In vitro activity of gallium maltolate against Staphylococci in logarithmic, stationary, and biofilm growth phases: comparison of conventional and calorimetric susceptibility testing methods.

Ga(3+) is a semimetal element that competes for the iron-binding sites of transporters and enzymes. We investigated the activity of gallium maltolate (GaM), an organic gallium salt with high solubility, against laboratory and clinical strains of methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), methicillin-susceptible Staphylococcus epidermidis (MSSE), and methicillin-resistant S. epidermidis (MRSE) in logarithmic or stationary phase and in biofilms. The MICs of GaM were higher for S. aureus (375 to 2000 microg/ml) than S. epidermidis (94 to 200 microg/ml). Minimal biofilm inhibitory concentrations were 3,000 to >or=6,000 microg/ml (S. aureus) and 94 to 3,000 microg/ml (S. epidermidis). In time-kill studies, GaM exhibited a slow and dose-dependent killing, with maximal action at 24 h against S. aureus of 1.9 log(10) CFU/ml (MSSA) and 3.3 log(10) CFU/ml (MRSA) at 3x MIC and 2.9 log(10) CFU/ml (MSSE) and 4.0 log(10) CFU/ml (MRSE) against S. epidermidis at 10x MIC. In calorimetric studies, growth-related heat production was inhibited by GaM at subinhibitory concentrations; and the minimal heat inhibition concentrations were 188 to 4,500 microg/ml (MSSA), 94 to 1,500 microg/ml (MRSA), and 94 to 375 microg/ml (MSSE and MRSE), which correlated well with the MICs. Thus, calorimetry was a fast, accurate, and simple method useful for investigation of antimicrobial activity at subinhibitory concentrations. In conclusion, GaM exhibited activity against staphylococci in different growth phases, including in stationary phase and biofilms, but high concentrations were required. These data support the potential topical use of GaM, including its use for the treatment of wound infections, MRSA decolonization, and coating of implants.

Efeito de concentração espermática e período de incubação oócitoespermatozóides na fecundação in vitro em bovinos da raça Gir

Avaliou-se o efeito de concentrações espermáticas e períodos de incubação na fecundação in vitro com sêmen de touros Gir (Bos indicus). Oócitos maturados in vitro foram fecundados com sêmen de dois touros, nas concentrações de 1, 2 ou 4x10(6) espermatozóides/mL, por 12 ou 18 horas, e avaliada a taxa de fecundação e polispermia. Posteriormente, foi avaliada a taxa de clivagem com 72 horas e de blastocisto no oitavo dia pósfecundação. Um segundo experimento verificou o efeito de 1, 2 e 4x10(6) espermatozóides/mL por 18 horas, utilizando sêmen de três touros separadamente, bem como o efeito de 12 e 18 horas de incubação, na concentração de 1 a 2x10(6) espermatozóides/mL. Observou-se maior (P<0,05) polispermia na concentração de 4x10(6) espermatozóides/mL e que a associação dessa concentração com 18 horas aumentou a taxa de clivagem (P<0,05). No segundo experimento, o aumento do período de incubação melhorou (P<0,05) a taxa de clivagem para um dos touros e, quando comparados entre si, os animais produziram taxa de clivagem e de blastocistos diferentes (P<0,05) em uma mesma concentração ou período de incubação. Os resultados revelaram que a polispermia é o principal efeito do aumento da concentração espermática e que a eficiência entre touros na fecundação in vitro é dependente da concentração e período de incubação.

Release of vancomycin and teicoplanin from a plasticized and resorbable gelatin sponge: in vitro investigation of a new antibiotic delivery system with glycopeptides.

BACKGROUND: The aim of this study was to evaluate the efficacy of sustained release of vancomycin and teicoplanin from a resorbable gelatin glycerol sponge, in order to establish a new delivery system for local anti-infective therapy. MATERIALS AND METHODS: 60 plasticized glycerol gelatin sponges containing either 10 or 20% gelatin (w/v) were incubated in vancomycin or teicoplanin solution at 20 degrees C for either 1 or 24 h. In vitro release properties of the sponges were investigated over a period of 1 week by determining the levels of vancomycin and teicoplanin eluted in plasma using fluorescent polarization immunoassay. The rate constant and the half-life for the antibiotic release of each group were calculated by linear regression assuming first order kinetics. RESULTS: Presoaking for 24 h was associated with a significant increase in the total antibiotic release in all groups opposed to 1 h of incubation, except for the 10% sponges presoaked in teicoplanin. Doubling the gelatin content of the sponges from 10 to 20% significantly increased the total release of antibiotic load only in teicoplanin-containing sponges after 24 h incubation. In all corresponding groups investigated, release of vancomycin was more prolonged compared to teicoplanin, which allowed a gradual release beyond 5 days. The half-life (h +/- SEM) of both types of vancomycin-containing sponges was significantly prolonged by 24 h incubation in comparison to 1 h incubation (29.1 +/- 5.9 vs 5.9 +/- 1.0; p < 0.001, 30.0 +/- 2.1 vs 11.1 +/- 1.9; p < 0.001). However, neither doubling the gelatin content of the sponges nor a prolonged incubation was associated with a significantly prolonged delivery of teicoplanin. CONCLUSION: This study demonstrated a better diffusion-controlled release of vancomycin-impregnated glycerol gelatin sponges compared to those pretreated with teicoplanin. The plasticized glycerol gelatin sponge may be a promising carrier for the application of vancomycin to infected wounds for local anti-infective therapy.

