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Current treatment of cutaneous leishmaniasis includes pentavalent antimonials as first-line drugs, but this therapy has shown severe adverse effects. An alternative to minimize this issue is based on combination therapy scheme with other drugs.
Pereira Lage,c, Anderson Miyoshi b, Vasco Azevedo,.Biologia Celular e Molecular (Luiz Carlos Ucha Junqueira e Jos Carneiro). Histologia Bsica (Luiz Carlos Ucha Junqueira.Pertussis is biologia celular e molecular carlos azevedo pdf a contagious disease that has reappeard in the recent years as a public health problem in our country.
In this study we analyzed the potential of the association of meglumine antimoniate (MA) with the oxiranes epoxy-α-lapachone (LAP) or epoxymethyl-lawsone (LAW). Results demonstrated that association between these drugs enhanced leishmanicidal activity on Leishmania (Leishmania) amazonensis infection. The compounds were tested in monotherapy or in combinations (3:1; 1:1 and 1:3) and reduced intracellular parasite numbers, measured by the endocytic index, in all tested conditions. The most effective combination regimens were MA/LAP or MA/LAW in 3:1 ratio, which achieved a reduction of 98.3% and 93.6% in the endocytic index, respectively. BALB/c mice challenged with L. (L.) amazonensis showed significant reduction in lesion size and parasite load in both footpad and lymph nodes, after four weeks of treatment. Although, MA, LAP or LAW monotherapy were able to control the evolution of lesions when compared to untreated animals (30%, 40% and 40% of reduction, respectively), the combination of MA/LAP and LAW in 3:1 ratio showed better results reducing 61.7 and 54.4%, respectively.
The results indicate that the association of meglumine antimoniate to oxiranes lead to an increment in the antileishmanial activity and represent a promising approach for the cutaneous leishmaniasis treatment. 1. IntroductionCutaneous leishmaniasis (CL) is an infectious disease, transmitted by female sandflies, which presents high incidence and morbidity rates in developing countries, where access to health services is often precarious. This poverty related disease lead to great social and economic burden. The World Health Organization estimates that global prevalence is approximately 12 million cases per year, occurring in 98 countries, but nearly 90% of the cutaneous and mucocutaneous cases are concentrated in seven developing countries, including Brazil. Leishmaniasis is considered an important neglected tropical disease due to its extensive distribution in tropical areas of the globe and risk to produce deformities.Despite almost seven decades of researches assaying alternative therapies to replace pentavalent antimonials, these drugs remain the first-line treatment for most forms of leishmaniasis worldwide. Pentavalent antimonials therapy is associated with mild to severe adverse effects, being often accompanied by pain and swelling at site of the intramuscular application and several systemic symptoms, which include nausea, vomiting, weakness and myalgia, abdominal colic, diarrhea, skin rashes and variable transient increase of transaminases and/or amylases (; ). The most serious adverse effect associated with pentavalent antimonials therapy is undoubtedly cardiotoxicity, characterized by ventricular repolarization disorders (as alterations in T wave and ST segment) and arrhythmia, which can lead to sudden death (; ).
To date, the precise mechanism of action whereby pentavalent antimonials kill Leishmania parasites remains unknown. However, it seems to be multifactorial, as with other heavy metals, rather than being due to a single route (, ). At the molecular level, it is proposed that the antimonial react with sulfhydryl groups present in proteins, causing structural changes that would lead to an impairment of its function. There is evidence that these compounds may inhibit certain phases of the energetic metabolism cycle of amastigotes.
Experimental observations indicated that in vitro exposure of L. (L.) mexicana to sodium stibogluconate resulted in a dose-dependent reduction of both cellular viability and the production of CO 2 from glucose and palmitate in parasite cultures. Data indicate that the depletion of intracellular ATP levels is due to an inhibition of glycolytic enzymes and other components of the fatty acid pathway, but not from the hexose monophosphate pathway and the citric acid cycle. Furthermore, studies have shown that the trivalent form (Sb 3+) is able to cause inhibition of the trypanothione reductase activity in L. (L.) donovani, an essential enzyme for the parasite survival within macrophages, and it may be responsible for part of the leishmanicidal effects (; ). Chemical structure of tested drugs.
