Synthesis and characterization of an experimental primer containing chitosan nanoparticles - Effect on the inactivation of metalloproteinases, antimicrobial activity and adhesive strength.

OBJECTIVE: The aim of this study was to synthesize and characterize an experimental primer containing cationic lipid nanoparticles (NPL-chitosan) and to evaluate its properties. DESIGN: The NPL-chitosan were synthesized by emulsion and sonication method. The experimental primers were applied in dentin surface of fifty human molars. The experimental groups were: 1) application of commercial primer; 2) Primer containing 2% of Chlorhexidine (CHX) 3); Primer with 2% NPL-chitosan 4); Primer with 0.6 % of NPL-chitosan 5); Primer with 0.4 % of NPL-chitosan. A composite resin plateau was used for the analysis, where sections were made for making the dentin beams. The effect of experimental primer with cationic nanoparticles in the inhibition of matrix metalloproteinase (MMP) activity was carrying out by in situ zymography. For the Resin-Dentin Adhesive Strength and in situ Zymography analysis, was used the One-way analysis of variance (ANOVA) with significance level of 95 %. RESULTS: Spherical NPL-chitosan presented size below 220 nm, polydispersity index of 0.179 and zeta potential positive and was stable over 75 days. These nanoparticles showed antibacterial activity agsainst S. mutans with MIC of the 0.4 % and MBC of 0.67 %. In the Microtensile Strength, no statistical difference was observed between the experimental groups (p = 0.9054). The in situ zymography assay showed that the group with 2% of NPL-chitosan presented higher inactivation activity of MMPs compared to the other groups (p < 0.05). CONCLUSION: The experimental primer containing NPL-chitosan has antimicrobial activity, does not alter the adhesive resistance and inactivates MMPs present in dentin.

Biosynthesis and characterization of gold nanoparticles using Brazilian red propolis and evaluation of its antimicrobial and anticancer activities.

Gold nanoparticles (AuNPs) are highlighted due to their low toxicity, compatibility with the human body, high surface area to volume ratio, and surfaces that can be easily modified with ligands. Biosynthesis of AuNPs using plant extract is considered a simple, low-cost, and eco-friendly approach. Brazilian Red Propolis (BRP), a product of bees, exhibits anti-inflammatory, anti-tumor, antioxidant, and antimicrobial activities. Here, we described the biosynthesis of AuNPs using BRP extract (AuNPextract) and its fractions (AuNPhexane, AuNPdichloromethane, AuNPethyl acetate) and evaluated their structural properties and their potential against microorganisms and cancer cells. AuNPs showed a surface plasmon resonance (SPR) band at 535 nm. The sizes and morphologies were influenced by the BRP sample used in the reaction. FTIR and TGA revealed the involvement of bioactive compounds from BRP extract or its fractions in the synthesis and stabilization of AuNPs. AuNPdichloromethane and AuNPhexane exhibited antimicrobial activities against all strains tested, showing their efficacy as antimicrobial agents to treat infectious diseases. AuNPs showed dose-dependent cytotoxic activity both in T24 and PC-3 cells. AuNPdichloromethane and AuNPextract exhibited the highest in vitro cytotoxic effect. Also, the cytotoxicity of biogenic nanoparticles was induced by mechanisms associated with apoptosis. The results highlight a potential low-cost green method using Brazilian red propolis to synthesize AuNPs, which demonstrated significant biological properties.

Targeted uptake of folic acid-functionalized polymeric nanoparticles loading glycoalkaloidic extract in vitro and in vivo assays.