In vitro combination of human and bovine free secretory component with IgA of various species.

The free form of the secretory component usually associated with secretory IgA can be isolated from human and bovine milk. These free secretory components of different origin combine in vitro with human polymeric myeloma IgA, with mouse myeloma IgA, and with the serum IgA of nine different mammalian species.

Freqüência de híbridos em cruzamentos controlados de citros: cultivo de sementes versus cultivo in vitro de embriões

Os objetivos deste trabalho foram estudar a freqüência de híbridos oriundos de cruzamentos controlados em citros, comparando a germinação de sementes em canteiros com o cultivo in vitro de embriões, e analisar o efeito da remoção dos tegumentos da semente na germinação. Como parentais femininos foram utilizadas as tangerineiras 'Clementina Palazelli' e 'Sunki', os limoeiros 'Cravo Santa Bárbara' e 'Volkameriano Catânia 2' e as laranjeiras Azedas 'Comum' e 'Double Calice'. Os parentais masculinos compreenderam Poncirus trifoliata e híbridos desta espécie. No cultivo das sementes em canteiros foi usado o delineamento experimental inteiramente casualizado, considerando-se os tratamentos de semente: íntegra; sem o tegumento externo (testa); e sem os tegumentos externo e interno (tégmen). O cultivo in vitro de embriões foi empregado como controle. Avaliou-se: velocidade de germinação das sementes, porcentagem de germinação e de poliembrionia, tamanho e número de embriões por semente, e porcentagem de híbridos. A remoção dos tegumentos das sementes acelerou o início de germinação de todas as variedades; afetou favorável e significativamente a porcentagem de germinação somente em tangerineira 'Sunki' e em limoeiro 'Volkameriano'. A porcentagem de indivíduos zigóticos resgatados foi maior no cultivo in vitro de embriões. A vantagem de uso desta técnica é diretamente proporcional ao grau de poliembrionia dos parentais femininos empregados nos cruzamentos.

Nanomedicines for active targeting: physico-chemical characterization of paclitaxel-loaded anti-HER2 immunonanoparticles and in vitro functional studies on target cells.

Paclitaxel (Tx)-loaded anti-HER2 immunonanoparticles (NPs-Tx-HER) were prepared by the covalent coupling of humanized monoclonal anti-HER2 antibodies (trastuzumab, Herceptin) to Tx-loaded poly (dl-lactic acid) nanoparticles (NPs-Tx) for the active targeting of tumor cells that overexpress HER2 receptors. The physico-chemical properties of NPs-Tx-HER were compared to unloaded immunonanoparticles (NPs-HER) to assess the influence of the drug on anti-HER2 coupling to the NP surface. The immunoreactivity of sulfo-MBS activated anti-HER2 mAbs and the in vitro efficacy of NPs-Tx-HER were tested on SKOV-3 ovarian cancer cells that overexpress HER2 antigens. Tx-loaded nanoparticles (NPs-Tx) obtained by a salting-out method had a size of 171+/-22 nm (P.I.=0.1) and an encapsulation efficiency of about of 78+/-10%, which corresponded to a drug loading of 7.8+/-0.8% (w/w). NPs-Tx were then thiolated and conjugated to activated anti-HER2 mAbs to obtain immunonanoparticles of 237+/-43 nm (P.I.=0.2). The influence of the activation step on the immunoreactivity of the mAbs was tested on SKOV-3 cells using 125I-radiolabeled mAbs, and the activity of the anti-HER2 mAbs was minimally affected after sulfo-MBS functionalization. Approximately 270 molecules of anti-HER2 mAbs were bound per nanoparticle. NPs-Tx-HER exhibited a zeta potential of 0.2+/-0.1 mV. The physico-chemical properties of the Tx-loaded immunonanoparticles were very similar to unloaded immunonanoparticles, suggesting that the encapsulation of the drug did not influence the coupling of the mAbs to the NPs. No drug loss was observed during the preparation process. DSC analysis showed that encapsulated Tx is in an amorphous or disordered-crystalline phase. These results suggest that Tx is entrapped in the polymeric matrix and not adsorbed to the surface of the NPs. In vitro studies on SKOV-3 ovarian cancer cells demonstrated the greater cytotoxic effect of NPs-Tx-HER compared to other Tx formulations. The results showed that at 1 ng Tx/ml, the viability of cells incubated with drug encapsulated in NP-Tx-HER was lower (77.32+/-5.48%) than the viability of cells incubated in NPs-Tx (97.4+/-12%), immunonanoparticles coated with Mabthera, as irrelevant mAb (NPs-Tx-RIT) (93.8+/-12%) or free drug (92.3+/-9.3%).