(A) 2,2-dimethyl-3,4-dihydrospirobenzogchromene-10,2′-oxiran-5-one, also known as epoxy-α-lapachone (C 16H 16O 3, 256.296 g/mol; CID 12000280) , (B) 2-methoxy-4 H-spironaphthalene-1,2′-oxiran-4-one, also known as epoxymethyl-lawsone (C 12H 10O 3, 202.21 g/mol), and (C) meglumine antimoniate known commercially as Glucantime (C 7H 18NO 8Sb, 365.98 g/mol) (Adapted from ).LAP led to a dose- and time-dependent decrease in the growth rate of Leishmania (Viannia) braziliensis and L. (L.) amazonensis promastigote cultures, killed amastigotes inside human macrophages , and reduced the mean lesion size in BALB/c mice infected by L. (L.) amazonensis. LAW affected promastigotes viability after 24 h of exposure and demonstrated an activity on intracellular amastigotes similar to the reference drug meglumine antimoniate.
LAW was also able to hinder lesion growth in L. (L.) amazonensis infected BALB/c mice.Studies aim to identify promising leishmanicidal drug associations with high activity in experimental infection, since it may lead to improved treatment efficacy of treatments and/or reduce dose regimen and toxicity (; ). Thereby, following this research line, the objective of the present study is to determine potential of the combination therapy using meglumine antimoniate (MA) associated with epoxy-α-lapachone or epoxymethyl-lawsone against experimental infection. Herein, we demonstrate an enhanced leishmanicidal effect produced by such combination treatments on L. (L.) amazonensis, either in macrophage cultures infected by this parasite or in experimentally challenged BALB/c mice. Activity against intracellular amastigotesMacrophages were co-incubated (4 h, 37 °C) with promastigotes in a proportion of 5:1 (parasite:cell) in Lab-Tek chamber slides, followed by washing with PBS and addition of RPMI medium containing 5% FCS. After, cultures were incubed (37 °C, 24 h) with meglumine antimoniate, epoxy-α-lapachone and epoxymethyl-lawsone in monotherapy at the concentrations corresponding to its IC 50 values previously determined (2.0 μM, 5.0 μM and 7.5 μM, respectively), and in combinations of MA:LAP or MA:LAW in fixed ratios 3:1, 1:1, 1:3, where 3:1 refers to 3 parts of MA IC 50 value with 1 part of LAP IC 50 or LAW IC 50 value (75% of MA + 25% of LAP or LAW = total dose).
Similarly, 1:1 and 1:3 ratios correspond to 1 part of MA IC 50 plus LAP IC 50 or LAW IC 50 (50%: 50%), and 1 part of MA IC 50 plus 3 parts of LAP IC 50 or LAW IC 50 (25%: 75%), respectively. Then, slides were fixed with 100% methanol and Giemsa-stained. The endocytic index was determined by counting intracellular parasites in at least 300 random cells. The results are expressed as the mean and standard deviation of three independent assays.
Toxicity of compounds and its combinations to BALB/c mice macrophagesMacrophages seeded on 96-well plates (2 × 10 5 per well) were exposed (37 °C, 72 h) to meglumine antimoniate, epoxy-α-lapachone and epoxymethyl-lawsone in monotherapy at a concentration equivalent to half of IC 50 previously defined (2.0 μM, 5.0 μM and 7.5 μM, respectively) and in combinations that followed the ratio 3: 1, 1: 1 and 1: 3, as described above. Then, macrophages viability was determined by addition of CellTiter-Glo™ (20μL/well) and incubation (25 °C, 3 min) under agitation.
