Redirecting anti-viral CTL against cancer cells by surface targeting of monomeric MHC class I-viral peptide conjugated to antibody fragments.

To combine the advantage of both the tumor targeting capacity of high affinity monoclonal antibodies (mAbs) and the potent killing properties of cytotoxic T lymphocytes (CTL), we investigated the activity of conjugates made by coupling single Fab' fragments, from mAbs specific for tumor cell surface antigens, to monomeric HLA-A2 complexes containing the immunodominant influenza-matrix peptide 58-66. In solution, the monovalent 95 kDa Fab-HLA-A2/Flu conjugates did not activate influenza-specific CTL. However, when targeted to tumor cells expressing the relevant tumor-associated antigen, the conjugates induced CTL activation and efficient tumor cell lysis, as a result of MHC/peptide surface oligomerization. The highly specific and sensitive in vitro cytotoxicity results presented suggest that injection of Fab-MHC/peptide conjugates could represent a new form of immunotherapy, bridging antibody and T lymphocyte attack on cancer cells.

Ocular distribution, spectrum of activity, and in vivo viral neutralization of a fully humanized anti-herpes simplex virus IgG Fab fragment following topical application.

Herpes simplex ocular infection is a major cause of corneal blindness. Local antiviral treatments exist but are associated with corneal toxicity, and resistance has become an issue. We evaluated the biodistribution and efficacy of a humanized anti-herpes simplex virus (anti-HSV) IgG FAb fragment (AC-8; 53 kDa) following repeated topical administration. AC-8 was found in the corneal epithelium, anterior stroma, subepithelial stromal cells, and retinal glial cells, with preferential entry through the ocular limbus. AC-8 was active against 13 different strains of HSV-1, with 50% and 90% mean effective concentrations (MEC(50) and MEC(90), respectively) ranging from 0.03 to 0.13 μg/ml, indicating broad-spectrum activity. The in vivo efficacy of AC-8 was evaluated in a mouse model of herpes-induced ocular disease. Treatment with low-dose AC-8 (1 mg/ml) slightly reduced the ocular disease scores. A greater reduction of the disease scores was observed in the 10-mg/ml AC-8-treated group, but not as much as with trifluridine (TFT). AC-8 treatment reduced viral titers but less than trifluridine. AC-8 did not display any toxicity to the cornea or other structures in the eye. In summary, topical instillation of an anti-HSV FAb can be used on both intact and ulcerated corneas. It is well tolerated and does not alter reepithelialization. Further studies to improve the antiviral effect are needed for AC-8 to be considered for therapeutic use.

Three-dimensional models of 14α-sterol demethylase (Cyp51A) from Aspergillus lentulus and Aspergillus fumigatus: an insight into differences in voriconazole interaction.

Aspergillus lentulus, an Aspergillus fumigatus sibling species, is increasingly reported in corticosteroid-treated patients. Its clinical significance is unknown, but the fact that A. lentulus shows reduced antifungal susceptibility, mainly to voriconazole, is of serious concern. Heterologous expression of cyp51A from A. fumigatus and A. lentulus was performed in Saccharomyces cerevisiae to assess differences in the interaction of Cyp51A with the azole drugs. The absence of endogenous ERG11 was efficiently complemented in S. cerevisiae by the expression of either Aspergillus cyp51A allele. There was a marked difference between azole minimum inhibitory concentration (MIC) values of the clones expressing each Aspergillus spp. cyp51A. Saccharomyces cerevisiae clones expressing A. lentulus alleles showed higher MICs to all of the azoles tested, supporting the hypothesis that the intrinsic azole resistance of A. lentulus could be associated with Cyp51A. Homology models of A. fumigatus and A. lentulus Cyp51A protein based on the crystal structure of Cyp51p from Mycobacterium tuberculosis in complex with fluconazole were almost identical owing to their mutual high sequence identity. Molecular dynamics (MD) was applied to both three-dimensional protein models to refine the homology modelling and to explore possible differences in the Cyp51A-voriconazole interaction. After 20ns of MD modelling, some critical differences were observed in the putative closed form adopted by the protein upon voriconazole binding. A closer study of the A. fumigatus and A. lentulus voriconazole putative binding site in Cyp51A suggested that some major differences in the protein's BC loop could differentially affect the lock-up of voriconazole, which in turn could correlate with their different azole susceptibility profiles.

