Acetate-induced apoptosis in colorectal carcinoma cells involves lysosomal membrane permeabilization and cathepsin D release.

Colorectal carcinoma (CRC) is one of the most common causes of cancer-related mortality. Short-chain fatty acids secreted by dietary propionibacteria from the intestine, such as acetate, induce apoptosis in CRC cells and may therefore be relevant in CRC prevention and therapy. We previously reported that acetic acid-induced apoptosis in Saccharomyces cerevisiae cells involves partial vacuole permeabilization and release of Pep4p, the yeast cathepsin D (CatD), which has a protective role in this process. In cancer cells, lysosomes have emerged as key players in apoptosis through selective lysosomal membrane permeabilization (LMP) and release of cathepsins. However, the role of CatD in CRC survival is controversial and has not been assessed in response to acetate. We aimed to ascertain whether LMP and CatD are involved in acetate-induced apoptosis in CRC cells. We showed that acetate per se inhibits proliferation and induces apoptosis. More importantly, we uncovered that acetate triggers LMP and CatD release to the cytosol. Pepstatin A (a CatD inhibitor) but not E64d (a cathepsin B and L inhibitor) increased acetate-induced apoptosis of CRC cells, suggesting that CatD has a protective role in this process. Our data indicate that acetate induces LMP and subsequent release of CatD in CRC cells undergoing apoptosis, and suggest exploiting novel strategies using acetate as a prevention/therapeutic agent in CRC, through simultaneous treatment with CatD inhibitors.

DODAB:monoolein-based lipoplexes as non-viral vectors for transfection of mammalian cells.

DNA/Cationic liposome complexes (lipoplexes) have been widely used as non-viral vectors for transfection. Neutral lipids in liposomal formulation are determinant for transfection efficiency using these vectors. In this work, we studied the potential of monoolein (MO) as helper lipid for cellular transfection. Lipoplexes composed of pDNA and dioctadecyldimethylammonium bromide (DODAB)/1-monooleoyl-rac-glycerol (MO) at different molar ratios (4:1, 2:1 and 1:1) and at different cationic lipid/DNA ratios were investigated. The physicochemical properties of the lipoplexes (size, charge and structure), were studied by Dynamic Light Scattering (DLS), Zeta Potential (ζ) and cryo-transmission electron microscopy (cryo-TEM). The effect of MO on pDNA condensation and the effect of heparin and heparan sulphate on the percentage of pDNA release from the lipoplexes were also studied by Ethidium Bromide (EtBr) exclusion assays and electrophoresis. Cytotoxicity and transfection efficiency of these lipoplexes were evaluated using 293T cells and compared with the golden standard helper lipids 1,2-dioleoyl-sn-glycero-3-hosphoethanolamine (DOPE) and cholesterol (Chol) as well as with a commercial transfection agent (Lipofectamine™ LTX). The internalization of transfected fluorescently-labeled pDNA was also visualized using the same cell line. The results demonstrate that the presence of MO not only increases pDNA compactation efficiency, but also affects the physicochemical properties of the lipoplexes, which can interfere with lipoplex-cell interactions. The DODAB:MO formulations tested showed little toxicity and successfully mediated in vitro cell transfection. These results were supported by fluorescence microscopy studies, which illustrated that lipoplexes were able to access the cytosol and deliver pDNA to the nucleus. DODAB:MO-based lipoplexes were thus validated as non-toxic, efficient lipofection vectors for genetic modification of mammalian cells. Understanding the relation between structure and activity of MO-based lipoplexes will further strengthen the development of these novel delivery systems.

Free radicals in the regulation of damage and cell death - basic mechanisms and prevention.

Reactive oxygen (ROS) and nitrogen (RNS) species are known to accumulate intracellularly due to both exogenous and/or endogenous factors. In normal physiological conditions, these reactive species are maintained in an equilibrium state by the cells' antioxidant defence systems. In addition, they are recognised to play important roles in several physiological functions. However, when an imbalance in the equilibrium between oxidants and antioxidants occurs in favour of the former, we come to a situation defined as oxidative stress. ROS/RNS can cause damage to all biomolecules (namely proteins, lipids and DNA) and ultimately participate in the regulation of mechanisms leading to cell death, being implicated in the etiology of several pathologies (like neurodegenerative and cardiovascular diseases). To cope with oxidative stress, cells possess effective enzymatic (e.g. superoxide dismutase, catalase, glutathione peroxidase) and non-enzymatic (e.g. glutathione, thioredoxin, coenzyme Q) antioxidant systems. In addition, several compounds present in plants and vegetables (e.g. vitamins C and E, polyphenols) have been described to react with free radicals. However, some drawbacks associated to these natural compounds are in part responsible for the undergoing development of novel synthetic compounds capable of acting as antioxidants and protect cells against oxidative stress-induced cell death. Here, we review the basic mechanisms of ROS/RNS formation, as well as their interaction with biomolecules and regulation of cell death, in order to identify possible drug targets. We also report the importance of natural antioxidant systems and the ongoing research leading to the development of more powerful and effective antioxidant drugs.