Luminescence was measured using a FlexStation 3 reader (Molecular Devices, Sunnyvale, CA, USA). Mice treatment schedulesTo determine doses to be applied in the combination assays, BALB/c mice were previously treated (five animals per group) with three different doses of each drug, defined here as low, intermediate and high dose, as follow: meglumine antimoniate (0.23 mg of Sb 5+/Kg/day, 2.27 mg of Sb 5+/Kg/day and 22.7 mg of Sb 5+/Kg/day); epoxy-α-lapachone (0.23 mg/kg/day, 2.27 mg/kg/day and 22.7 mg/kg/day); and epoxymethyl-lawsone (0.11 mg/kg/day, 1.14 mg/kg/day and 11.4 mg/kg/day), starting six weeks after infection, as previously described (; ).
Briefly, oxiranes were diluted in a mixture of DMSO: propylene glycol: saline (1: 12: 7) because of their low solubility in water, then drugs were administrated daily by subcutaneous route (100 μL per animal), from Monday to Friday until 20 doses. Negative-control group was treated with vehicle used to dissolve the oxirane compounds. The lesions were evaluated weekly by measuring the height and width of the paw, and lesion volumes were obtained by multiplying these measures in mm 3 with a digital caliper. The intermediate doses of each drug were selected as its efficacy was similar to a high dose. Meglumine antimoniate, epoxy-α-lapachone and epoxymethyl-lawsone were tested in monotherapy (2.27 mg of Sb 5+/Kg/day, 2.27 mg/kg/day and 1.14 mg/kg/day, respectively) and in combinations following fixed-proportion of 3: 1; 1: 1 and 1: 3, as already described in 2.4. Parasite kinetoplast DNA quantification by real-time PCR (qPCR)DNA from the footpad and draining lymph nodes of 3 animals per group were extracted following a standard phenol/chloroform protocol. DNA was quantified in a NanoDrop 2000c spectrophotometer (ThermoScientific) and diluted to 10 ng/μL of total DNA.
Real time PCR was performed in QuantStudio™ 3 Real Time PCR System equipment using fast cycle with GoTaq™ qPCR Master Mix, 2 μL of DNA sample at a final volume of 10 μL. Primers were target for the parasite kDNA1 (forward: 5′-GGGTAGGGGCGTTCTGC-3′, reverse: 5′-TACACCAACCCCCAGTTTGC-3’; accession number: ), and mouse β-actin (forward: 5′-AGAGGGAAATCGTGCGTGAC-3′, reverse: 5′-CAATAGTGATGACCTGGCCGT-3’; accession number: ) was used as an endogenous control.
Parasite load was estimated from a standard curve (1.7 × 10 −1 to 1.7 × 10 4 parasites) of known amounts of Leishmania parasite obtained from in vitro culture, considering size of the genome sequence of L. (L.) amazonensis and kinetoplast DNA replication (;; ). 3. Results and discussionLeishmaniases represent a major public health issue in the Americas due to their distribution in several countries and high prevalence.
Treatment is a pivotal step to control the disease; however, effectiveness of current options has been questioned due to the increasing numbers of resistance and toxicity, pointing toward the need of novel drugs. In recent years, efforts have been performed to improve antimonial chemotherapy. Several studies added further information on its chemical structure , mechanisms of action, new methods of preparation and potential incorporation into different formulations (reviewed in ). In this context, a promising strategy is the association of pentavalent antimonials with oxiranes, compounds that showed significant antileishmanial activity; such association could reduce drug doses and toxicity, while maintain or improve the baseline efficacy of pentavalent antimonials. In addition, a combination of two drugs with rather different mechanisms of action over the parasites may have the benefit of hindering the emergence of resistant strains.
Therefore, we devised in the present study a new approach for the CL chemotherapy based on the combination treatment of meglumine antimoniate with epoxy-α-lapachone or epoxymethyl-lawsone. Effects of drug combinations against intracellular amastigotes of Leishmania (L.) amazonensisInitially, we investigated the combination effects of MA with oxiranes LAP and LAW on peritoneal macrophages of BALB/c mice infected in vitro with L. (L.) amazonensis. The drugs were tested alone (at a concentration equivalent to half of IC 50 values previously determined) or in combinations following fixed-ratios of 3: 1; 1: 1 and 1: 3. All drugs as well as its combinations exhibited impact over the macrophage-infecting parasites, as determined by the endocytic index (EI): treated cultures had EI values lower than untreated cultures.
