Suplementação com óleos ricos em ácidos graxos poli-insaturados na dieta de leitões na fase de creche: efeitos no desempenho, na resposta inflamatória, no perfil lipídico e no "status" oxidativo / Supplementation with oils rich in polyunsaturated fatty acids in the diet of pigs in the nursery phase: effects on performance, inflammatory response, lipid profile and oxidative "status"

Nos sistemas de produção, os produtos naturais vêm se destacando na substituição a produtos sintéticos, dentre eles podemos ressaltar os óleos vegetais ricos em ácido graxos poli-insaturados (PUFA), que são conhecidos, popularmente, por seus efeitos benéficos. Dessa forma, o objetivo com este trabalho foi avaliar os efeitos da suplementação dos óleos de pequi (Caryocar brasiliense) e girassol (Helianthus annus) sobre parâmetros fisiológicos em leitões na fase de creche. Foram utilizados 180 leitões alojados em granja comercial, distribuídos em três grupos (n=60) conforme suplementação alimentar: óleo de pequi, óleo de girassol e controle negativo. Amostras de sangue e o peso dos animais foram obtidos nos intervalos de quatro trocas de rações da fase de creche para avaliação do desempenho, da resposta inflamatória, do perfil lipídico e do "status" oxidativo. Os parâmetros fisiológicos mensurados demonstraram que os óleos interferiram positivamente na resposta inflamatória sistêmica, por meio dos leucócitos totais e da relação neutrófilo/linfócito (P<0,05); no equilíbrio oxidante-antioxidante, por mensuração de óxido nítrico e do malondialdeído (P<0,05); e no metabolismo lipídico, com a avaliação de colesterol total e triglicérides (P<0,05). Por outro lado, esses óleos vegetais interferiram no ganho de peso e no consumo de ração (P<0,05). Assim, concluí-se que a suplementação com óleos de pequi e girassol melhora a saúde dos animais, mas tem impacto negativo no desempenho zootécnico de leitões na fase de creche.(AU)

In production systems, natural products have been outstanding in replacing synthetic products, among them, we can highlight the vegetable oils rich in polyunsaturated fatty acids (PUFA) that are popularly known for their beneficial effects. Thus, the objective of this study was to evaluate the effects of pequi (Caryocar brasiliense) and sunflower (Helianthus annus) oil supplementation on nursery physiological parameters. We used 180 piglets housed in a commercial farm, distributed in 3 groups (n=60) according to food supplementation: pequi oil, sunflower oil and negative control. Blood samples and animal weight were obtained at intervals of four nursery phase rations to evaluate performance, inflammatory response, lipid profile and oxidative status. The physiological parameters measured showed that the oils positively interfered in the systemic inflammatory response through total leukocytes and neutrophil / lymphocyte ratio (P<0.05), in the oxidant-antioxidant balance by measuring nitric oxide and malondialdehyde (P<0.05). and lipid metabolism with the assessment of total cholesterol and triglycerides (P<0.05). On the other hand, these vegetable oils interfered with weight gain and feed intake (P<0.05). Thus, it can be concluded that supplementation with pequi and sunflower oils improves animal health, but has a negative impact on the piglet's performance in the nursery phase.(AU)

Avaliação do estresse e do desempenho de suínos na fase de creche, empregando-se técnicas de enriquecimento ambiental / Evaluation of stress and performance of pigs during the nursery phase, using environmental enrichment techniques