Antitumor and Antiviral Activity of Colombian Medicinal Plant Extracts

Extracts of nine species of plants traditionally used in Colombia for the treatment of a variety of diseases were tested in vitro for their potential antitumor (cytotoxicity) and antiherpetic activity. MTT (Tetrazolium blue) and Neutral Red colorimetric assays were used to evaluate the reduction of viability of cell cultures in presence and absence of the extracts. MTT was also used to evaluate the effects of the extracts on the lytic activity of herpes simplex virus type 2 (HSV-2). The 50% cytotoxic concentration (CC50) and the 50% inhibitory concentration of the viral effect (EC50) for each extract were calculated by linear regression analysis. Extracts from Annona muricata, A. cherimolia and Rollinia membranacea, known for their cytotoxicity were used as positive controls. Likewise, acyclovir and heparin were used as positive controls of antiherpetic activity. Methanolic extract from Annona sp. on HEp-2 cells presented a CC50 value at 72 hr of 49.6x103mg/ml. Neither of the other extracts examined showed a significant cytotoxicity. The aqueous extract from Beta vulgaris, the ethanol extract from Callisia grasilis and the methanol extract Annona sp. showed some antiherpetic activity with acceptable therapeutic indexes (the ratio of CC50 to EC50). These species are good candidates for further activity-monitored fractionation to identify active principles.

Multiple isolates from Aids patients: aspects of an analysis by a genotypic marker and antimicrobial susceptibilities variations

Twenty-one Mycobacterium avium multisolates, from ten human immunodeficiency virus-infected patients, were typed by restriction fragment length polymorphism using as marker the IS1245 and characterized by minimum inhibitory concentration for nine different antibiotics. Two out of four patients harboring multisolates with different fingerprint profile, were therefore considered as having a polyclonal infection, since their isolates were taken from sterile site. This result confirms that polyclonal infection caused by M. avium occurs with a nonnegligenciable frequency. Analyzing the multisolates susceptibility profile of each patient it was observed that most of them were infected with strains having appreciably different antimicrobial susceptibility patterns, no matter what the genotypic pattern of the strains was. These results have strong implication for the treatment of the patients.

Cytotoxic and Antiviral Activities of Colombian Medicinal Plant Extracts of the Euphorbia genus

Forty-seven plant extracts of 10 species of the genus Euphorbia (Euphorbiaceae) used by Colombian traditional healers for the treatment of ulcers, cancers, tumors, warts, and other diseases, were tested in vitro for their potential antitumour (antiproliferative and cytotoxic) and antiherpetic activity. To evaluate the capacity of the extracts to inhibit the lytic activity of herpes simplex virus type 2 (HSV-2) and the reduction of viability of infected or uninfected cell cultures, the end-point titration technique (EPTT) and the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay were used, respectively. The therapeutic index of the positive extracts for the antiviral activity was determined by calculating the ratio CC50 (50% cytotoxic concentration) over IC50 (50% inhibitory concentration of the viral effect). Five of the 47 extracts (11%) representing 3 out of 10 Euphorbia species (30%) exhibited antiherpetic action; the highest activity was found in the leaf/stem water-methanol extracts from E. cotinifolia and E. tirucalli. The therapeutic indexes of these two plant species were > 7.1; these extracts exhibited no cytotoxicity. Six extracts (13%) representing 4 plant species (40%) showed cytotoxic activity. The highest cytotoxicity was found in the dichloromethane extract obtained from E. cotinifolia leaves and the CC50 values for the most susceptible cell lines, HEp-2 and CHO, were 35.1 and 18.1 µg/ml, respectively.

