospecific biofilm, adherent to the surfaces of the samples, was obtained. A colorimetric technique (MTT assay), based on the reduction of a yellow tetrazolium salt to a purple formazan, was used to evaluate the biomass adherent to the specimen surfaces. ANOVA and Scheffé’s tests were used to statistically analyze the results. Results Two-way ANOVA demonstrated there was no interaction between curing-time factor and curing-power factor ( p = 0.970); one-way ANOVA was used to analyze separately the data obtained from each curing-power level. Both levels showed highly significant differences ( p < 0.0001) among the different curing time groups. The non-parametric test for trend showed in both levels the existence of a highly significant trend ( p < 0.0001) for bacterial colonization reduction as curing time increases. Significance A reduced curing time seems to be responsible for increased in vitro colonization of composite surfaces by MS; this phenomenon is likely to be related to the presence of unpolymerized monomers on the material surface.

Medicinal plants from the Yanesha (Peru): Evaluation of the leishmanicidal and antimalarial activity of selected extracts

Aim of the study Ninety-four ethanolic extracts of plants used medicinally by the Yanesha, an Amazonian Peruvian ethnic group, for affections related to leishmaniasis and malaria were screened in vitro against Leishmania amazonensis amastigotes and against a Plasmodium falciparum chloroquine resistant strain. Materials and methods The viability of Leishmania amazonensis amastigote stages was assessed by the reduction of tetrazolium salt (MTT) while the impact on Plasmodium falciparum was determined by measuring the incorporation of radio-labelled hypoxanthine. Results and conclusions Six plant species displayed good activity against Plasmodium falciparum chloroquine resistant strain (IC 50 < 10 μg/ml): a Monimiaceae, Siparuna aspera (Ruiz & Pavon), A. DC., two Zingiberaceae, Renealmia thyrsoidea (Ruiz & Pavon) Poepp. & Endl. and Renealmia alpinia (Rottb.), two Piperaceae ( Piper aduncum L. and Piper sp.) and the leaves of Jacaranda copaia (Aubl.) D. Don (Bignoniaceae). Eight species displayed interesting leishmanicidal activities (IC 50 < 10 μg/ml): Carica papaya L. (Caricaceae), Piper dennisii Trel (Piperaceae), Hedychium coronarium J. König (Zingiberaceae), Cestrum racemosum Ruiz & Pav. (Solanaceae), Renealmia alpinia (Rottb.) Zingiberaceae, Lantana sp. (Verbenaceae), Hyptis lacustris A. St.-Hil. ex Benth. (Lamiaceae) and Calea montana Klat. (Asteraceae). Most of them are used against skin affections by Yanesha people. Results are discussed herein, according to the traditional use of the plants and compared with data obtained from the literature.

Phenylpyrazole insecticides induce cytotoxicity by altering mechanisms involved in cellular energy supply in the human epithelial cell model Caco-2

Phenylpyrazoles are relatively new insecticides designed to manage problematic insect resistance and public health hazards encountered with older pesticide families. In vitro cytotoxicity induced by the phenylpyrazole insecticides, Ethiprol and Fipronil, and Fipronil metabolites, sulfone and sulfide, was studied in Caco-2 cells. This cellular model was chosen because it made possible to mimic the primary site of oral exposure to xenobiotics, the intestinal epithelium. Assessment of the barrier function of Caco-2 epithelium was assessed by TEER measurement and showed a major loss of barrier integrity after exposure to Fipronil and its metabolites, but not to Ethiprol. The disruption of the epithelial barrier was attributed to severe ATP depletion independent of cell viability, as revealed by LDH release. The origin of energetic metabolism failure was investigated and revealed a transient enhancement of tetrazolium salt reduction and an increase in lactate production by Caco-2 cells, suggesting an increase in glucose metabolism by pesticides. Cellular symptoms observed in these experiments lead us to hypothesize that phenylpyrazole insecticides interacted with mitochondria.