O enriquecimento ambiental é uma ferramenta importante dentro dos sistemas de produção, a fim de promover o bem-estar e favorecer a saúde dos animais. Portanto, o objetivo deste trabalho foi avaliar o efeito do enriquecimento ambiental sobre o estresse de suínos na fase de creche. Foram utilizados 32 leitões, alojados em granja experimental, distribuídos em quatro grupos (n= 8): corda, corrente, garrafa PET e controle negativo. Amostras de sangue foram coletadas no início e no final do experimento para contagem de leucócitos circulantes e determinação de antioxidantes não enzimáticos, óxido nítrico, malondialdeído, e de saliva para avaliação do cortisol. Foi aplicado etograma e fez-a ganho médio de peso diário e a conversão alimentar. Os parâmetros avaliados no primeiro dia do experimento não variaram entre os grupos (P>0,05). No último dia do experimento, os valores de neutrófilos e da relação neutrófilo/linfócito foram mais elevados nos leitões do grupo corrente, assim como os valores de cortisol salivar (P<0,05). O ácido úrico apresentou-se mais elevado nos leitões do grupo corrente e o malondialdeído (MDA) nos do grupo garrafa (P< 0,05). Os enriquecimentos ambientais estimularam comportamentos positivos nos leitões, tendo a corda se destacado como o mais atrativo. Por outro lado, a corrente apresentou efeito negativo sobre a fisiologia dos animais, gerando estresse, assim como a garrafa, que induziu a peroxidação lipídica e um menor ganho de peso nos leitões.(AU)

Environmental enrichment is an important tool within production systems to promote welfare and animal health. The aim of this study was to evaluate the effect of enrichment objects on stress of piglets in nursery phase. 32 piglets housed in experimental farm were distributed in 4 groups (n= 8): rope, chain, pet bottle and negative control. Blood samples were collected at the beginning and end of the experiment to count circulating leukocytes and determine non-enzymatic antioxidants, nitric oxide, malondialdehyde, and saliva to evaluate cortisol. At the same time, an etogram and evaluation of mean daily gain and feed conversion were applied. The evaluated parameters on first day of the experiment did not vary between groups (P> 0.05). On the last day, neutrophil and neutrophil /lymphocyte ratios were higher in chain group piglets, as were salivary cortisol values (P< 0.05). Uric acid was higher in chain group and MDA in bottle group (P< 0.05). Environmental enrichment stimulated positive behaviors in piglets, where the rope stood out as the most attractive. On the other hand, chain had a negative effect on animal physiology, generating stress, as well as the bottle that induced lipid peroxidation and a lower weight gain in piglets.(AU)

[Chronicity and primary care: the role of prison health]. / Cronicidad y atención primaria: rol de la sanidad penitenciaria.

The Prison Primary Health Care Teams in Catalonia have been integrated into the Catalan Health Institute. This integration shall facilitate¹ training and updating, while eliminating the existing differences between the health services belonging to prison institutions and those of the Catalan Health Service. It shall enable team work and coordination between Primary Health Care Teams in the community and the PHCTs in prisons within the same geographical area by sharing ongoing training, multi-sector work teams and territory-based relations, thereby facilitating continuance in care and complete and integrated treatment of chronicity. The existing information systems in Primary Health Care and the shared clinical history in Catalonia are key factors for this follow up process. Support tools for clinical decision making shall also be shared, which shall contribute towards an increase in quality and clinical safety. These tools include electronic clinical practice guides, therapeutic guides, prescription alert systems, etc. This shall be an opportunity for Prison Health Care Teams to engage in teaching and research, which in turn shall have an indirect effect on improvements in health care quality and the training of professionals in this sector. The critical factor for success is the fact that a unique chronicity health care model shall be shared, where measures for health promotion prevention can be taken, along with multi-sector monitoring of pathologies and with health care information shared between professionals and levels throughout the patient's life, both in and out of the prison environment.

Current concepts on diagnosis and treatment of mastocytosis.

Mastocytosis is a heterogeneous group of disorders characterized by a clonal proliferation and accumulation of mast cells in one or more organ, primarily in the skin and bone marrow. The clinical spectrum of the disease varies from relatively benign forms with isolated skin lesions to very aggressive variants with extensive systemic involvement and poor prognosis. The growth and proliferation of clonal mast cells is caused by an activating mutation of the tyrosine kinase receptor Kit for Stem Cell Factor, the main growth factor for mast cells. Clinical symptoms are related to mast-cell mediator release, to the tissue mast cell infiltration or both. The degree of infiltration and cell activation determines the highly variable clinical and morphological features. Current treatment of mastocytosis includes symptomatic, antimediator drugs and cytoreductive targeted therapies.