Antibacterial xanthones from Kielmeyera variabilis mart. (Clusiaceae)

The bioassay-guided fractionation of stems from Kielmeyera variabilis, traditionally used in Brazilian folk medicine, yielded assiguxanthone-B (1), kielcorin (4), 2,5-dihydroxybenzoic acid (3), and a mixture of xanthones containing assiguxanthone-B (1) and 1,3,5,6-tetrahydroxy-2-prenylxanthone (2) (1:1 w/w). The xanthone mixture inhibited Staphylococcus aureus and Bacillus subtilis at a concentration of 6.25 µg/ml. When tested alone, the minimal inhibitory concentration of assiguxanthone-B was 25 µg/ml against B. subtilis. Kielcorin and 2,5-dihydroxybenzoic acid were inactive against both strains. None of the fractions was active against Escherichia coli or Pseudomonas aeruginosa. Viable cells of S. aureus were reduced by a 1-3 log CFU/ml within 12 h after exposure of one to eight times the MIC of the xanthone mixture. It is not known whether the tetrahydroxy-2-prenylxanthone or other components of the xanthone mixture are responsible for the main antibacterial activity or whether additive or synergistic action is involved

In vitro susceptibility characteristics of Cryptococcus neoformans varieties from AIDS patients in Goiânia, Brazil

Sixty clinical isolates of Cryptococcus neoformans from AIDS from Goiânia, state of Goiás, Brazil, were characterized according to varieties, serotypes and tested for antifungal susceptibility. To differentiate the two varieties was used L-canavanine-glycine-bromothymol blue medium and to separate the serotypes was used slide agglutination test with Crypto Check Iatron. The Minimal Inhibitory Concentration (MIC) of fluconazole, itraconazole, and amphotericin B were determined by the National Committee for Clinical Laboratory Standards macrodilution method. Our results identified 56 isolates as C. neoformans var. neoformans serotype A and 4 isolates as C. neoformans var. gattii serotype B. MIC values for C. neoformans var. gattii were higher than C. neoformans var. neoformans. We verified that none isolate was resistant to itraconazole and to amphotericin B, but one C. neoformans var. neoformans and three C. neoformans var. gattii isolates were resistant to fluconazole. The presence of C. neoformans var. gattii fluconazole resistant indicates the importance of determining not only the variety of C. neoformans infecting the patients but also measuring the MIC of the isolate in order to properly orient treatment.

Antimicrobial activity in vitro of plumbagin isolated from Plumbago species

Plumbagin is a naturally occurring naphthoquinone isolated from roots of Plumbago scandens. The plant was collected at the Campus of Fundação Oswaldo Cruz, Rio de Janeiro, Brazil. P. scandens is used as a traditional medicine for the treatment of several diseases. The antimicrobial activity of plumbagin was evaluated using the macrodilution method. The compound exhibited relatively specific activity against bacteria and yeast. The minimum inhibitory concentration test showed the growth inhibiton of Staphylococcus aureus at a concentration of 1.56 µg/ml and of Candida albicans at a concentration of 0.78 µg/ml. These results suggest the naphthoquinone plumbagin as a promising antimicrobial agent.

Antibacterial activity of extracts and neolignans from Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck

The evaluation of the activity of the aqueous and ethyl acetate extracts of the leaves of Piper regnellii was tested against gram-positive and gram-negative bacteria. The aqueous extractdisplayed a weak activity against Staphylococcus aureus and Bacillus subtilis with minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of 1000 µg/ml. The ethyl acetate extract presented a good activity against S. aureus and B. subtilis with MIC and MBC at 15.62 µg/ml. In contrast to the relative low MICs for gram-positive bacteria, gram-negative bacteria were not inhibited by the extracts at concentrations < 1000 mg/ml. The ethyl acetate extract was fractionated on silica gel into nine fractions. The hexane and chloroform fractions were active against S. aureus (MIC at 3.9 µg/ml) and B. subtilis (MIC at 3.9 and 7.8 µg/ml, respectively). Using bioactivity-directed fractionation, the hexane fraction was rechromatographed to yield the antimicrobial compounds 1, 2, 5, and 6identified as eupomatenoid-6, eupomatenoid-5, eupomatenoid-3, and conocarpan, respectively. The pure compounds 1 and 2 showed a good activity against S. aureus with MIC of 1.56 µg/ml and 3.12 µg/ml, respectively. Both compounds presented MIC of 3.12 µg/ml against B. subtilis. The pure compound 6 named as conocarpan was quite active against S. aureus and B. subtilis with MIC of 6.25 µg/ml. The antibacterial properties of P. regnellii justify its use in traditional medicine for the treatment of wounds, contaminated through bacteria infections.

Antimicrobial susceptibility determined by the E test, Löwenstein-Jensen proportion, and DNA sequencing methods among Mycobacterium tuberculosis isolates discrepancies, preliminary results

Mycobacterium tuberculosis strains resistant to streptomycin (SM), isoniazid (INH), and/or rifampin (RIF) as determined by the conventional Löwenstein-Jensen proportion method (LJPM) were compared with the E test, a minimum inhibitory concentration susceptibility method. Discrepant isolates were further evaluated by BACTEC and by DNA sequence analyses for mutations in genes most often associated with resistance to these drugs (rpsL, katG, inhA, and rpoB). Preliminary discordant E test results were seen in 75% of isolates resistant to SM and in 11% to INH. Discordance improved for these two drugs (63%) for SM and none for INH when isolates were re-tested but worsened for RIF (30%). Despite good agreement between phenotypic results and sequencing analyses, wild type profiles were detected on resistant strains mainly for SM and INH. It should be aware that susceptible isolates according to molecular methods might contain other mechanisms of resistance. Although reproducibility of the LJPM susceptibility method has been established, variable E test results for some M. tuberculosis isolates poses questions regarding its reproducibility particularly the impact of E test performance which may vary among laboratories despite adherence to recommended protocols. Further studies must be done to enlarge the evaluated samples and looked possible mutations outside of the hot spot sequenced gene among discrepant strains.

Real-time antifungal susceptibility testing of Mucor spp., Fusarium spp. and Scedosporium spp. by isothermal microcalorimetry

Objective: Aspergillus species are the main pathogens causing invasive fungal infections but the prevalence of other mould species is rising. Resistance to antifungals among these new emerging pathogens presents a challenge for managing of infections. Conventional susceptibility testing of non-Aspergillus species is laborious and often difficult to interpret. We evaluated a new method for real-time susceptibility testing of moulds based on their of growth-related heat production.Methods: Laboratory and clinical strains of Mucor spp. (n = 4), Scedoporium spp. (n = 4) and Fusarium spp. (n = 5) were used. Conventional MIC was determined by microbroth dilution. Isothermal microcalorimetry was performed at 37 C using Sabouraud dextrose broth (SDB) inoculated with 104 spores/ml (determined by microscopical enumeration). SDB without antifungals was used for evaluation of growth characteristics. Detection time was defined as heat flow exceeding 10 lW. For susceptibility testing serial dilutions of amphotericin B, voriconazole, posaconazole and caspofungin were used. The minimal heat inhibitory concentration (MHIC) was defined as the lowest antifungal concentration, inhbiting 50% of the heat produced by the growth control at 48 h or at 24 h for Mucor spp. Susceptibility tests were performed in duplicate.Results: Tested mould genera had distinctive heat flow profiles with a median detection time (range) of 3.4 h (1.9-4.1 h) for Mucor spp, 11.0 h (7.1-13.7 h) for Fusarium spp and 29.3 h (27.4-33.0 h) for Scedosporium spp. Graph shows heat flow (in duplicate) of one representative strain from each genus (dashed line marks detection limit). Species belonging to the same genus showed similar heat production profiles. Table shows MHIC and MIC ranges for tested moulds and antifungals.Conclusions: Microcalorimetry allowed rapid detection of growth of slow-growing species, such as Fusarium spp. and Scedosporium spp. Moreover, microcalorimetry offers a new approach for antifungal susceptibility testing of moulds, correlating with conventional MIC values. Interpretation of calorimetric susceptibility data is easy and real-time data on the effect of different antifungals on the growth of the moulds is additionally obtained. This method may be used for investigation of different mechanisms of action of antifungals, new substances and drug-drug combinations.