Green Tea Catechin Epigallocatechin-3-Gallate (EGCG) Induces Cell Death in Acute Myeloid Leukemic Cells Via DAPK2 and Potentiates ATRA-Induced Neutrophil Differentiation

Acute promyelocytic leukemia (APL) patients are currently treated with all-trans retinoic acid (ATRA) successfully resolving the differentiation block. However, concurrent chemotherapy is still necessary in combination with ATRA and novel less toxic therapeutic approaches are a major demand. Epigallocatechin-3-gallate (EGCG), the main polyphenolic compound present in green tea, has been reported to have chemopreventive and chemotherapeutic effects in different neoplasms. EGCG inhibits cell growth in vitro and in vivo and induces apoptosis in cancer cells without adversely affecting normal cells. Also, EGCG has been found in earlier studies to effectively kill AML blasts, but it has never been applied in combination with ATRA. We have previously shown that expression of the death-associated protein kinase 2 (DAPK2) enhanced ATRA-induced neutrophil development, and recently it was shown that EGCG specifically killed multiple myeloma cells while inducing DAPK2. Therefore, we first tested whether EGCG treatment induces DAPK2 expression in myeloid leukemic blast cells and whether this will lead to cell death. EGCG treatment of HL60 and NB4 acute myeloid leukemic cells led to a dose-dependent increase of DAPK2 mRNA with a maximum of 5.5- and 3.9-fold, respectively. In parallel, DAPK2 protein was markedly upregulated in both cell lines accompanied by a 72% increase of cell death after 24h, as measured by reduction of tetrazolium salt (XTT assay), and by hallmarks of apoptosis: activation of caspase-3 and phosphatidylserine exposure on the plasma membrane surface. EGCG-induced cytotoxicity was reduced by 48% in HL60 and by 46% in NB4 cells upon stable short hairpin RNA-mediated silencing of DAPK2. Moreover, combined ATRA and EGCG treatment of HL60 cells resulted in cooperative DAPK2 induction and potentiated myeloid differentiation as measured by CD11b and CD15 expression using flow cytometry (Figure A) as well as by C/EBPε and G-CSFR mRNA levels using quantitative RT-PCR. Enhanced differentiation was significantly reversed by knocking down DAPK2. Moreover, EGCG toxicity of NB4 and HL60 cells correlated with 67 kDa laminin receptor (67LR) protein expression which is downregulated during ATRA treatment of HL60 and NB4 cells but not in the ATRA-resistant sublines HL60-R and NB4-R. In line, HL60-R and NB4-R cells are still susceptible to EGCGinduced cell killing, whereas pretreatment of parental HL60 and NB4 cells with ATRA, causing downregulation of 67LR, rendered these cells resistant to EGCG mediated killing. Likewise, susceptibility of primary AML (n=8) samples to EGCG treatment was closely associated with 67LR expression. Moreover, neutrophils and PBMCs from healthy donors did not express 67LR and hence were resistant to EGCG treatment (Figure B). [Display omitted] In summary, we found that DAPK2 is essential for EGCG-induced cell death in myeloid leukemic cells, and that a combination of ATRA and EGCG treatment significantly boosted neutrophil differentiation. This enhanced differentiation is most likely due to preferred EGCG-induced killing of ATRA-resistant leukemic cells expressing the EGCG receptor 67LR. We thus conclude that simultaneous ATRA and EGCG treatment might improve differentiation therapies for APL. In addition, other AML subtypes with high 67LR expression might also benefit from a combined chemotherapy and EGCG regimen.

Blocking the Autophagy Gene 5 (ATG5) Impairs ATRA-Induced Myeloid Differentiation, and ATG5 Is Downregulated in AML