Conservação in vitro de germoplasma de cana-de-açúcar

O objetivo deste trabalho foi avaliar o efeito da temperatura, de sacarose, manitol e sorbitol, como fontes de carbono e reguladores osmóticos, e do ácido abscísico, como regulador de crescimento na conservação in vitro de germoplasma de cana-de-açúcar. Foram utilizadas, como material vegetal, gemas apicais de plantas de 10 meses de idade, do banco de germoplasma in vivo, da Universidade Federal de Alagoas. Brotos da quarta repicagem, no estádio de multiplicação in vitro, foram a fonte de explantes para três experimentos. Houve efeito positivo da diminuição da temperatura e da utilização da sacarose como fonte de carbono e regulador osmótico na manutenção da viabilidade dos explantes conservados in vitro. O ácido abscísico (1 mg/L) foi essencial para manter os explantes em crescimento mínimo por 12 meses (52 semanas). O uso das concentrações de 1 mg/L de ácido abscísico e de 20 g/L de sacarose associadas às condições de temperatura reduzida (15°C) demonstraram que os brotos permaneceram viáveis por um ano no mesmo meio de cultura, sem a necessidade de serem subcultivados.

Efeito de fungicidas na germinação in vitro de conídios de Claviceps africana

A cultura do sorgo não enfrentava problemas sérios de doenças nas condições de cultivo no Brasil, até o ano de 1995, quando se registrou a ocorrência da doença-açucarada, causada por Claviceps africana, que afeta as panículas e reduz a quantidade e a qualidade dos grãos; os conídios constituem a principal fonte de inóculo fúngico. Este trabalho teve como objetivo estudar o efeito dos fungicidas tebuconazole, propiconazole, triadimenol, triadimefon, flutriafol e mancozeb na inibição in vitro da germinação dos conídios. Os fungicidas mancozeb, triadimenol e propiconazole foram os mais eficientes na inibição da germinação dos conídios.

A new self-expanding aortic stent valve with annular fixation: in vitro haemodynamic assessment.

OBJECTIVE: Balloon-expandable stent valves require flow reduction during implantation (rapid pacing). The present study was designed to compare a self-expanding stent valve with annular fixation versus a balloon-expandable stent valve. METHODS: Implantation of a new self-expanding stent valve with annular fixation (Symetis, Lausanne, Switzerland) was assessed versus balloon-expandable stent valve, in a modified Dynatek Dalta pulse duplicator (sealed port access to the ventricle for transapical route simulation), interfaced with a computer for digital readout, carrying a 25 mm porcine aortic valve. The cardiovascular simulator was programmed to mimic an elderly woman with aortic stenosis: 120/85 mmHg aortic pressure, 60 strokes/min (66.5 ml), 35% systole (2.8 l/min). RESULTS: A total of 450 cardiac cycles was analysed. Stepwise expansion of the self-expanding stent valve with annular fixation (balloon-expandable stent valve) resulted in systolic ventricular increase from 120 to 121 mmHg (126 to 830+/-76 mmHg)*, and left ventricular outflow obstruction with mean transvalvular gradient of 11+/-1.5 mmHg (366+/-202 mmHg)*, systolic aortic pressure dropped distal to the valve from 121 to 64.5+/-2 mmHg (123 to 55+/-30 mmHg) N.S., and output collapsed to 1.9+/-0.06 l/min (0.71+/-0.37 l/min* (before complete obstruction)). No valve migration occurred in either group. (*=p<0.05). CONCLUSIONS: Implantation of this new self-expanding stent valve with annular fixation has little impact on haemodynamics and has the potential for working heart implantation in vivo. Flow reduction (rapid pacing) is not necessary.