Fitoterápicos na odontologia: estudo etnobotânico na cidade de Manaus / Phytotherapics in Odontology: ethnobotanical study in Manaus

O reconhecimento oficial da fitoterapia na odontologia no Brasil veio acompanhado de diversas lacunas na pesquisa científica e na utilização de plantas medicinais, especificamente para espécies vegetais com aplicação nas doenças da cavidade oral. O estado do Amazonas, especificamente a cidade de Manaus, não possui um diagnóstico da aplicabilidade de plantas medicinais nos serviços de atenção Odontológica. Esse estudo propôs realizar um estudo do tipo quali-quantitativo, descritivo e exploratório visando o levantamento da comercialização de plantas medicinais e o estudo etnobotânico para identificação das principais plantas medicinais indicadas e utilizadas nas patologias orais nos atendimentos odontológicos ambulatoriais na cidade de Manaus. Foram entrevistados 197 usuários do serviço odontológico, 150 Cirurgiões-Dentistas, e 47 comerciantes credenciados na prefeitura Municipal de Manaus. A amostragem foi realizada por acessibilidade ou conveniência para os Cirurgiões-Dentistas, enquanto os usuários foram selecionados através de amostragem aleatória simples, e os comerciantes de plantas medicinais foram entrevistados em sua totalidade. Os resultados demonstraram a existência de comercialização de plantas medicinais para patologias orais, destacando-se as seguintes espécies: Pedra ume cãa (Aulomyrcia sphareocarpa), Crajiru (Arrabidae chica), além da planta, sem identificação botânica, conhecida popularmente como Sara tudo. Entre os Cirurgiões-Dentistas e entre os pacientes, apenas 8% e 7,61%, respectivamente, utilizaram plantas medicinais para alterações patológicas orais. Os autores concluíram que as plantas medicinais comercializadas na cidade de Manaus são utilizadas de maneira empírica e que, apesar da Política Nacional de Práticas Integrativas e Complementares (PNPIC), novas políticas públicas de saúde devem inserir plantas medicinais e fitoterápicos de uso oral na rede pública de saúde na cidade de Manaus.

The official recognition of herbal medicine in dentistry in Brazil was accompanied by several gaps in scientific research and the use of medicinal plants, specifically for plant species applied to diseases of the oral cavity. The state of Amazonas, specifically the city of Manaus, does not have a diagnosis of applicability of medicinal plants in Dental care services. This research intended to conduct a qualitative and quantitative-type, descriptive and exploratory study in order to conduct a survey on the commercialization of medicinal plants and an ethnobotanical study to identify the main medicinal plants indicated and used in oral pathologies in outpatient dental care in the city of Manaus. We interviewed 197 users of dental services, 150 dentists, and 47 accredited traders in the Municipality of Manaus. The sampling was performed by accessibility or convenience for surgeon-dentists, while users were selected through simple random sampling, and all traders of medicinal plants were interviewed. The results demonstrated that medicinal plants for oral diseases are commercialized, among which the following species are highlighted: Pedra ume Caa (Aulomyrcia sphareocarpa), Crajiru (Arrabidaea chica), in addition to an unidentified botanical plant popularly known as Sara Tudo. 8% of dentists and 7.61% of patients used medicinal plants for oral pathological changes. The authors concluded that medicinal plants commercialized in the city of Manaus are used empirically, and that, despite the rules from the National Policy on Integrative and Complementary Practices (PNPIC), new public health policies must add medicinal plants and herbal medicines for oral use to the public health system of the city of Manaus.

Rosuvastatin displays anti-atherothrombotic and anti-inflammatory properties in apoE-deficient mice.