Di(hetero)arylamines in the benzo[b]thiophene series as novel potent antioxidants.

The damaging consequences of oxidative stress are known to be involved in several pathologies. So, the development of new drugs that can aid cells to cope with excessive levels of free radicals still assumes great relevance. Here, we investigated the antioxidant properties of four novel di(hetero)arylamines (named MJQ1, MJQ3, MJQ4 and MJQ5), sharing a common benzo[b]thiophene nucleus (an indole analogue), against oxidative damage induced to H9c2 myoblasts. Cell proliferation, evaluated by the sulforhodamine B assay, was not compromised by the presence of any of these compounds for concentrations below 50 µM (at 24 h) and 1 µM (72 h). Moreover, all of them showed a dose-dependent protective effect against tert-butylhydroperoxide (t-BHP)-induced cell death for concentrations in the nanomolar range. Their ability to scavenge free radicals seems to account for their protective effects, as they were able to prevent almost completely, at 25 nM, t-BHP-induced intracellular ROS formation, assessed by DCF fluorescence. Furthermore, their relatively high partition coefficient values are indicative of their ability to easily permeate lipid membranes and act intracellularly. Additionally, these novel diarylamines led to a reduction, between 60-70%, of the amount of DNA strand breaks induced by t-BHP, evaluated by the Comet assay, and lipid peroxidation (TBARS assay) induced by the oxidant pair ascorbate/iron. In all these parameters, which show their ability to prevent the oxidation of the main biomolecules, their protective role was superior to the traditional antioxidant Trolox. Although the mechanisms underlying the action of these diarylamines are currently under investigation, the data obtained so far reveals their high pharmacological potential as antioxidant molecules.

Materials in particulate form for tissue engineering. 2. Applications in bone.

Materials in particulate form have been the subjects of intensive research in view of their use as drug delivery systems. While within this application there are still issues to be addressed, these systems are now being regarded as having a great potential for tissue engineering applications. Bone repair is a very demanding task, due to the specific characteristics of skeletal tissues, and the design of scaffolds for bone tissue engineering presents several difficulties. Materials in particulate form are now seen as a means of achieving higher control over parameters such as porosity, pore size, surface area and the mechanical properties of the scaffold. These materials also have the potential to incorporate biologically active molecules for release and to serve as carriers for cells. It is believed that the combination of these features would create a more efficient approach towards regeneration. This review focuses on the application of materials in particulate form for bone tissue engineering. A brief overview of bone biology and the healing process is also provided in order to place the application in its broader context. An original compilation of molecules with a documented role in bone tissue biology is listed, as they have the potential to be used in bone tissue engineering strategies. To sum up this review, examples of works addressing the above aspects are presented.

In vivo response to starch-based scaffolds designed for bone tissue engineering applications.

Our purpose was to evaluate the in vivo endosseous response to three starch-based scaffolds implanted in rats (n = 54). We implanted the three scaffold groups; a 50/50 (wt %) blend of corn starch and ethylene-vinyl alcohol (SEVA-C), the same composition coated with a biomimetic calcium phosphate (Ca-P) layer (SEVA-C/CaP), and a 50/50 (wt %) blend of corn starch and cellulose acetate (SCA), all produced by extrusion with blowing agents, into distal femurs proximal to the epiphyseal plate, for 1, 3, or 6 weeks. Our results showed that at 1 week considerable reparative bone formed around all scaffold groups, although the bone was separated from the scaffold by an intervening soft tissue interfacial zone that comprised two distinct compartments: the surface of the scaffold was occupied by multinucleate giant cells and the compartment between these cells and the surrounding bone was occupied by a streaming fibrous-like tissue. The extracellular matrix of the latter was continuous with the extracellular bone matrix itself, labeled positively for osteocalcin and appeared mineralized by back-scattered electron imaging. All three scaffolds showed a similar tissue response, with the soft tissue interface diminishing with time. No bone contact was observed with SEVA-C at any time point, only transitory bone contact was observed with SEVA-C/CaP at 3 weeks, but SCA exhibited direct bone contact at 6 weeks where 56.23 +/- 6.46% of the scaffold surface was occupied by bone. We conclude that all materials exhibited a favorable bony response and that the rapidly forming initial "connective tissue" seen around all scaffolds was a very early form of bone formation.