Antirotavirus immunoglobulin A neutralizes virus in vitro after transcytosis through epithelial cells and protects infant mice from diarrhea.

Rotaviruses are the major cause of severe diarrhea in infants and young children worldwide. Due to their restricted site of replication, i.e., mature enterocytes, local intestinal antibodies have been proposed to play a major role in protective immunity. Whether secretory immunoglobulin A (IgA) antibodies alone can provide protection against rotavirus diarrhea has not been fully established. To address this question, a library of IgA monoclonal antibodies (MAbs) previously developed against different proteins of rhesus rotavirus was used. A murine hybridoma "backpack tumor" model was established to examine if a single MAb secreted onto mucosal surfaces via the normal epithelial transport pathway was capable of protecting mice against diarrhea upon oral challenge with rotavirus. Of several IgA and IgG MAbs directed against VP8 and VP6 of rotavirus, only IgA VP8 MAbs (four of four) were found to protect newborn mice from diarrhea. An IgG MAb recognizing the same epitope as one of the IgA MAbs tested failed to protect mice from diarrhea. We also investigated if antibodies could be transcytosed in a biologically active form from the basolateral domain to the apical domain through filter-grown Madin-Darby canine kidney (MDCK) cells expressing the polymeric immunoglobulin receptor. Only IgA antibodies with VP8 specificity (four of four) neutralized apically administered virus. The results support the hypothesis that secretory IgA antibodies play a major role in preventing rotavirus diarrhea. Furthermore, the results show that the in vivo and in vitro methods described are useful tools for exploring the mechanisms of viral mucosal immunity.

In vitro and in vivo evaluation of sunitinib eluting beads

Background: Chemoembolization is used to treat liver malignancies. However recurrence occurs frequently, possibly because of neoangiogenesis triggered by ischemia caused by the embolic agent. In this context, the combination of an embolic agent with an anti-angiogenic drug seems appealing. This study characterizes the in vitro loading and release profile of sunitinib eluting beads of different sizes and their pharmacokinetic profile in a rabbit model. Methods: 70-150 μm and 100-300 μm drug eluting beads (DC Bead, Biocompatibles UK) were loaded by incubation in a sunitinib hydrochloride solution. Drug was quantified by spectrophotometry at 430 nm. Drug release was measured over one-week periods and normalized using an internal standard in 30% ethanol in NaCl 0.9%. New-Zealand white rabbits were used. Eight animals received 0.2 ml of 100-300 μm DC Bead loaded with 6 mg of sunitinib in the hepatic artery (group 1) and 4 animals received 6 mg of sunitinib p.o. (group 2). Half of the animals were sacrificed after 6 hours and half after24 hours. Liver enzymes were measured at 0, 6 and 24 hours in both groups. Plasmatic sunitinib concentration was determined by tandem mass spectroscopy (LC MS/MS) at 0, 1, 2, 3, 4, 5, 6 and 24 hours. At sacrifice, the livers were harvested and sunitinib concentration in liver tissue was assessed by LC MS/MS. Results: High drug loading was obtained for both microsphere bead sizes. Particle shrinking was observed with adsorption of sunitinib. Almost complete release of sunitinib was detected under physiological conditions, with very similar release for 70-150 μm and 100-300 μm (t50%=1.2 h) DC Bead. Conclusions: Sunitinib eluting beads are well tolerated by rabbits when administered in the hepatic artery. No unexpected toxicity was observed. Very high drug concentration can be obtained at the site of embolization with minimal systemic passage.

Propagação in vitro e avaliação de parâmetros morfofisiológicos de porta-enxertos de videira

A micropropagação de genótipos selecionados pode contribuir para atender a demanda de plantas matrizes e mudas de qualidade genética e sanitária comprovadas de videira (Vitis spp.) no Estado de Santa Catarina. O objetivo deste trabalho foi estabelecer e multiplicar in vitro porta-enxertos de videira e avaliar parâmetros morfofisiológicos fundamentais à micropropagação e aclimatização. Os porta-enxertos VR043-43, VR039-16, Paulsen 1103, R110, SO4 e Kober 5BB foram estabelecidos e multiplicados in vitro pelo método de gemas axilares em meio de cultura DSD1. Quarenta e dois por cento dos explantes foram estabelecidos in vitro. Houve variabilidade de crescimento, área foliar e matéria seca entre os genótipos. O porta-enxerto Paulsen 1103 foi numericamente superior aos demais no desenvolvimento in vitro em comprimento de caule (6,2 cm), produção de biomassa (34,8 mg) e área foliar (18,1 cm²) in vitro. O teor de clorofila total variou entre os porta-enxertos e o ambiente de cultura, com 0,7 e 2,8 mg/g de matéria fresca do R110 (in vitro) e VR043-43 (ex vitro), respectivamente. A maior (216,4/mm²) e a menor (119,2/mm²) densidade estomática foram apresentadas pelo VR039-16 in vitro e pelo SO4 ex vitro, respectivamente. A taxa de sobrevivência de plantas na aclimatização foi em média 90,3±1,1% por genótipo. Os porta-enxertos de videira avaliados apresentaram características morfofisiológicas apropriadas para a propagação in vitro e a transferência ex vitro.