Inflammation contributes importantly to all stages of atherosclerosis, including the onset of acute thrombotic complications. In clinical trials, statins are beneficial in the primary and secondary prevention of coronary heart disease. Moreover, statins have been shown to possess several pleiotropic properties independent of cholesterol lowering in experimental settings. Based on these premises, we investigated the anti-inflammatory and anti-atherothrombotic properties of rosuvastatin in vivo, testing its effect on cholesterol and monocyte accumulation, and on adhesion molecules and tissue factor (TF) expression. ApoE-deficient female mice were fed a cholesterol-rich diet containing rosuvastatin (0, 1, 2 or 10 mg kg(-1)d(-1)) for 12 weeks. Treatment with rosuvastatin did not significantly affect either body weight gain or plasma total cholesterol (C) and triglyceride levels. However, rosuvastatin treatment dose-dependently reduced ICAM-1 expression in the aortic valves (V) (up to 40% inhibition, p<0.05) and in the proximal segment of the ascending aorta (AA) (-50%, p<0.001). Similarly, rosuvastatin inhibited VCAM-1 expression in the V (-40%) and in the AA (-35%, p<0.05). Moreover, there was a reduced accumulation of macrophages in the V in a dose-dependent and statistically significant manner (-45%, p<0.01). These anti-inflammatory effects were reflected in a reduction of cholesterol deposition in the entire aorta, both in the free and in the esterified form. Finally, the expression of tissue factor, the most potent pro-thrombogenic agent, was consistently reduced in AA by rosuvastatin treatment (-71%, p<0.001). Altogether, these data demonstrate that rosuvastatin has anti-inflammatory and anti-atherothrombotic activities in apoE-deficient mice that could translate in a beneficial effect on atherogenesis.

Myxoid monophasic synovial sarcoma: case report of an unusual histological variant.

We report the case of a 61-year-old woman who presented a recurrent myxoid synovial sarcoma involving the right ankle and foot. This tumor, defined as a synovial sarcoma showing more than 50% myxoid change in the stroma, has only recently been described as a rare histological variant. The histological diagnosis is particularly difficult in such cases, as the one we are describing, where the tumor is entirely myxoid and monophasic raising the possibility of other myxoid soft tissue neoplasms, such as extraskeletal myxoid chondrosarcoma, malignant peripheral nerve sheath tumor or leiomyosarcoma. On the basis of morphological and immunophenotypical findings, the diagnosis of myxoid synovial sarcoma should be properly established, especially in view of its unusual clinical course and treatment. Furthermore, we will discuss the clinicopathological and immunohistochemical features observed in our case.

A structure-activity study for the inhibition of metalloproteinase-9 activity and gene expression by analogues of gallocatechin-3-gallate.

Catechins are able to modulate the gelatinolytic activity of matrix metalloproteinase-9 (MMP-9) by reducing its release from macrophages. Gallocatechins decrease MMP-9 secretion by lowering MMP-9 promoter activity and mRNA levels. The effect appears to be dependent on some structural and stereochemical requirements. In this study, the relationship between chemical structure and activity was studied by testing the effect of analogues of (+/-)-gallocatechin-3-gallate (+/-)-GCG, selectively deprived of hydroxyl groups, on MMP-9 activity, transcription, and secretion. Our results indicate that (+/-)-GCG and (+/-)-catechin-3-gallate are characterized by a substitution pattern compatible with direct inhibition of MMP-9 activity. Conversely, when transcription was the target, (+/-)-trans-3-flavanol-3-benzoate, lacking all the hydroxyl groups, was the most effective both in lowering MMP-9 promoter activity and consequently protein secretion, and in inhibiting nuclear-factor-kappaB-driven transcription. Our results suggest that the structural requirements for enzyme inhibition are different from those necessary for targeting gene expression.

Improved survival in stage III melanoma patients with GM2 antibodies: a randomized trial of adjuvant vaccination with GM2 ganglioside.

PURPOSE: To perform a double-blind randomized trial with American Joint Commission on Cancer (AJCC) stage III melanoma patients for the following reasons: (1) to confirm our previous finding that patients with antibodies against the melanoma differentiation antigen GM2 have an improved prognosis, and (2) to demonstrate clinical benefit from GM2 antibody induction. PATIENTS AND METHODS: One hundred twenty-two patients with AJCC stage III melanoma who were free of disease after surgery were randomized: 58 to receive treatment with the GM2/BCG vaccine, and 64 to receive treatment with bacille Calmette-Guèrin (BCG) alone. All patients were pretreated with low-dose cyclophosphamide (Cy). RESULTS: GM2 antibody was detected in 50 of 58 patients treated with GM2/BCG and seven of 64 patients treated with BCG alone. With a minimum follow-up period of 51 months, there was a highly significant increase in the disease-free interval (P = .004) and a 17% increase in overall survival (P = .02) in these 57 antibody-positive patients, confirming our earlier experience. Exclusion of all patients with preexisting GM2 antibodies (one in the GM2/BCG group and five in the BCG group) from statistical analysis resulted in a 23% increase in disease-free interval (P = .02) and a 14% increase in overall survival (P = .15) at 51 months for patients treated with the GM2/BCG vaccine. However, when all patients in the two treatment groups were compared as randomized, these increases were 18% for disease-free interval and 11% for survival in the GM2/BCG treatment group, with neither result showing statistical significance. CONCLUSION: (1) Vaccination with GM2/BCG induced immunoglobulin M (IgM) antibodies in most patients. (2) GM2 antibody production was associated with a prolonged disease-free interval and survival. (3) Comparison of the two arms of this trial as randomized fails to show a statistically significant improvement in disease-free interval or survival for patients treated with GM2/BCG vaccines.