Autophagy describes a process of membrane trafficking where specialized compartments (autophagosomes) engulf damaged or dispensable organelles and target them to lysosomic degradation. Thus, autophagy has cytoprotective functions e.g. during starvation, but autophagy can also lead to caspase-independent cell death (PCD type II). ATG5 is a central player in autophagy and inactivating ATG5 severely impairs normal development. In hematopoiesis, ATG5 is essential for maturation of B lymphocytes as well as for T cell survival and proliferation. In recent years, several differentiation and death-inducing agents were shown to activate autophagy in acute myeloid leukemia (AML) cell line models, but the mechanisms involved are still poorly defined. We therefore decided to investigate the role of ATG5 and autophagy in all-trans retinoic acid (ATRA)-induced neutrophil differentiation of AML cells. We found that ATG5 mRNA was upregulated 5.9-, 3.8- and 3.4-fold after 6 days of ATRA-treatment in HL60, NB4 and HT93 AML cells, respectively. In contrast, PMA-induced macrophage differentiation of HL60 and U937 cells only slightly induced ATG5 mRNA by 1.3- and 1.5-fold, respectively, indicating a specific role for ATG5 in granulocyte differentiation. In line with the above observation in leukemic cell lines, we found that ATG5 mRNA levels were increased in 5/5 APL patients upon ATRA therapy. In addition, ATG5-ATG12 conjugates, hallmarks of the autophagic process, were markedly activated in ATRA-treated NB4 and HL60 cells compared to control cells as measured by Western blotting. In agreement, we found increased autophagic activity during myeloid differentiation as evidenced by two additional autophagy markers, i.e. conversion of light chain 3 B (LC3B)-I into LC3B-II and degradation of sequestosome 1 (SQSTM1, p62) protein by Western blotting. Inhibition of autophagy during ATRA treatment of NB4 and HL60 cells with 3-Methyladenine or Bafilomycin A significantly impaired neutrophil differentiation by 80% as measured by CD11b surface expression. Similarly, lentivirus-driven short hairpin (sh)RNA-mediated silencing of ATG5 in NB4 cells resulted in an 85% reduction of ATRA-induced neutrophil differentiation as measured by CD11b surface expression and by quantitative RT-PCR of the myeloid differentiation markers GCSFR, C/EBPε and lactotransferrin. Interestingly, inhibition of autophagy increased overall cell death during the myeloid differentiation process rather than reducing it as measured by reduction of tetrazolium salt (XTT assay). Enhanced cell death and reduced myeloid differentiation upon blocking ATG5 would suggest that autophagy is needed for maintaining myeloid differentiation. Further support for our hypothesis that ATG5 is needed for neutrophil development stems from our survey of ATG5 mRNA expression in primary hematopoietic cells. We found significantly higher ATG5 mRNA levels in granulocytes and macrophages (n=7) as compared to CD34+ hematopoietic progenitor cells from healthy donors (n=4; p=0.0424) as well as compared to primary AML patient samples at diagnosis (n=76; p=0.0003). ATG5 mRNA expression in CD34+ and AML patients was not significantly different (p=0.6669). In summary, we show a correlation of high ATG5 expression with terminal myeloid differentiation and of low ATG5 expression with a myeloid leukemic phenotype. Using chemical inhibitors of autophagy as well as RNAi technology to knockdown ATG5, we further provide evidence that ATG5 and consequently autophagy are essential for neutrophil development.

Synthesis and Characterization of New Binaphthyl‐Linked Phenanthroline‐, Bipyridine‐, or Pyridine‐Derived Ligands, and the Study of Their Cytotoxic Activity

AbstractIn the present work, we describe the synthesis and characterization of five new versatile acyclic or macrocyclic ligands containing binaphthyl‐linked pyridine, bipyridine, or phenanthroline groups in their framework (see Schemes 1–4). The structures of the ligands were elucidated on the basis of elemental analyses, IR, 1H‐NMR, 13C‐NMR, and FAB mass spectra. The cytotoxicity of these compounds was tested in vitro by using the tetrazolium salt reduction (MTT) assay on A549 (human lung carcinoma epithelial like) cells. All of the tested compounds induced time‐ and concentration‐dependent cytotoxic effect.

Biocompatibility Properties of ZrO&lt;sub&gt;2&lt;/sub&gt; Ceramics and ZrO&lt;sub&gt;2&lt;/sub&gt;-TiN Composites

In order to study bicompability properties of fully dense ZrO2 ceramics containing 2-3 mol% Y2O3 stabilizer, and ZrO2-TiN composites containing 1-1.75 mol Y2O3 and 1 mol% Nd2O3 stabilizers, and electrical conductive TiN particles (40-60 vol%), cell viability was evaluated based on the reduction of tetrazolium salt to formazan crystals by living cells, the alkaline phosphatase production was investigated, and nitrite quantification was done for indirect evaluation of osteoblast NO production. The samples were defined biocompatible since there is not any significant change observed in the physiological functions of the cells when incubated together.