Solanum lycocarpum fruits contain two major glycoalkaloids (GAs), solamargine (SM) and solasonine (SS). These compounds are reported as cytotoxic. However, they have poor water solubility and low bioavailability. To overcome these disadvantages and getting an efficient formulation the current study aimed to develop, characterize, and test the effectiveness of a nanotechnology-based strategy using poly(D,L-lactide) (PLA) nanoparticles functionalized with folate as delivery system of glycoalkaloidic extract (AE) for bladder cancer therapy. The strategic of adding folic acid into nanoformulations can increase the selectivity of the compounds to the cancer cells reducing the side effects. Our results revealed the successful preparation of AE-loaded folate-targeted nanoparticles (NP-F-AE) with particle size around 177 nm, negative zeta potential, polydispersity index <0.20, and higher efficiency of encapsulation for both GAs present in the extract (>85 %). To investigate the cellular uptake, the fluorescent dye coumarin-6 was encapsulated into the nanoparticle (NP-F-C6). The cell studies showed high uptake of nanoparticles by breast (MDA-MB-231) and bladder (RT4) cancer cells, but not for normal keratinocytes cells (HaCaT) indicating the target uptake to cancer cells. The cytotoxicity of nanoparticles was evaluated on RT4 2D culture model showing 2.16-fold lower IC50 than the free AE. Furthermore, the IC50 increased on the RT4 spheroids compared to 2D model. The nanoparticles penetrated homogeneously into the urotheliumof porcine bladder. These results showed that folate-conjugated polymeric nanoparticles are potential carriers for targeted glycoalkaloidic extract delivery to bladder cancer cells.

The NAD(+) salvage pathway modulates cancer cell viability via p73.

The involvement of the nicotinamide adenine dinucleotide (NAD(+)) salvage pathway in cancer cell survival is poorly understood. Here we show that the NAD(+) salvage pathway modulates cancer cell survival through the rarely mutated tumour suppressor p73. Our data show that pharmacological inhibition or knockdown of nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme in the NAD(+) salvage pathway, enhances autophagy and decreases survival of cancer cells in a p53-independent manner. Such NAMPT inhibition stabilizes p73 independently of p53 through increased acetylation and decreased ubiquitination, resulting in enhanced autophagy and cell death. These effects of NAMPT inhibition can be effectively reversed using nicotinamide mononucleotide (NMN), the enzymatic product of NAMPT. Similarly, knockdown of p73 also decreases NAMPT inhibition-induced autophagy and cell death, whereas overexpression of p73 alone enhances these effects. We show that the breast cancer cell lines (MCF-7, MDA-MB-231 and MDA-MB-468) harbour significantly higher levels of NAMPT and lower levels of p73 than does the normal cell line (MCF-10A), and that NAMPT inhibition is cytotoxic exclusively to the cancer cells. Furthermore, data from 176 breast cancer patients demonstrate that higher levels of NAMPT and lower levels of p73 correlate with poorer patient survival, and that high-grade tumours have significantly higher NAMPT/p73 mRNA ratios. Therefore, the inverse relationship between NAMPT and p73 demonstrable in vitro is also reflected from the clinical data. Taken together, our studies reveal a new NAMPT-p73 nexus that likely has important implications for cancer diagnosis, prognosis and treatment.

Reference values for high-density lipoprotein particle size and volume by dynamic light scattering in a Brazilian population sample and their relationships with metabolic parameters.

BACKGROUND: Current data indicate that the size of high-density lipoprotein (HDL) may be considered an important marker for cardiovascular disease risk. We established reference values of mean HDL size and volume in an asymptomatic representative Brazilian population sample (n=590) and their associations with metabolic parameters by gender. METHODS: Size and volume were determined in HDL isolated from plasma by polyethyleneglycol precipitation of apoB-containing lipoproteins and measured using the dynamic light scattering (DLS) technique. RESULTS: Although the gender and age distributions agreed with other studies, the mean HDL size reference value was slightly lower than in some other populations. Both HDL size and volume were influenced by gender and varied according to age. HDL size was associated with age and HDL-C (total population); non- white ethnicity and CETP inversely (females); HDL-C and PLTP mass (males). On the other hand, HDL volume was determined only by HDL-C (total population and in both genders) and by PLTP mass (males). CONCLUSIONS: The reference values for mean HDL size and volume using the DLS technique were established in an asymptomatic and representative Brazilian population sample, as well as their related metabolic factors. HDL-C was a major determinant of HDL size and volume, which were differently modulated in females and in males.

Hyperbaric oxygen therapy as salvage treatment for sudden sensorineural hearing loss: a prospective controlled study.