Effect of starch-based biomaterials on the in vitro proliferation and viability of osteoblast-like cells.

The cytotoxicity of starch-based polymers was investigated using different methodologies. Poly-L-lactic acid (PLLA) was used as a control for comparison purposes. Extracts of four different starch-based blends (corn starch and ethylene vinyl alcohol (SEVA-C), corn starch and cellulose acetate (SCA), corn starch and polycaprolactone (SPCL) and starch and poly-lactic acid (SPLA70) were prepared in culture medium and their toxicity was analysed. Osteoblast-like cells (SaOs-2) were incubated with the extracts and cell viability was assessed using the MTT test and a lactate dehydrogenase (LDH) assay. In addition DNA and total protein were quantified in order to evaluate cell proliferation. Cells were also cultured in direct contact with the polymers for 3 and 7 days and observed in light and scanning electron microscopy (SEM). LDH and DNA quantification revealed to be the most sensitive tests to assess respectively cell viability and cell proliferation after incubation with starch-based materials and PLLA. SCA was the starch blend with higher cytotoxicity index although similar to PLLA polymer. Cell adhesion tests confirmed the worst performance of the blend of starch with cellulose acetate but also showed that SPCL does not perform as well as it could be expected. All the other materials were shown to present a comparable behaviour in terms of cell adhesion showing slight differences in morphology that seem to disappear for longer culture times. The results of this study suggest that not only the extract of the materials but also their three-dimensional form has to be biologically tested in order to analyse material-associated parameters that are not possible to consider within the degradation extract. In this study, the majority of the starch-based biomaterials presented very promising results in terms of cytotoxicity, comparable to the currently used biodegradable PLLA which might lead the biocompatibility evaluation of those novel biomaterials to other studies.

Physical properties and biocompatibility of chitosan/soy blended membranes.

Blends of polysaccharides and proteins are a source for the development of novel materials with interesting and tailorable properties, with potential to be used in a range of biomedical applications. in this work a series of blended membranes composed by chitosan and soy protein isolate was prepared by solvent casting methodology. in addition, cross-linking was performed in situ with glutaraldehyde solutions in the range 5x10(-3)-0.1 M. Furthermore, the influence of the composition and cross-linking on the degradation behaviour, water uptake and cell adhesion was investigated. The obtained results showed that the incorporation of chitosan, associated to network formation by cross linking, promoted a slight decrease of water absorption and a slower degradability of the membranes. Moreover, direct contact biocompatibility studies, with L929 cells, indicate that the cross-linking enhances the capability of the material to support cell growth.

Lues maligna in an HIV-infected patient.

We report such a case of malignant syphilis in a 42-year-old HIV-infected man, co-infected with hepatitis B virus, who presented neurolues and the classical skin lesions of lues maligna. The serum VDRL titer, which was 1:64 at presentation, increased to 1:2,048 three months after successful therapy with penicillin, decreasing 15 months later to 1:8.

Lues maligna in an HIV-infected patient

Descrevemos um caso de sífilis maligna em um paciente de 42 anos com infecção pelo HIV e pelo vírus da hepatite B. O paciente, com lesões cutâneas clássicas de lues maligna e VDRL positivo no soro e no líquor, teve uma resposta excelente ao tratamento com penicilina cristalina. O VDRL sérico, que no diagnóstico era de 1:64, aumentou três meses depois para 1:2.048 e diminuiu para 1:8 após 15 meses.

Biological response to pre-mineralized starch based scaffolds for bone tissue engineering.