Pore-forming peptides induce rapid phospholipid flip-flop in membranes.

A kinetic model for pore-mediated and perturbation-mediated flip-flop is presented and used to characterize the mechanism of peptide-induced phospholipid flip-flop in bilayers. The model assumes that certain peptides can bind to and aggregate within the membrane. When the aggregate attains a critical size (M peptides), a channel is created that results in a fast flip-flop of phospholipids. In addition, certain peptides induce flip-flop through perturbation of the membrane without forming a pore. Donor phospholipid vesicles with an asymmetrical distribution of the fluorescent phospholipid 1-oleoyl-2-[12-[(7-nitro-1,2,3-benzoxadiazol-4- yl)amino]dodecanoyl]phosphatidylcholine (NBD-PC) were used to measure the extent of flip-flop by quantitating the decrease in fluorescence as the NBD-PC exchanged from the donor vesicles to acceptor vesicles that contained a quencher of the NBD fluorescence. Flip-flop curves generated at lipid/peptide ratios ranging from 30/1 to 300000/1 could be well-simulated by the model. Pore-forming peptides, such as melittin or the synthetic peptide GALA (WEAALAEALAEALAEHLAEALAEALEALAA), induce rapid phospholipid flip-flop with half-times for flip-flop of seconds at low peptide/vesicle ratios. The deduced pore sizes are M = 10 +/- 2 for GALA and M = 2 - 4 for melittin. The synthetic peptide LAGA (WEAALAEAEALALAEHEALALAEAELALAA) can catalyze flip-flop via bilayer perturbation. In contrast, hydrophobic peptides such as gramicidin A and valinomycin intercalate into the membrane, but induce little flip-flop. Modeling of the kinetics of phospholipid translocation supports pore formation as the key factor in accelerating phospholipid flip-flop. Thus, amphipathic segments from membrane proteins may account for non-energy-dependent phospholipid flip-flop in biological membranes.

Zinc supplementation restores plasma concentrations of zinc and thymulin in patients with Crohn's disease.

The aim of this work was to evaluate whether oral supplementation with zinc sulphate (ZnSO4) could restore thymic endocrine function in patients with Crohn's disease who showed decreased plasma concentrations of zinc and active thymulin, a zinc-dependent thymic hormone. Twenty-seven patients in clinical remission were randomly assigned to receive, for 3 months, one of the following treatments: 60 mg/day ZnSO4; 200 mg/day ZnSO4 or placebo. Plasma thymulin activity and zinc concentrations significantly increased only in patients treated with 200 mg/day ZnSO4. Lymphocyte subpopulations, within the range of normality before zinc supplementation, were unaffected by any of the administered treatments. In conclusion, low plasma concentrations of zinc and thymulin in Crohn's disease patients were restored by the administration of high doses of zinc.

Secondary structure and orientation of the amphipathic peptide GALA in lipid structures. An infrared-spectroscopic approach.