The most commonly used treatment for sensorineural sudden hearing loss (SSHL) in clinical practice is the administration of steroids; however, a favorable result is not always obtained. We studied 58 patients who failed to recover after primary treatment with IV steroids, 44 of these met our inclusion criteria (mean age 50.7, 27 males, range 30-74). We treated 23 patients (mean age 47.3, 16 males, age range 22-74) with hyperbaric oxygen therapy (HBO) (2.5 ATA for 60 min for 15 treatments), while 21 (mean age 54.5, 11 males, age range 22-71) patients refused to be treated and served as a non-randomized control group. Patients treated with HBO had a mean improvement of 15.6 dB (SD ± 15.3), with 1 of them completely healed, 5 with a good recovery, 10 with a fair recovery and 7 unchanged. Patients who were not treated had a spontaneous mean improvement of 5.0 dB (SD ± 11.4) with 3 patients with a good recovery, 1 patient with a fair recovery and 17 patients unchanged. Mean improvement was significantly better in patients treated with HBO compared to controls (p = 0.0133). Patients with worst hearing had the greater degree of improvement whether or not they were treated in the first 10 days after the onset of the hearing loss or between 11 and 30 days. In conclusion, hyperbaric oxygen therapy can lead to significant improvement of pure tone hearing thresholds in patients with SSHL who failed primary corticosteroid treatment and are within 4 weeks of the onset of deafness.

Cisplatin Properties in a Nanobiotechnological Approach to Cancer: A Mini-Review.

For many years, cisplatin has been used to treat many types of cancer, including urogenital, skin and lung cancers. Unfortunately, treatment with this drug causes serious side effects, such as severe toxicity; including nephrotoxicity, neurotoxicity, gastrointestinal toxicity, peripheral neuropathy, ototoxicity, asthenia and hematological toxicity.Therefore, the clinical use of cisplatin has been hampered.The incidence of nephrotoxicity frequently prevents the use of high enough doses to maximize the antineoplastic effects, and strict attention must be given to the hydration of cisplatin-treated patients to minimize kidney damage.Nanobiotechnology, or nanomedicine, was developed to mitigate, or even eliminate,the toxic effects of pharmaceutical compounds; for example, drug-targeting systems were developed to enable site specificity and to control the delivery drug. Therefore, biomedical nanotechnology researchers attempted to develop nanostructures not only to deliver chemotherapeutics to the desired treatment site but also to control when and how quickly the compounds are released. To achieve these ends, a drug can either be encapsulated in a matrix or attached to a particle surface. Studies concerning the encapsulation of cisplatin in liposomes, polymeric nanoparticles, solid lipid nanoparticles and carbon nanotubes, as well as the immobilization of cisplatin on metallic nanoparticles, have already been published. The association of cancer treatment, particularly chemotherapeutics, with nanotechnology is currently one of the most exciting areas of research. In this mini-review, cisplatin will be discussed in terms of its efficacy against many cancers, including bladder cancer. Additionally, established nanostructure-based drug delivery systems for cisplatin and their efficacy against different types of cancer will be reviewed. Because cisplatin is a standard treatment with good performance statistics and with an effective renal function-glomerular filtration rate, we expect that this review will be helpful for future research.

Gemcitabine enhances the efficacy of reovirus-based oncotherapy through anti-tumour immunological mechanisms.

BACKGROUND: Reovirus preferentially infects and kills cancer cells and is currently undergoing clinical trials internationally. While oncolysis is the primary mode of tumour elimination, increasing evidence illustrates that reovirus additionally stimulates anti-tumour immunity with a capacity to target existing and possibly relapsing cancer cells. These virus-induced anti-tumour immune activities largely determine the efficacy of oncotherapy. On the other hand, anti-viral immune responses can negatively affect oncotherapy. Hence, the strategic management of anti-tumour and anti-viral immune responses through complementary therapeutics is crucial to achieve the maximum anti-cancer benefits of oncotherapy. METHODS: Intra-peritoneal injection of mouse ovarian surface epithelial cells (ID8 cells) into wild-type C57BL/6 mice was treated with a therapeutic regimen of reovirus and/or gemcitabine and then analysed for prolonged survival, disease pathology, and various immunological parameters. Furthermore, in vitro analyses were conducted to assess apoptosis, viral spread, and viral production during reovirus and/or gemcitabine treatment. RESULTS: We demonstrate that reovirus and gemcitabine combination treatment postpones peritoneal carcinomatosis development and prolongs the survival of cancer-bearing hosts. Importantly, these anti-cancer benefits are generated through various immunological mechanisms, including: (1) inhibition of myeloid-derived suppressor cells recruitment to the tumour microenvironment, (2) downmodulation of pro-MDSC factors, and (3) accelerated development of anti-tumour T-cell responses. CONCLUSION: The complementation of reovirus with gemcitabine further potentiates virus-initiated anti-cancer immunity and enhances the efficacy of oncotherapy. In the context of ongoing clinical trials, our findings represent clinically relevant information capable of enhancing cancer outcomes.