Measurement of Proliferative Responses of Cultured Lymphocytes

Measurement of proliferative responses of human lymphocytes is a fundamental technique for the assessment of their biological responses to various stimuli. Most simply, this involves measurement of the number of cells present in a culture before and after the addition of a stimulating agent. This unit contains several different prototype protocols to measure the proliferative response of lymphocytes following exposure to mitogens, antigens, allogeneic or autologous cells, or soluble factors. Each of these protocols can be used in conjunction with an accompanying support protocol which contains methods for pulsing cultures with [3H]thymidine and determining incorporation of [3H]thymidine into DNA or assessing cell proliferation by nonradioactive methods, e.g., reduction of tetrazolium salts (MTT). The protocols described here provide an estimate of DNA synthesis and cell proliferation in an entire cell population, but do not provide information on the proliferation of individual cells. A protocol for CFSE labeling allows specific subpopulations of cells to be separated viably for further analysis.

Pulse radiolysis of tetrazolium violet in aqueous and aqueous-alcoholic solutions under oxidative and reductive conditions

The radiolytic reduction of colourless tetrazolium salts to coloured formazans in liquid and solid state is suggested for dosimetry purposes. In order to clarify the reaction mechanism, a pulse radiolysis study was conducted in aqueous and aqueous-alcoholic solutions under oxidative and reductive conditions. Under reducing conditions, fast formation of the electron adduct tetrazolinyl radical was observed: coloured formazan final product formed during the decay of electron adduct. Both the decay of the tetrazolinyl radical and the formation of the formazan were found to be second order. The spectra of the formazan were similar in neutral and alkaline solutions, but with higher absorbance in the latter solutions due to the higher molar absorption coefficient. Under oxidative conditions formazan did not form; hydroxylated products through OH-adducts were observed in the pH range studied.

Cell cycle arrest and differentiation induction by 5,7-dimethoxycoumarin in melanoma cell lines

In the present study we investigated the antiproliferative activity of 5,7-dimethoxycoumarin on the murine B16 and human A375 melanoma cell lines. The inhibitory concentration 50 (IC50) was estimated for each cell line by preliminary assay of tetrazolium salt reduction (MTT). With Trypan blue exclusion test we detected a cytostatic but not cytotoxic effect of the treatment in melanoma cells: 5,7-dimethoxycoumarin significantly reduced cell proliferation in a time- and dose-dependent manner, blocking the cell cycle in the G0/G1 phase both in B16 and A375 cells. Melanoma growth reduction was coupled to a differentiation process detected by monitoring some specific markers: i) morphological changes with development of dendrite-like projections from the cell surface; ii) melanin synthesis; and iii) PpIX accumulation. Induction of the differentiation process was more significant in murine melanoma cells, where the treatment irreversibly reduced cell growth. Consistent with G0/G1 arrest and melanogenesis in B16 cells, 5,7-dimethoxycoumarin strongly decreased activation of the mitogen-activated protein kinase extracellular signal-related kinase 1/2, which is upregulated in many types of cancer. These findings suggest that 5,7-dimethoxycoumarin should be further investigated through studies both in vitro, to identify the binding partners for this compound, and in preclinical animal models.

196 CURCUMIN INHIBITS INTERLEUKIN-6, -8, NITRIC OXIDE AND PROSTAGLANDIN E 2 SYNTHESIS BY BOVINE CHONDROCYTES

CONCLUSIONS Altogether, these in vitro results indicate that curcumin may reduce inflammation and pain in OA by reducing the production of inflammatory mediators by chondrocytes. These findings provide a preclinical basis for the in vivo testing of curcumin and suggest that this natural compound could be helpful to alleviate symptoms in OA patients. AIMS OF THE STUDY Curcumin (Curc) [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] derived from the plant Curcuma longa possesses anti-inflammatory and antioxidant properties. Inflammatory mediators such as interleukin (IL)-6 and -8, nitric oxide (NO), and prostaglandin (PG) E2 play a key role in the pathogenesis and progression of osteoarthritis (OA). This study aims to investigate the effects of Curc, on the production of IL-6, IL-8, NO, and PGE2 by primary bovine chondrocytes. METHODS Primary bovine chondrocytes were cultured in monolayer until confluence and then incubated for 24h in the absence (basal condition) or in the presence of IL-1β (10-11M) and with or without Curc, indomethacin (Ind) and celecoxib (Cel) at a concentration ranged between 1 to 30 μM. Cell viability was determined by measuring MTT tetrazolium salt reduction and lactate deshydrogenase release. NO production was assessed by quantifying nitrite in the culture supernatants using the Griess spectrophotometric method. PGE2 was measured in the culture supernatants by a specific radioimmunoassay. Inducible NO synthase (iNOS), cyclooxygenase COX-2, IL-6 and IL-8 gene expression were determined by real-time RT-PCR. RESULTS •Curc had no significant effects on DNA content, MTT reduction and LDH release. •Curc inhibited IL-1β induced NO production and iNOS gene expression.