GALA, a synthetic, amphipathic 30-amino-acid peptide, based upon a Glu-Ala-Leu-Ala motive, was designed to mimic the behavior of viral fusion proteins. GALA is a water-soluble peptide with an aperiodic conformation at neutral pH, and becomes an amphipathic alpha helix as the pH is lowered to 5, where it interacts with phospholipid bilayers. Attenuated total-reflection infrared spectroscopy, using polarized light, provides information on the structure and orientation of the peptide and the lipids, which is not subject to artifacts due to light scattering with large particles. H/2H-exchange rate of the amide N-H group and analysis of the shape of the amide I' by Fourier self-deconvolution and curve fitting indicate that the alpha-helical content increases from 19% to 69%, on lowering the pH. A further increase to 100% alpha helix is observed after interaction with palmitoyloleoylglycerophosphocholine (PamOleGroPCho) vesicles. Dichroism data obtained with oriented bilayers of the PamOleGroPCho-GALA complex demonstrate that PamOleGroPCho hydrocarbon chains and the peptide alpha helical axis are essentially perpendicular (+/- 15) to the membrane plane. At neutral pH, in the presence of dimyristoylglycerophosphocholine (Myr2GroPCho), GALA is known to form discoidal structures similar to those formed under the same conditions by apolipoproteins AI and AII. In these discoidal complexes, the alpha-helical content was estimated to be 65%, with the rest of the structure being essentially unordered. No significant modification of the all-trans conformation of the hydrocarbon chain of Myr2GroPCho was detected upon disc formation. Dichroism measurements show that the alpha-helical axis is essentially parallel to the hydrocarbon chains. These data support a model in which a discoidal patch of the bilayer is surrounded by amphipathic helices which shield the hydrophobic region of the bilayer from the aqueous environment. The infrared spectrum of GALA in this complex was found to be very similar to those of apolipoproteins AI and AII which form discoidal complexes with Myr2GroPCho, but the spectrum is quite different from that of apolipoprotein B100 in low-density lipoproteins, which does not form discoidal complexes.

Association of a pH-sensitive peptide with membrane vesicles: role of amino acid sequence.

The solution properties and bilayer association of two synthetic 30 amino acid peptides, GALA and LAGA, have been investigated at pH 5 and 7.5. These peptides have the same amino acid composition and differ only in the positioning of glutamic acid and leucine residues which together compose 47% of each peptide. Both peptides undergo a similar coil to helix transition as the pH is lowered from 7.5 to 5.0. However, GALA forms an amphipathic alpha-helix whereas LAGA does not. As a result, GALA partitions into membranes to a greater extent than LAGA and can initiate leakage of vesicle contents and membrane fusion which LAGA cannot (Subbarao et al., 1987; Parente et al., 1988). Membrane association of the peptides has been studied in detail with large phosphatidylcholine vesicles. Direct binding measurements show a strong association of the peptide GALA to vesicles at pH 5 with an apparent Ka around 10(6). The single tryptophan residue in each peptide can be exploited to probe peptide motion and positioning within lipid bilayers. Anisotropy changes and the quenching of tryptophan fluorescence by brominated lipids in the presence of vesicles also indicate that GALA can interact with uncharged vesicles in a pH-dependent manner. By comparison to the peptide LAGA, the membrane association of GALA is shown to be due to the amphipathic nature of its alpha-helical conformation at pH 5.

Mechanism of leakage of phospholipid vesicle contents induced by the peptide GALA.

The synthetic, amphipathic peptide GALA undergoes a pH-dependent conformational change and induces leakage of contents from large unilamellar phosphatidylcholine vesicles when in a helical conformation. The kinetics of this process have been investigated over a wide range of pH and lipid and peptide concentrations. Leakage from lipid vesicles is rapidly initiated (within 2 s) when the pH is lowered below 6 and is rapidly terminated when the pH is raised to 7.5. The leakage shows a selectivity to the size of the entrapped molecules and occurs by an all or none mechanism; vesicles either leak or retain all of their contents. Using this experimental data, we have developed a mathematical description of the kinetics of leakage induced by GALA. This model assumes that GALA becomes incorporated into the vesicle bilayer and aggregates to form a pore. Leakage occurs when a critical number of peptides assemble into a supramolecular aggregate in the bilayer. Leakage curves generated at lipid/peptide ratios ranging from 500/1 to 30000/1 can be well described by this formalism. On the basis of the results and the model, we suggest that GALA forms a transbilayer channel composed of 8-12 monomers. The channel diameter ranges from 5 to 10 A. To the best of our knowledge, this is the first model that can predict the leakage kinetics of solutes entrapped in lipid vesicles induced by a pore-forming peptide. The analysis may be of general use in defining the kinetics and state of aggregation of similarly acting peptides and proteins which form multimeric assemblies in membranes.

pH-dependent fusion of phosphatidylcholine small vesicles. Induction by a synthetic amphipathic peptide.