It is known that calcium-phosphate (Ca-P) coatings are able not only to improve the bone bonding behaviour of polymeric materials, but at the same time play a positive role on enhancing cell adhesion and inducing the differentiation of osteoprogenitor cells. Recently an innovative biomimetic methodology, in which a sodium silicate gel was used as a nucleative agent, was proposed as an alternative to the currently available biomimetic coating methodologies. This methodology is especially adequate for coating biodegradable porous scaffolds. In the present work we evaluated the influence of the referred to treatment on the mechanical properties of 50/50 (wt%) blend of corn starch/ethylene-vinyl alcohol (SEVA-C) based scaffolds. These Ca-P coated scaffolds presented a compressive modulus of 224.6 +/- 20.6 and a compressive strength of 24.2 +/- 2.20. Cytotoxicity evaluation was performed according ISO/EN 10993 part 5 guidelines and showed that the biomimetic treatment did not have any deleterious effect on L929 cells and did not inhibit cell growth. Direct contact assays were done by using a cell line of human osteoblast like cells (SaOS-2). 3 x 10(5) cells were seeded per scaffold and allowed to grow for two weeks at 37( composite function)C in a humidified atmosphere containing 5% CO(2). Total protein quantification and scanning electron microscopy (SEM) observation showed that cells were able to grow in the pre-mineralized scaffolds. Furthermore cell viability assays (MTS test) also show that cells remain viable after two weeks in culture. Finally, protein expression studies showed that after two weeks osteopontin and collagen type I were being expressed by SaOS-2 cells seeded on the pre-mineralized scaffolds. Moreover, alkaline phosphatase (ALP) activity was higher in the supernatants collected from the pre-mineralized samples, when compared to the control samples (non Ca-P coated). This may indicate that a faster mineralization of the ECM produced on the pre-mineralized samples was occurring. Consequently, biomimetic pre-mineralization of starch based scaffolds can be a useful route for applying these materials on bone tissue engineering.

Entrapment ability and release profile of corticosteroids from starch-based microparticles.

We previously described the synthesis of starch-based microparticles that were shown to be bioactive (when combined with Bioactive Glass 45S5) and noncytotoxic. To further assess their potential for biomedical applications such as controlled release, three corticosteroids with a similar basic structure-dexamethasone (DEX), 16alpha-methylprednisonole (MP), and 16alpha-methylprednisolone acetate (MPA)-were used as models for the entrapment and release of bioactive agents. DEX, MP, and MPA were entrapped into starch-based microparticles at 10% wt/wt of the starch-based polymer and the loading efficiencies, as well as the release profiles, were evaluated. Differences were found for the loading efficiencies of the three corticosteroids, with DEX and MPA being the most successfully loaded (82 and 84%, respectively), followed by MP (51%). These differences might be explained based on the differential distribution of the molecules within the matrix of the microparticles. Furthermore, a differential burst release was observed in the first 24 h for all corticosteroids with DEX and MP being more pronounced (around 25%), whereas only 12% of MPA was released during the same time period. Whereas the water uptake profile can account for this first stage burst release, the subsequent slower release stage was mainly attributed to degradation of the microparticle network. Differences in the release profiles can be explained based on the structure of the molecule, because MPA, a more bulky and hydrophobic molecule, is released at a slower rate compared with DEX and MP. In this work, it is shown that these carriers were able to sustain a controlled release of the entrapped corticosteroids over 30 days, which confirms the potential of these systems to be used as carriers for the delivery of bioactive agents.

Preparation and characterisation in simulated body conditions of glutaraldehyde crosslinked chitosan membranes.

Chitosan membranes, aimed at biomedical applications, were prepared by a solvent casting methodology. Crosslinking was previously performed in acetic acid solution with glutaraldehyde, in order to obtain different degrees of crosslinking. Some membranes were neutralised in a NaOH solution. Mechanical tensile tests comprised quasi-static experiments at constant stress rate and temperature sweep dynamic mechanical analysis tests. This included measurements with the samples immersed in isotonic saline solution at 37 degrees C, in order to simulate physiological conditions, that were performed using a specific liquid container. It was observed that for higher crosslinking levels the membranes become stiffer but their strength decreases; these results are in agreement with swelling tests, also performed at body temperature. All the membranes exhibited similar and significant damping properties in wet conditions, which were stable in a broad temperature range. Weight loss measurements showed that the developed membranes degrade slowly up to 60 days. Cytotoxicity screening, using cell culture tests, showed that eventually such materials could be adequate for use in biomedical applications.

Synthesis and evaluation of novel bioactive composite starch/bioactive glass microparticles.

The aim of the development of composite materials is to combine the most desired properties of two or more materials. In this work, the biodegradable character, good controlled-release properties, and natural origin of starch-based biomaterials are combined with the bioactive and bone-bonding properties of bioactive glass (BG). Novel, bioactive composite starch-BG microparticles were synthesized starting from a blend of starch and polylactic acid (50%/50% wt) with BG 45S5 powder using a simple emulsion method. Morphological and chemical characterization showed that these particles exhibited a spherical morphology with sizes up to 350 microm and that BG 45S5 was incorporated successfully into the composite particles. Upon immersion in a solution simulating body fluids, for periods up to 3 weeks, their bioactive nature was confirmed, as a calcium-phosphate layer resembling biological apatite was formed onto their surface. The short-term cytotoxicity of these materials was also tested by placing 24-h leachables of the materials extracted in culture medium in contact with a fibroblastic cell line (L929) up to 72 h. At this time period, two biochemical tests--MTT and total protein quantification--were performed. The results showed that these materials are not cytotoxic. These results constitute the basis of future encapsulation studies using bone-acting therapeutic agents such as bone morphogenetic proteins or other bone-relevant factors. The particles developed here may be very useful for applications in which controlled release, degradability, and bone-bonding ability are the main requirements.