Brain Spectrins 240/235 and 240/235E: Differential Expression During Development of Chicken Dorsal Root Ganglia in vivo and in vitro.

Brain spectrin, a membrane-related cytoskeletal protein, exists as two isoforms. Brain spectrin 240/235 is localized preferentially in the perikaryon and axon of neuronal cells and brain spectrin 240/235E is found essentially in the neuronal soma and dendrites and in glia (Riederer et al., 1986, J. Cell Biol., 102, 2088 - 2097). The sensory neurons in dorsal root ganglia, devoid of any dendrites, make a good tool to investigate such differential expression of spectrin isoforms. In this study expression and localization of both brain spectrin isoforms were analysed during early chicken dorsal root ganglia development in vivo and in culture. Both isoforms appeared at embryonic day 6. Brain spectrin 240/235 exhibited a transient increase during embryonic development and was first expressed in ventrolateral neurons. In ganglion cells in situ and in culture this spectrin type showed a somato - axonal distribution pattern. In contrast, brain spectrin 240/235E slightly increased between E6 and E15 and remained practically unchanged. It was localized mainly in smaller neurons of the mediodorsal area as punctate staining in the cytoplasm, was restricted exclusively to the ganglion cell perikarya and was absent from axons both in situ and in culture. This study suggests that brain spectrin 240/235 may contribute towards outgrowth, elongation and maintenance of axonal processes and that brain spectrin 240/235E seems to be exclusively involved in the stabilization of the cytoarchitecture of cell bodies in a selected population of ganglion cells.

Toxicidade de antibióticos no cultivo in vitro da batata em meios semi-sólido e líquido

O objetivo deste trabalho foi avaliar os efeitos fitotóxicos de antibióticos no crescimento e na taxa de multiplicação in vitro da batata. Brotações da cultivar Baronesa foram cultivadas em meio de multiplicação de consistência semi-sólida e líquida. O meio de multiplicação foi formado pelos sais e vitaminas de MS ao qual adicionou-se um dos seguintes antibióticos: ampicilina, cloranfenicol, estreptomicina e tetraciclina, previamente selecionados em razão da ação bactericida sobre contaminantes da cultura, nas concentrações de 0, 32, 64, 128, 256, 512 e 1.024 mg L-1. Por 21 dias os materiais foram mantidos em sala de crescimento a 25±2°C, 16 horas de luz e fluxo de radiação de 35 µmol m-2 s-1. Nos tratamentos em que se utilizou meio de cultura líquido, os frascos foram mantidos sob constante agitação em mesa agitadora do tipo orbital. A ampicilina foi o único antibiótico que não afetou a sobrevivência e o desenvolvimento dos explantes de batata em meio de multiplicação, podendo ser indicada para trabalhos de descontaminação in vitro dessa espécie. O aumento das concentrações de cloranfenicol, estreptomicina e tetraciclina no meio de cultura apresentou efeitos fitotóxicos severos sobre o crescimento e taxa de multiplicação do material vegetal.

Metabolismo de plantas de cana-de-açúcar cultivadas in vitro sob diferentes concentrações de nitrogênio

Variedades de cana-de-açúcar (RB 732577, RB 83102, RB 75126, CB 45-3, Caiana e Co 997) foram cultivadas in vitro em meio nutritivo básico MS com diferentes concentrações de N (9,83 mM, 7,37 mM, 4,92 mM, 2,46 mM, 0,49 mM e 0,0 mM), com o objetivo de avaliar a eficiência no processo de assimilação desse elemento. Após 45 dias de cultivo, foram determinados os ganhos de biomassa e os teores de amônio, aminoácidos e açúcares solúveis. A variedade Co 997 apresentou maior ganho de biomassa e maior quantidade de N incorporado em aminoácidos livres em relação às demais variedades, sugerindo maior eficiência no processo de assimilação. Os teores mais elevados de amônio foram observados nas variedades RB 83102 e RB 75126, quando cultivadas em meio desprovido de N, mostrando-se menos eficientes no processo de assimilação uma vez que apresentaram os menores valores de aminoácidos livres nas mesmas condições de cultivo.