A synthetic, amphipathic 30-amino acid peptide with the major repeat unit Glu-Ala-Leu-Ala (GALA) was designed to mimic the behavior of the fusogenic sequences of viral fusion proteins. GALA is a water-soluble peptide with an aperiodic conformation at neutral pH and becomes an amphipathic alpha-helix as the pH is lowered to 5.0 where it interacts with bilayers. Fluorescence energy transfer measurements indicated that GALA induced lipid mixing between phosphatidylcholine small unilamellar vesicles but not large unilamellar vesicles. This lipid mixing occurred only at pH 5.0 and not at neutral pH. Concomitant with lipid mixing, the vesicles increased in diameter from 500 to 750 to 1000 A as measured by dynamic light scattering and internal volume determination. GALA induced leakage of small molecules (Mr 450) at pH 5.0 was too rapid to permit detection of contents mixing. However, retention of larger molecules (Mr 4100) under the same conditions suggests that vesicle fusion is occurring. For a 100/1 lipid/peptide ratio all vesicles fused just once, whereas for a 50/1 ratio higher order fusion products formed. A mass action model gives good simulation of the kinetics of increase in fluorescence intensity and yields rate constants of aggregation and fusion. As the lipid to peptide ratio decreases from 100/1 to 50/1 both rate constants of aggregation and fusion increase, indicating that GALA is a genuine inducer of vesicle fusion. The presence of divalent cations which can alter GALAs conformation at pH 7.5 had little effect on its lipid mixing activity. GALA was modified by altering the sequence while keeping the amino acid composition constant or by shortening the sequence. These peptides did not have any lipid mixing activity nor did they induce an increase in vesicle size. Together, these results indicate that fusion of phosphatidylcholine small unilamellar vesicles induced by GALA requires both a peptide length greater than 16 amino acids as well as a defined topology of the hydrophobic residues.

pH-dependent bilayer destabilization by an amphipathic peptide.

A 30-residue amphipathic peptide was designed to interact with uncharged bilayers in a pH-dependent fashion. This was achieved by a pH-induced random coil-alpha-helical transition, exposing a hydrophobic face in the peptide. The repeat unit of the peptide, glutamic acid-alanine-leucine-alanine (GALA), positioned glutamic acid residues on the same face of the helix, and at pH 7.5, charge repulsion between aligned Glu destabilized the helix. A tryptophan was included at the N-terminal as a fluorescence probe. The rate and extent of peptide-induced leakage of contents from large, unilamellar vesicles composed of egg phosphatidylcholine were dependent on pH. At pH 5.0 with a lipid/peptide mole ratio of 500/1, 100% leakage of vesicle contents occurred within 1 min. However, no leakage of vesicle contents occurred at pH 7.5. Circular dichroism measurements indicated that the molar ellipticity at 222 nm changed from about -4000 deg cm2 dmol-1 at pH 7.6 to -11,500 deg cm2 dmol-1 at pH 5.1, indicating a substantial increase in helical content as the pH was reduced. Changes in molar ellipticity were most significant over the same pH range where a maximum change in the extent and rate of leakage occurred. The tryptophan fluorescence emission spectra and the circular dichroism spectra of the peptide, in the presence of lipid, suggest that GALA did not associate with the bilayer at neutral pH. A change in the circular dichroism spectrum and a blue shift of the maximum of the tryptophan fluorescence emission spectra at pH 5.0, in the presence of lipid, indicated an association of GALA with the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)

Rate and extent of poly(ethylene glycol)-induced large vesicle fusion monitored by bilayer and internal contents mixing.