Novel starch-based scaffolds for bone tissue engineering: cytotoxicity, cell culture, and protein expression.

Starch-based biomaterials and scaffolds have been proposed for several biomedical applications. In the present work new scaffolds based on a 50/50 (wt%) blend of corn starch/ethylene-vinyl alcohol (SEVA-C) were studied. These scaffolds were processed by a melt-based technology, which has been used before with other starch-based materials but never with SEVA-C. Scanning electron microscopy (SEM) observation showed that the developed porous structures were 60% porous with pore size between 200 and 900 microm and a reasonable degree of interconnectivity. Moreover, scaffolds presented a compressive modulus of 117.50 +/- 3.7 MPa and a compressive strength of 20.8 +/- 2.4 MPa. Cytotoxicity evaluation was performed according to ISO/EN 10993 part 5 guidelines, and revealed that the developed scaffolds were nontoxic and did not inhibit cell growth. Direct contact assays were also carried out by use of a cell line of human osteoblast-like cells (SaOS-2). Cells were seeded (3 x 10(5) per scaffold) and allowed to grow for 4 weeks at 37 degrees C, in a humidified atmosphere containing 5% CO(2). Total protein assay showed that the cells were able to grow for the 4 weeks of the experiment. These data were further confirmed by SEM. Moreover, a cell viability assay (MTS test) demonstrated that cells were perfectly viable after the 4 weeks of culture, showing the adequacy of the developed structure in supporting them. Finally, Western blot analysis revealed that osteopontin was being actively expressed by the cells, which, in association with collagen deposition observed by SEM, seems to indicate that bone extracellular matrix was being deposited. Consequently it is believed that starch-based scaffolds should be considered as an alternative for bone tissue-engineering applications in the near future.

In vitro degradation and cytocompatibility evaluation of novel soy and sodium caseinate-based membrane biomaterials.

Soy- and casein-based membranes are newly proposed materials disclosing a combination of properties that might allow for their use in a range of biomedical applications. Two of the most promising applications are drug delivery carrier systems and wound dressing membranes. As for all newly proposed biomaterials, a cytotoxic scanning must be performed as a preliminary step in the process of the determination of the compatibility with biological systems (biocompatibility). In this study, the cytotoxicity of both soy- and casein-based protein biomaterials has been evaluated and correlated with the materials degradation behavior. It was possible to show, through morphological and biochemical tests that these natural origin materials do not exert any cytotoxic effect over cells, and in some cases can in fact enhance cell proliferation. The different treatments to which the membranes were subjected during their processing (that include crosslinking with glyoxal and tannic acid, and physical modification by thermal treatment) seemed to have a clear effect both on the materials mechanical properties and on their in vitro biological behavior.

[Vasculitic neuropathy in Wegener granulomatosis: case report]. / Neuropatia vasculítica na granulomatose de Wegener: relato de caso.

The neurologic involvement during Wegener granulomatosis reaches 15-50% of patients, however as initial symptomatology is not commented in the literature. We describe a patient with Wegener's granulomatosis and mononeuritis multiplex, emphasizing the fact that neurologic manifestations anticipated systemic symptoms, and also focusing on the diagnostic contribution of sural biopsy and the good outcome of neurologic disease.

Child sexual abuse in Matabeleland, Zimbabwe.

The extent, nature, causes and consequences of child sexual abuse in Matabeleland. Zimbabwe, are explored by an intersectoral working group consisting of health, legal and AIDS prevention workers who were struck in the course of their work by the regularity with which they saw sexually abused children infected with HIV and STDs. Methods used in this study are record review, focus group discussions, structured and in-depth interviews. Child sexual abuse cases form between 40-60% of the rape cases brought to the attention of hospitals, police and court and many more are believed to remain unreported. Half of the sexual abuse in children is detected through STDs and some have HIV. The majority of offenders are mature men known to the child. Factors influencing child sexual abuse are male dominance in society, men's professed inability to control sexual desire, and magic beliefs. Victims are traumatized by the abuse itself as well as by subsequent problems in family, health and in court. Since child sexual abuse may endanger the life and well-being of the child, it is a serious problem that requires urgent action.