Acute salinity stress alters the haemolymph metabolic profile of Penaeus monodon and reduces immunocompetence to white spot syndrome virus infection

Influence of acute salinity stress on the immunological and physiological response of Penaeus monodon to white spot syndrome virus (WSSV) infection was analysed. P. monodon maintained at 15‰ were subjected to acute salinity changes to 0‰ and 35‰ in 7 h and then challenged orally with WSSV. Immune variables viz., total haemocyte count, phenol oxidase activity (PO), nitroblue tetrazolium salt (NBT) reduction, alkaline phosphatase activity (ALP), acid phosphatase activity (ACP) and metabolic variables viz., total protein, total carbohydrates, total free amino acids (TFAA), total lipids, glucose and cholesterol were determined soon after salinity change and on post challenge days 2 (PCD2) and 5 (PCD5). Acute salinity change induced an increase in metabolic variables in shrimps at 35‰ except TFAA. Immune variables reduced significantly (P<0.05) in shrimps subjected to salinity stress with the exception of ALP and PO at 35‰ and the reduction was found to be more at 0‰. Better performance of metabolic and immune variables in general could be observed in shrimps maintained at 15‰ that showed significantly higher post challenge survival following infection compared to those under salinity stress. Stress was found to be higher in shrimps subjected to salinity change to lower level (0‰) than to higher level (35‰) as being evidenced by the better immune response and survival at 35‰. THC (P<0.001), ALP (P<0.01) and PO (P<0.05) that together explained a greater percentage of variability in survival rate, could be proposed as the most potential health indicators in shrimp haemolymph. It can be concluded from the study that acute salinity stress induces alterations in the haemolymph metabolic and immune variables of P. monodon affecting the immunocompetence and increasing susceptibility to WSSV, particularly at low salinity stress conditions.

Evaluation of the leishmanicidal activity of plants used by Peruvian Chayahuita ethnic group

Aim of the study A total of 27 ethanolic plant extracts from 27 species were screened for leishmanicidal activity in vitro against Leishmania amazonensis. Most of the selected species (19) are traditionally used by the Chayahuitas, an Amazonian Peruvian ethnic group, to treat skin affections and/or leishmaniasis. Material and Methods A colorimetric method based on the reduction of tetrazolium salt (MTT) was used to measure the viability of Leishmania amazonensis promastigote and amastigote stages. Results and Conclusions Only the leaves of two species of the Piperaceae family ( Piper hispidum Sw., and Piper strigosum Trel.) showed good leishmanicidal activities (IC 50 < 10 μg/ml against amastigotes). Roots of Tabernaemontana sananho Ruiz & Pav. (Apocynaceae), together with bark of Vismia tomentosa Ruiz & Pav. (Clusiaceae), fruits of Solanum straminifolium var straminifolium Jacq. (Solanaceae), and stems of Zamia lindenii Regel ex André (Cycadaceae) showed low activity against amastigote stage (IC 50 around 50 μg/ml). Of those only Tabernaemontana sananho displayed also good activity on promastigotes (IC 50 < 10 μg/ml). Results are discussed herein, in relation with the traditional use of the plants and compared with other data from the relevant literature.

Serum albumin leads to false-positive results in the XTT and the MTT assay

Tetrazolium salts like 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) or sodium 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) that form formazans after reduction are widely used to investigate cell viability. Besides cellular enzymes, some constituents of cell media and other substances reduce tetrazolium salts, thereby interfering with these assays. We describe here that different preparations of serum albumin from bovine or human origin can lead to a concentration-dependent increase in the signals of the XTT assay; therefore leading to an overestimation of cell numbers and to an underestimation of potential cytotoxic effects of compounds to be tested. The same effect was seen in the MTT assay with human serum albumin (HSA). We demonstrate that this reductive activity cannot be inactivated by proteolytic digestion, but that it is due to the free cysteine residue in albumin, and is also observed when cysteine or glutathione (GSH) are used. Binding of N-ethylmaleimide (NEM) to the free cysteine residue leads to a decrease of the albumin interference in the XTT assay.