Stabilisation of p53 enhances reovirus-induced apoptosis and virus spread through p53-dependent NF-κB activation.

BACKGROUND: Naturally oncolytic reovirus preferentially kills cancer cells, making it a promising cancer therapeutic. Mutations in tumour suppressor p53 are prevalent in cancers, yet the role of p53 in reovirus oncolysis is relatively unexplored. METHODS: Human cancer cell lines were exposed to Nutlin-3a, reovirus or a combination of the two and cells were processed for reovirus titration, western blot, real-time PCR and apoptosis assay using Annexin V and 7-AAD staining. Confocal microscopy was used to determine translocation of the NF-κB p65 subunit. RESULTS: We show that despite similar reovirus replication in p53(+/+) and p53(-/-) cells, stabilisation of p53 by Nutlin-3a significantly enhanced reovirus-induced apoptosis and hence virus release and dissemination while having no direct effect on virus replication. Enhanced apoptosis by Nutlin-3a was not observed in p53(-/-) or p53 knockdown cells; however, increased expression of Bax and p21 are required. Moreover, elevated NF-κB activation in reovirus-infected cells following Nutlin-3a treatment was necessary for enhanced reovirus-induced apoptosis, as synergistic cytotoxicity was overcome by specific NF-κB inhibitors. CONCLUSION: Nutlin-3a treatment enhances reovirus-induced apoptosis and virus spread through p53-dependent NF-κB activation, and combination of reovirus and Nutlin-3a might represent an improved therapy against cancers harbouring wild-type p53.

Nanostructured polymer and lipid carriers for sunscreen. Biological effects and skin permeation.

The interest in developing new sunscreens is increasing due to the harmful effects of UV radiation on the skin, such as erythema, accelerated skin ageing (photoageing) and the induction of skin cancer. However, many molecular sunscreens penetrate into the skin causing photoallergies, phototoxic reactions and skin irritation. Thus, the aim of this work was the preparation and characterization of polymeric and solid lipid nanoparticles to act carriers of benzophenone-3 (BZ3), aiming to improve the safety of sunscreen products by increasing the sun protection factor (SPF), decreasing BZ3 skin penetration and decreasing BZ3 concentration in sunscreen formulation. BZ3 was encapsulated in poly(epsilon-caprolactone) (PCL) nanoparticles by the nanoprecipitation method and in solid lipid nanoparticles (SLN) by the hot high pressure homogenization method. The particles were stable for 40 days. The BZ3 encapsulated in PCL nanoparticles was released faster than BZ3 encapsulated in SLN. The sun protection factor increased when BZ3 was encapsulated in both nanostructures. However, BZ3 encapsulated in PCL nanoparticles decreased its skin permeation more than SLN-BZ3. Furthermore, BZ3 encapsulated in SLN did not exhibit cytotoxic or phototoxic effects in human keratinocytes (HaCaT cells) and BABL/c 3T3 fibroblasts, whereas PCL nanoparticles with BZ3 showed phototoxic potential in HaCaT cells. Nevertheless, BZ3 free and encapsulated in PCL nanoparticles or in SLN did not show allergic reactions in mice. Our results suggest that these nanostructures are interesting carriers for sunscreen.

Histopathologic classification of 171 cases of canine and feline non-Hodgkin lymphoma according to the WHO.