Poly(ethylene glycol) (PEG) of average molecular weight 8000 was used to mediate the fusion of large unilamellar vesicles composed of dipalmitoylphosphatidylcholine. Fusion was monitored by fluorescence assays of lipid mixing and aqueous contents mixing. The extent of lipid mixing, as monitored by DPHpPC fluorescence lifetime, indicated that large unilamellar vesicles underwent a single fusion cycle when incubated with PEG and subsequently diluted into buffer. The ANTS/DPX assays for contents mixing and leakage indicated that, while addition and dilution of PEG were accompanied by extensive contents leakage, this occurred on a much different time scale as compared to contents mixing. Both the lipid-mixing and contents-mixing assays gave comparable estimates for the number of rounds of fusion that occurred in a given time following PEG addition, although the contents-mixing assay always yielded an estimate 10-15% larger than the lipid-mixing assay. These assays were used to evaluate several factors purported to influence PEG-induced fusion. First, the initial rate of fusion was found to be dependent on PEG concentration in the range of 0-35 wt %, while the extent of fusion was not. In addition, a substantial rate enhancement occurred when vesicles were incubated with greater than 26% PEG. Second, the creation of an osmotic gradient upon dilution of vesicle-PEG mixtures was shown to have no effect on either the extent or the initial rate of fusion. Consistent with this observation, both contents and lipid mixing were found to occur prior to and independent of the dilution of the PEG-vesicle suspension. Third, impurities, either present in our commercially available PEG or added to vesicle-PEG mixtures, also had no effect on the rate or extent of fusion. Fourth, another dehydrating polymer, dextran (average mol wt 9000), was capable of promoting fusion, though at a much lower rate than PEG. These results suggest that even partial bilayer dehydration accompanied by vesicle collapse and close interbilayer contact may be sufficient to induce vesicle fusion.

Phase I trial of taxol given as a 3-hour infusion every 21 days.

Taxol is a unique plant product that promotes in vitro assembly of microtubules. In a phase I trial, adults with advanced solid tumors were given taxol (formulated with cremophor EL and dehydrated alcohol) as a 3-hour iv infusion every 21 days. The total dose administered ranged from 15 to 230 mg/m2 in nine escalation steps. Leukopenia, thrombocytopenia, nausea and vomiting, alopecia, stomatitis, transient rashes, increases in serum triglyceride levels, and hypersensitivity reactions were observed. Hypersensitivity reactions characterized by acute dyspnea, generalized erythema, and hypotension immediately following the initiation of the taxol infusion occurred in three of five patients receiving greater than or equal to 190 mg/m2 (18% of patients overall). No antitumor activity was observed. Hypersensitivity reactions constituted a treatment-limiting toxicity for this preparation of taxol given on this schedule over the dosage range examined. With the severity and unpredictability of the hypersensitivity reactions, further usage of taxol is not indicated with this drug formulation on this administration schedule. Further studies are warranted to uncover ways to permit the safe administration of taxol.

Phase II trial of gallium nitrate in patients with advanced malignant melanoma.

A phase II trial of gallium nitrate (250-300 mg/m2/day for 7 consecutive days) was conducted in patients with metastatic melanoma. Therapy was well-tolerated, but only one of 31 evaluable patients experienced a partial regression. Further evaluation of gallium nitrate at this dose and schedule is not warranted in patients with malignant melanoma.

Particular features of cell-mediated immunity in patients with anaplastic gliomas. A comparison with kidney and bladder cancer patients.

The authors studied 24 patients affected by anaplastic gliomas in regard to the killer (Antibody-Dependent Cellular Cytotoxicity) and natural killer (Spontaneous Lymphocyte-Mediated Cytotoxicity) immunological functions, by counting the Cr51 release in Change liver and K 562 cell cultures, respectively. These parameters were also evaluated in 24 healthy donors as control, in 24 patients affected by bladder cancer and in nine cases of kidney cancer. Our data show, pre-operatively, a statistically significant impairment of ADCC and SLMC activity in glioma patients as compared both with controls, bladder and kidney cancer patients. The particular impairment of K and NK functions in gliomas is discussed with regard to the specific features of Central Nervous System malignancies. An improvement of ADCC activity was also found in the post-operative samples. This finding confirms other reports about partial restoring of altered immunocompetence after surgery, suggesting a link between extention of tumor mass and impaired immunological reactions.