The effects of MA or LAW monotherapy on parasites showed similar values; the EI was reduced 82% and 80%, respectively. LAP alone showed the lowest impact over the parasites (EI reduced 75%). Some of the drug combinations were sensibly more effective over the intracellular parasites: MA/LAP or MA/LAW at 3: 1 ratio led to 98.3% and 93.6% reduction on the EI of the control, respectively. In relation to respective drugs alone, the ratio combinations showed reductions of 90.5% and 93.2% (MA/LAP) and 64.6% and 67.7% (MA/LAW). No significant difference in 1:3 and 1:1 ratios were detected.
Cytotoxicity assays performed using either the drugs monotherapy or its combinations, with ratios established above, indicated no significant change in the viability of treated macrophage cultures when compared to untreated controls. Control groups were cultivated with medium only or 0.8% DMSO, used as solvent for compounds, (data not shown).
This set of results established the feasibility of proceeding to in vivo experiments, aiming to analyze the effects of combination treatments on the progression of lesions in experimentally challenged mice. Effects of combination treatments with epoxy-α-lapachone and meglumine antimoniate (A) and epoxymethyl-lawsone and meglumine antimoniate (B) on the endocytic index in peritoneal murine macrophages infected in vitro with Leishmania (L.) amazonensis. Cultures of peritoneal macrophages isolated from BALB/c mice were infected with L.
(L.) amazonensis and incubated (24 h, 37 °C) with either meglumine antimoniate (MA), epoxy-α-lapachone (LAP) or epoxymethyl-lawsone (LAW) in monotherapy at the concentrations corresponding to its IC 50 values previously determined (2.0 μM, 5.0 μM and 7.5 μM, respectively), and in combinations of MA: LAP 3: 1 (1.5 μM + 1.25 μM), 1: 1 (1.0 μM + 2.5 μM), 1: 3 (0.5 μM + 3.75 μM) and MA: LAW in fixed ratios 3: 1 (1.50 μM + 1.88 μM), 1: 1 (1.0 μM + 3.75 μM), 1: 3 (0.5 μM + 5.63 μM). Control cultures (white bars) were treated with 0.8% DMSO. The data are expressed as the mean and standard deviation (±) of three independent assays. Difference between groups was analyzed with one-way ANOVA, Bonferroni post-test and Tukey HSD.
(#) Statistically significance between control and treated groups (p ≤ 0.001). (.) Difference between treated groups and the most active combination (3:1) (p ≤ 0.001).Previous data from our research group indicated that treatment of BALB/c mice challenged with L.
(L.) amazonensis with either MA, LAP and LAW (in three distinct dosages) led to a measurable reduction in the mean size of parasite-associated lesions, pointing to a control of the in vivo infection. The tenth week post-infection was settled as the chosen time point for lesion size measurements.
Considering that no dose-response pattern was observed for any of the applied dosages of LAW and LAP and that the highest dosage of these compounds elicited significant signs of tissue toxicity , the intermediate dosage was elected to perform the combination treatment assays. Effects of drug combinations treatment on experimental cutaneous lesions caused by Leishmania (L.) amazonensisAs previously observed, the administration of MA, LAP or LAW prevented lesion development, leading to a reduction in mean lesion size in treated challenged mice when compared to untreated animals after the fourth week of treatment. Similar results were observed in mice treated with combinations of the compounds (A and C). The mean lesion volume measured in untreated animals 10 weeks post-infection was 10.7 ± 1.25 mm 3. MA presented the lowest effect on reducing mean lesions size, leading to a 30% decrease in lesions size, whereas both LAP and LAW monotherapy presented better effects, decreasing 40% of lesions size (B and D). Results of drug combination revealed a better profile of MA/LAP or MA/LAW at 3: 1 ratio (75% of MA and 25% of LAP or LAW) achieving a lesion reduction of 61.7 and 54.4%, respectively.