A retrospective collection of 171 lymphoid neoplasms (123 dogs and 48 cats) was classified according to the Revised European-American Lymphoma (REAL) classification, adopted in 2002 by the World Health Organization (WHO), to evaluate the WHO system for categorization of canine and feline neoplasms. Microscopic examination was performed after standard hematoxylin-eosin staining and immunohistochemical labelling for B (CD79a) or T (CD3) cell phenotypes. B-cell lymphomas were prevalent in dogs and T-cell lymphomas in cats. B-Large cell lymphoma (B-LCL) frequently showed plasmacytoid differentiation; notably, two canine plasma cell tumours (PCT) expressed both CD79 and CD3. There were difficulties in differentiating B-lymphoblastic lymphoma (B-LBL) from Burkitt-type lymphoma. Furthermore, intestinal T-cell lymphoma (ITCL) exhibited a huge morphologic variability. Finally, multicentric mature small and thymic T-cell lymphomas were diagnosed, although these categories are not codified by the WHO classification.

Microencapsulation of antibiotic rifampicin in poly(3-hydroxybutyrate-co-3-hydroxyvalerate).

The aim of this study was the preparation of microparticles containing rifampicin using a biodegradable polymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) for oral administration produced by a bacteria. The poly(3-hydroxybutyrate-co-3-hydroxyvalerate) microparticles with and without rifampicin were prepared by the emulsification and solvent evaporation method, in which chloroform and polyvinyl alcohol are used as the solvent and emulsifier, respectively. Microparticles were obtained within a size range of 20-60 microm by changing the initial poly(3-hydroxybutyrate-co-3-hydroxyvalerate), polyvinyl alcohol and rifampicin concentrations. An encapsulation efficiency value of 14% was obtained. The optimized total yield of 60% of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/ rifampicin was obtained. A load of 0.035 mg/1 mg of PHBV was reached. Almost 90% of the drug loaded in the microparticles was released after 24 h. The size, encapsulation efficiency and ribampicin release of the microparticles varied as a function of the initial poly(3-hydroxybutyrate-co-3-hydroxyvalerate), polyvinyl alcohol and rifampicin concentrations. It was demonstrated that the microencapsulated rifampicin, although was not totally available in the medium, exhibited a similar inhibition value as free rifampicin at 24 h of incubation with S. aureus. Cytotoxicity assays demonstrated a reduction of the toxicity when rifampicin was microencapsulated in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) while maintaining its antibacterial activity.

Characterization of interstitial nephritis in pigs with naturally occurring postweaning multisystemic wasting syndrome.

Kidney samples with interstitial nephritis from 26 pigs affected by postweaning multisystemic wasting syndrome (PMWS) were selected. A histologic evaluation was carried out to describe the type of inflammation and its relationship with viral load, as assessed by in situ hybridization (ISH). Of 26 cases, 10 revealed a tubulointerstitial, lymphoplasmacytic nephritis, 11 an interstitial granulomatous nephritis, and 5 both types of inflammation (mixed type). In 4 cases of granulomatous inflammation, the pattern was not classically nodular, and a population of macrophages and lymphocytes was present (interstitial lymphohistiocytic nephritis). ISH confirmed the presence of porcine circovirus type 2 (PCV2) nucleic acid in all cases. The epithelium of the renal tubules was the most constantly ISH-positive structure. In tubulointerstitial nephritis, the higher the number of positive inflammatory cells, the more severe the inflammation. The ISH reaction was more heterogeneous and unpredictable in granulomatous nephritis, with some epithelioid and giant cells positive by ISH. To quantify macrophages distributed in the three patterns of nephritis, immunohistochemical methods using anti-major histocompatibility complex II (anti-MHC-II) and anti-lysozyme antibodies were undertaken, and semiquantitative evaluation was carried out. MHC-II was mainly expressed by lymphocytes in tubulointerstitial nephritis, but did not always stain macrophages in cases of granulomatous (including lymphohistiocytic) nephritis; the anti-lysozyme antibody revealed macrophages when present in tissues. The amount of PCV2 nucleic acid was not apparently associated with the pattern of inflammation (tubulointerstitial or granulomatous). PCV2 load seems to reflect the severity of the lymphoplasmacytic inflammation but not that of granulomatous and lympho histiocytic types.

Poly (epsilon-caprolactone)/propolis extract: microencapsulation and antibacterial activity evaluation.