Interactions of clinical and environmental Aeromonas isolates with Caco-2 and HT29 intestinal epithelial cells

Evaluation of adherence and invasion of Aeromonas spp. to human colon carcinoma cell lines Caco-2 and HT29 and assessment of cytotoxic activity. A number of 27 strains of Aeromonas caviae and 23 strains of Aeromonas hydrophila was analysed. All strains were capable to adhere to sub-confluent monolayers of Caco-2 and HT29 cell types, presenting aggregative and diffuse adherence patterns cells, respectively. In the cytotoxic assays all strains showed cytopathic and/or cytotoxic activities to Vero cells. The evaluation of the tetrazolium salt (MTT test) reduction capability was carried out in Vero, Caco-2, and HT29 cells. MTT test showed that Vero cell line was the most sensitive cell type. In the invasion test, 13 strains were analysed on Caco-2 and HT29 monolayers. Only two (15%) of the 13 strains, A. hydrophila and A. caviae species, both isolated from vegetables were invasive to Caco-2 cells. No strains were able to invade the HT29 cells. A. hydrophila and A. caviae isolated from human diarrhoeic faeces, vegetables, and water, were able to adhere to and produce cytotoxic/cytopathic effects in intestinal epithelial cell lines. The presence of Aeromonas spp. in food and water samples expressing virulence factors suggest that these sources may act as dissemination vehicles of human pathogen with implication in the public health.

Metal Ions May Suppress or Enhance Cellular Differentiation in Candida albicans and Candida tropicalis Biofilms

Candida albicans and Candida tropicalis are polymorphic fungi that develop antimicrobial-resistant biofilm communities that are characterized by multiple cell morphotypes. This study investigated cell type interconversion and drug and metal resistance as well as community organization in biofilms of these microorganisms that were exposed to metal ions. To study this, Candida biofilms were grown either in microtiter plates containing gradient arrays of metal ions or in the Calgary Biofilm Device for high-throughput susceptibility testing. Biofilm formation and antifungal resistance were evaluated by viable cell counts, tetrazolium salt reduction, light microscopy, and confocal laser scanning microscopy in conjunction with three-dimensional visualization. We discovered that subinhibitory concentrations of certain metal ions (CrO(4)(2-), Co(2+), Cu(2+), Ag(+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), AsO(2)(-), and SeO(3)(2-)) caused changes in biofilm structure by blocking or eliciting the transition between yeast and hyphal cell types. Four distinct biofilm community structure types were discerned from these data, which were designated "domed," "layer cake," "flat," and "mycelial." This study suggests that Candida biofilm populations may respond to metal ions to form cell-cell and solid-surface-attached assemblages with distinct patterns of cellular differentiation.

In vitro activity of eugenol against Candida albicans biofilms

Most manifestations of candidiasis are associated with biofilm formation occurring on the surfaces of host tissues and medical devices. Candida albicans is the most frequently isolated causative pathogen of candidiasis, and the biofilms display significantly increased levels of resistance to the conventional antifungal agents. Eugenol, the major phenolic component of clove essential oil, possesses potent antifungal activity. The aim of this study was to investigate the effects of eugenol on preformed biofilms, adherent cells, subsequent biofilm formation and cell morphogenesis of C. albicans. Eugenol displayed in vitro activity against C. albicans cells within biofilms, when MIC(50) for sessile cells was 500 mg/L. C. albicans adherent cell populations (after 0, 1, 2 and 4 h of adherence) were treated with various concentrations of eugenol (0, 20, 200 and 2,000 mg/L). The extent of subsequent biofilm formation were then assessed with the tetrazolium salt reduction assay. Effect of eugenol on morphogenesis of C. albicans cells was observed by scanning electron microscopy (SEM). The results indicated that the effect of eugenol on adherent cells and subsequent biofilm formation was dependent on the initial adherence time and the concentration of this compound, and that eugenol can inhibit filamentous growth of C. albicans cells. In addition, using human erythrocytes, eugenol showed low hemolytic activity. These results indicated that eugenol displayed potent activity against C. albicans biofilms in vitro with low cytotoxicity and therefore has potential therapeutic implication for biofilm-associated candidal infections.