Collectively this data indicates that oxiranes potentiate the leishmanicidal effects of MA. Additionally, the combination of MA/LAW at 1: 3 ratio also presented statistical difference compared to MA monotherapy (D). Treatment of mice challenged with Leishmania (L.) amazonensis. BALB/c mice were treated daily with meglumine antimoniate (MA), epoxy-α-lapachone (LAP) or epoxymethyl-lawsone (LAW) in monotherapy at doses previously defined (2.27 mg of Sb 5+/Kg/day; 2.27 mg/kg/day and 1.14 mg/kg/day, respectively), and in combinations of MA: LAP 3:1 (1.70 mg + 0.57 mg), 1: 1 (1.14 mg + 1.14 mg) and 1: 3 (0.57 mg + 1.70 mg) and MA: LAW 3:1 (1.70 mg + 0.29 mg), 1: 1 (1.14 mg + 0.57 mg) and 1: 3 (0.57 mg + 0.86 mg).
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Treatments were administrated daily until 20 doses and lesion sizes (mm 3) were measured weekly (A and C). The results are represented as mean and standard deviation (±) from three independent experiments. The endpoint was on the 10th week and the differences (%) of each treatment are shown (C and D). Difference between groups was analyzed using Mann-Whitney test.Statistically significant p ≤ 0.05,.Statistically significant p ≤ 0.01 and.Statistically significant p ≤ 0.001. Parasite kinetoplast DNA quantification in footpad and lymph nodes of treated miceThe effectiveness of the drugs and its combinations in experimental cutaneous leishmaniasis was also evaluated by measuring the parasite kinetoplast DNA (kDNA) quantification from footpad and lymph nodes after the last week of treatment (tenth week) by a quantitative real-time PCR (qPCR) assay.
The performance of the qPCR for Leishmania kDNA quantification was related with the presence of parasite in the lesion sites and lymph nodes and defined here as parasite load. The results show significant decrease of parasite load in footpad lesion and lymph nodes for all treatment modalities compared to control. LAP, LAW and MA treatments presented similar parasite load reductions in footpad (65.22, 69.75, and 84.25%) and in lymph nodes (74.59, 81.54, and 79.09%). Parasite load in footpad lesion and lymph node in BALB/c mice.
Animals infected with Leishmania (L.) amazonensis were treated or not (control) with oxiranes epoxy-α-lapachone (LAP) or epoxymethyl-lawsone (LAW) and meglumine antimoniate alone or in combinations at fixed-ratio of 3:1, 1: 1 and 1: 3. The Leishmania kDNA were quantified by real-time PCR (qPCR) assay. Parasite load quantification was performed in footpad (A and C) and lymph nodes (B and D) at the endpoint of 10 weeks of treatment. The results are expressed (parasite load/20 ng total DNA) as the mean and standard deviation of three independent assays.
Difference between groups was analyzed using Mann-Whitney test.Statistically significant p ≤ 0.05,.Statistically significant p ≤ 0.01 and.Statistically significant p ≤ 0.001.Although both treatment combinations showed a statistically significant reduction in relation to the control, MA with LAP showed the best results in the reduction of footpad lesion parasite load compared to MA and LAW combination. MA/LAP at 1: 1 and at 1: 3 ratios exhibited highest parasite elimination (96.81 and 96.65%), presenting significant decrease in relation to the drugs alone (p 0.05).The search for new drugs to treat leishmaniasis is still incipient. Drug development faces many obstacles and new drug candidates against CL often did not progress further to clinical trials. The few advances so far are limited to the development of new formulations for clinical available drugs or drug repurposing.
Despite the high incidence of adverse effects and the increasing numbers of unresponsive strains, pentavalent antimonials remain the basis of chemotherapy and in short-term there is no perspective of new chemical entity or formulation approval.Enhancement of effects due to combination therapies can occur basically in two ways: ( i) one drug may increase the activity of the other, or ( ii) the effect of two drugs may combine to produce an activity distinct to that observed for each one individually. The global increment in leishmancidal activity observed for treatment with MA/LAP or MA/LAW at 3:1 ratio, considering the reduction of lesion size was not confirmed by parasite load quantification. According to this measurement, the best results related to combination treatment were found in MA/LAP at 1: 1 and 1: 3 ratio.