Spherical and homogenous microparticles of poly(epsilon-caprolactone) (PCL), containing propolis were prepared by the emulsification-solvent evaporation technique. Using this method of preparation, a solid formulation of propolis, free of ethanol and suitable for manipulation and storage, was obtained from an ethanolic extract of propolis. The incorporation efficiency of propolis in the microparticles was almost 30% and around 60% of the substance was released in 48 h. In vitro propolis microparticles exhibited similar halo zones in the Petri plate test against Streptococcus mutans (GS5) with a 10-fold lower concentration than the free propolis extract showing that the encapsulated propolis in microparticles is more efficient as antibiotic.

Immunohistochemical MHC-II and interleukin 2-R (CD25) expression in lymph nodes of pigs with spontaneous postweaning multisystemic wasting syndrome (PMWS).

Immunohistochemical expression of immunocompetent cells bearing major histocompatibility complex (MHC-II) and interleukin 2-R (IL2-R) (CD25) molecules was performed on lymph nodes with spontaneous postweaning multisystemic wasting syndrome (PMWS). Control lymph nodes displayed intense diffuse immunoreactivity to MHC-II in both follicles and interfollicular areas. A marked reduction of follicular MHC-II immunoreactivity and inconsistent staining of histiocytes in interfollicular areas was observed in PMWS cases with a slight lymphoid depletion; in those cases with moderate to severe lymphoid depletion, there was a progressive decrease in MHC-II expression. In controls and in slightly depleted nodes, IL2-R was equally expressed in interfollicular tissue and in follicles, whereas in moderate and severe cases, it was detected in interfollicular remnants only. Immunohistochemical staining was scored semiquantitatively. The mean MHC-II score was significantly reduced in PMWS cases compared with controls (Spearman test), whereas there was no difference in the IL2-R score. The evident reduction of MHC-II immunoreactivity suggests an impairment in MHC-II linked antigen presenting cell expression.

Ten cases of feline mesothelioma: an immunohistochemical and ultrastructural study.

In the cat only 10 cases of mesothelioma, mainly of the peritoneum, have been previously reported. This paper describes a further 10 cases, eight pleural and two peritoneal, in males and females aged 1-17 years. Histologically, five tumours were epithelial, three fibrosarcomatous and two biphasic. Immunohistochemical markers used in human pathology for the identification of mesotheliomas include vimentin, cytokeratin (CK) AE1/AE3, HBME-1, CK 5/6, calretinin, thrombomodulin, carcinoembryonic antigen (CEA), CD15, E-cadherin and desmin. All 10 feline mesotheliomas were positive for vimentin and CK AE1/AE3, six were positive for HBME-1, two for CK5/6, three for CEA and four for E-cadherin. All were negative for desmin and calretinin. Antibodies to thrombomodulin and CD15 failed to cross-react with feline tissues. Electron microscopy, performed in four cases, revealed microvillar structures, desmosomes and intracytoplasmic lumina, confirming its value as a diagnostic tool. The study showed that mesothelial marker antibodies commonly used in human patients can be used for the diagnosis of feline mesothelioma, preferably as a panel of antibodies rather than only one.

Deciduoid peritoneal mesothelioma in a dog.

Deciduoid mesothelioma is a rare variant of epithelial mesothelioma, up to now only described in human pathology, which bears remarkable cytomorphologic resemblance to the endometrium of pregnancy, termed decidua. A case of peritoneal mesothelioma with deciduoid features in a 10-year-old, female dog is reported. Multiple whitish-gray nodules (1-5 mm in diameter) in parietal peritoneum and mesentery were histologically composed of large, proliferating, polygonal or ovoid cells with an abundant eosinophilic, glassy cytoplasm. Immunohistochemical evaluation indicated that the neoplastic cells coexpressed cytokeratin and vimentin with strong and diffuse cytoplasmic staining, and ultrastructural analysis showed long and slender mesothelial-type microvilli; these findings confirmed the mesothelial origin of the tumor.

Cytological diagnosis of mandibular salivary gland adenocarcinoma in a dog.

A case of mandibular salivary gland adenocarcinoma in a 9-year-old female dog is described. Material collected by fine needle aspiration underwent cytological examination and after the diagnosis of salivary carcinoma the mass was surgically excised and then was processed for histological examination. The aim of this work is to describe the cytopathological features of this carcinoma and to emphasize the usefulness of fine needle aspiration technique, which is an effective, inexpensive and minimally invasive method of diagnosis that can be performed before incisional biopsy or even before surgical excision.