Antifungal activity from Ocimum gratissimum L. towards Cryptococcus neoformans

Cryptococcal infection had an increased incidence in last years due to the explosion of acquired immune deficiency syndrome epidemic and by using new and effective immunosuppressive agents. The currently antifungal therapies used such as amphotericin B, fluconazole, and itraconazole have certain limitations due to side effects and emergence of resistant strains. So, a permanent search to find new drugs for cryptococcosis treatment is essential. Ocimum gratissimum, plant known as alfavaca (Labiatae family), has been reported earlier with in vitro activity against some bacteria and dermatophytes. In our work, we study the in vitro activity of the ethanolic crude extract, ethyl acetate, hexane, and chloroformic fractions, essential oil, and eugenol of O. gratissimum using an agar dilution susceptibility method towards 25 isolates of Cryptococcus neoformans. All the extracts of O. gratissimum studied showed activity in vitro towards C. neoformans. Based on the minimal inhibitory concentration values the most significant results were obtained with chloroformic fraction and eugenol. It was observed that chloroformic fraction inhibited 23 isolates (92%) of C. neoformans at a concentration of 62.5 µg/ml and eugenol inhibited 4 isolates (16%) at a concentration of 0.9 µg/ml. This screening may be the basis for the study of O. gratissimum as a possible antifungal agent.

Wet chemical silver treatment of endotracheal tubes to produce antibacterial surfaces.

Mechanically ventilated patients in hospitals are subjected to an increased risk of acquiring nosocomial pneumonia that sometimes has a lethal outcome. One way to minimize the risk could be to make the surfaces on endotracheal tubes antibacterial. In this study, bacterial growth was inhibited or completely prevented by silver ions wet chemically and deposited onto the tube surface. Through the wet chemical treatment developed here, a surface precipitate was formed containing silver chloride and a silver stearate salt. The identity and morphology of the surface precipitate was studied using x-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, scanning electron microscopy, and x-ray powder diffraction. Leaching of silver ions into solution was examined, and bacterial growth on the treated surfaces was assayed using Pseudomonas aeruginosa wild type (PAO1) bacteria. Furthermore, the minimum inhibitory concentration of silver ions was determined in liquid- and solid-rich growth medium as 23 and 18 microM, respectively, for P. aeruginosa.

Antibacterial and cytotoxic activity of Brazilian plant extracts - Clusiaceae

Twelve extracts obtained from nine plants belonging to six different genera of Clusiaceae were analyzed against Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and Enterococcus faecalis) bacteria using the microdilution broth assay. Tovomita aff. longifolia, T. brasiliensis, Clusia columnaris, Garcinia madruno, Haploclathra paniculata, and Caraipa grandifolia extracts showed significant results against the bacteria. The organic extract obtained from the leaves of T. aff. longifolia showed minimal inhibitory concentration (MIC) = 70 µg/ml and minimal bactericidal concentration (MBC) = 90 µg/ml against E. faecalis and the organic extract made with the stem of C. columnaris showed MIC = 180 µg/ml and MBC = 270 µg/ml against P. aeruginosa. None of the antibacterial extracts showed lethal activity against brine shrimp nauplii. On the other hand, both aqueous and organic extracts obtained from the aerial organs of Vismia guianensis that were cytotoxic to brine shrimp nauplii did not show a significant antibacterial activity in the assay.