The decrease of the parasitic kDNA in lymphatic nodes of all treated animals confirms the systemic leishmanicidal effect of the oxiranes tested. However, differences among its combinations with reference drug MA were not possible to demonstrate.
Beginning in, the land that is now Portugal became part of the vast Umayyad Caliphate's empire of, which stretched from the river in the Indian sub-continent up to the South of France, until its collapse in The cell cycle and the mechanisms of cell differentiation, ageing and programmed death. An mainly equipped with, a mainly equipped with tanks and APC and a consisting of, and Rangers. The synthesis, processing and destination of proteins and other biomolecules; 5. In addition to being a destination for, Portugal is also among the top places of origin for international students. Uploader:Date Added:9 February 2017File Size:17.37 MbOperating Systems:Windows NT/2000/XP/2003/2003/7/8/10 MacOS 10/XDownloads:44416Price:Free.Free Regsitration RequiredThe Assembly of the Republic is a unicameral body composed of up to deputies. Cellular and Molecular Biology Code: Portugal's last overseas territory, was peacefully handed over to the People's Republic of China PRC inunder the joint declaration that set the terms for Macau's handover from Portugal to the PRC.It is a new world evem more remarkable by its subtleness, intriguing by the complexity and conceptually and ethically demanding.
Biologia Celular Pdf
Integrated knowledge on molecular processes leading to production and use of biomoleculesin cells and organisms, particularly those having major nutritional value.While these users - small performance optimization. Pedro's reign saw the consolidation of national independence, imperial expansion, and investment in domestic production.Inreached and brought and its population of 1. The railway network is managed by the while the transport of passengers and goods are the responsibility of Moleculraboth public companies.Top origins for foreign-born naturalized citizens of Portugal InPortugal had 10, inhabitants of whom aboutwere legal. Key concepts in Molecular Biology 2. At the time of exiting the bailout the economy had contracted by 0. Some former state-owned entities are managed by state-run, which is a of several public and biolgoia companies.
It has essentially Executive powers, but has also limited Legislative powers.Since the turn of the 21st century, there has been a trend towards the development of a renewable biokogia industry and reduction of both consumption and use of fossil fuel resources. Particularly popular and distinguished is, recipient of the In May the country exited the bailout but reaffirmed its commitment to maintaining its reformist momentum.
BIOLOGIA CELULAR E MOLECULAR CARLOS AZEVEDO PDF DOWNLOADAs of Juneelectricity exports had outnumbered imports. On 1 Decemberthe entered into force, after it had been signed by the European Union member states on 13 December in the, in Lisbon, enhancing the efficiency and democratic legitimacy of the Union and improving the coherence of its action.The, signed on 22 April between Portugal and Spain, specified the anti-meridian to the line of demarcation specified in the Treaty of Tordesillas. The cytoplasm organelles and their functions; 6. Other episodes during this period of the Portuguese presence in Africa include the. The Portuguese government also agreed to eliminate its in which gave it veto power over vital decisions.
The Taifa period ended with the conquest of the who came from velular winning a decisive victory at the, followed a century later inafter the second period of Taifa, by the, also from. Molecular BiologyThus, the Portuguese empire held dominion over commerce in the and South Atlantic. Content tecla de atalho: Students participation during classes is encouraged; discussions with the students are maintained, and they are asked to propose hypothesis for solving specific questions. To raise or stimulate the joy to study beyond the class subjects, using textbooks, published articles, selected Internet sites and other media.New programs combine wind and water: The installation of space. In equestrian sports, Portugal won the only Horseball-Pato World Championship inachieved the azevedl position in the First World Cup organized in Ponte de Lima, Portugal, inand has achieved several victories in the European Championship.In water sports, Portugal has two major sports: Adventurer and poet c. On 1 Februarythe king Dom and his. It was believed by some scholars that early in the first millennium BC, several waves of invaded Portugal from and inter-married with the local populations, forming.A sign of the recent evolution is the power of molecular biology methods that departing from single cells or organisms, take only a short time to collect huge quantities of biological information that is stored in fast growing databases.The primary is, although many other domestic airlines provide services within bioloiga without the country.
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