BACKGROUND In addition to the limited therapeutic arsenal and the side effects of antileishmanial agents, drug resistance hinders disease control. In Brazil, Leishmania braziliensis causes atypical (AT) tegumentary leishmaniasis lesions, frequently refractory to treatment. OBJECTIVES The main goal of this study was to characterise antimony (Sb)-resistant (SbR) L. braziliensis strains obtained from patients living in Xakriabá indigenous community, Minas Gerais, Brazil. METHODS The aquaglyceroporin 1-encoding gene (AQP1) from L. braziliensis clinical isolates was sequenced, and its function was evaluated by hypo-osmotic shock. mRNA levels of genes associated with Sb resistance were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Atomic absorption was used to measure Sb uptake. FINDINGS Although clinical isolates presented delayed recovery time in hypo-osmotic shock, AQP1 function was maintained. Isolate 340 accumulated less Sb than all other isolates, supporting the 65-fold downregulation of AQP1 mRNA levels. Both 330 and 340 isolates upregulated antimony resistance marker (ARM) 56/ARM58 and multidrug resistant protein A (MRPA); however, only ARM58 upregulation was an exclusive feature of SbR field isolates. CA7AE seemed to increase drug uptake in L. braziliensis and represented a tool to study the role of glycoconjugates in Sb transport. MAIN CONCLUSIONS There is a clear correlation between ARM56/58 upregulation and Sb resistance in AT-harbouring patients, suggesting the use of these markers as potential indicators to help the treatment choice and outcome, preventing therapeutic failure.

A leishmaniose visceral canina (LVC) é uma zoonose com aspectos clínicos e laboratoriais variáveis. O objetivo deste trabalho foi identificar os principais achados hematológicos e bioquímicos em cães naturalmente infectados com Leishmania (Leishmania)infantum e associar esses parâmetros com as formas clínicas da LVC. Foram analizadas amostras sanguíneas provenientes de 51 cães, sendo 15 cães não infectados (grupo controle) e 36 infectados, os quais foram classificados clinicamente em três grupos: assintomáticos (n=12), oligossintomáticos (n=12) e sintomáticos (n=12). Todos os cães infectados apresentaram valores na relação albumina/globulina (A/G) abaixo do limite inferior de referência. Os valores médios de proteína total, uréia, α-2 globulina, globulina e A/G dos grupos de cães infectados permaneceram fora dos intervalos de referências e significativamente diferente quando comparados aos do grupo controle. Anemia foi registrada somente nos grupos de animais que manifestavam sinais clínicos da enfermidade, sendo que nas análises estatísticas constatou-se frequencia significativamente maior de alterações no eritrograma quando comparados ao grupo assintomático. Associação significativa foi observada entre anemia e a presença de sinais clínicos, onde os cães com os maiores valores de eritrograma apresentavam a melhor condição clínica. Os resultados fornecem evidência adicional que as formas clínicas da LVC podem refletir no eritrograma.

Canine visceral leishmaniasis (CVL) is a zoonotic disease that presents variable clinical and laboratory aspects. The aims of this study were to identify the main biochemical/hematological status of dogs naturally infected with Leishmania (Leishmania) infantum and to associate theses parameters with clinical forms of CVL. Blood samples were analyzed from 51 dogs, 15 uninfected (control group) and 36 infected, which were classified clinically in three groups: asymptomatic (n=12), oligosymptomatic (n=12) and symptomatic (n=12). All the infected dogs showed lower albumin/globulin ratios (A-G ratio) than the limit of reference. The mean values of total protein, urea, α-globulin 2, globulin and A-G ratio of infected dogs were outside the reference interval and differed significantly from those of the controls. Anemia was detected only in groups that showed clinical signs of the disease, and a statistical analysis indicated a significantly higher frequency of lower eritrogram in these groups than in the asymptomatic group. In addition, a significant association was observed between anemia and the presence of the symptoms, with dogs displaying higher erythrogram values showing better clinical conditions. These results provide additional evidence that the clinical forms of CVL may reflect on the erythrogram status.

O crescente uso das nanotecnologias nas terapias avançadas tem permitido a observação de reações adversas específicas relacionadas às nanoestruturas. A toxicidade de uma nova formulação lipossomal de antimoniato de meglumina após dose única foi avaliada em cães com leishmaniose visceral. Grupos de 12 animais receberam por via intravenosa uma dose única de antimoniato de meglumina lipossomal (grupo I [GI], 6,5 mg Sb/kg), lipossomas vazios (GII) ou solução salina isotônica (GIII). A avaliação de parâmetros hematológicos e bioquímicos não revelou alterações significativas quatro dias após a administração. Nenhum efeito indesejável foi registrado no GIII. No entanto, reações adversas foram observadas em 67,7% dos cães de ambos os grupos que receberam formulações lipossomais. Os efeitos colaterais iniciaram momentos após a administração em "bolus" e desapareceram no decurso dos primeiros 15 minutos após o tratamento. Prostração, sialorréia e defecação foram os sinais clínicos mais frequentes, registrados em 33,3% e 41,6% dos animais dos grupos GI e GII, respectivamente. Taquipnéia, midríase, miose, vômitos e cianose também foram registrados em ambos os grupos. As reações adversas observadas neste trabalho foram atribuídas à ativação do sistema complemento pelas vesículas lipídicas em fenômeno conhecido como Pseudoalergia Relacionada à Ativação do Complemento (PARAC). A influência das características físico-químicas da formulação lipossomal no desencadeamento de PARAC é abordada.

The increasing use of nanotechnologies in advanced therapies has allowed the observation of specific adverse reactions related to nanostructures. The toxicity of a novel liposome formulation of meglumine antimoniate in dogs with visceral leishmaniasis after single dose has been investigated. Groups of 12 animals received by the intravenous route a single dose of liposomal meglumine antimoniate (group I [GI], 6.5 mg Sb/kg), empty liposomes (GII) or isotonic saline (GIII). Evaluation of hematological and biochemical parameters showed no significant changes 4 days after administration. No undesired effects were registered in the GIII. However, adverse reactions were observed in 67.7% of dogs from both groups that received liposomal formulations. The side effects began moments after bolus administration and disappeared during the first 15 minutes after treatment. Prostation, sialorrhea and defecation were the most frequent clinical signs, registered in 33.3% and 41.6 % of animals from the groups GI and GII, respectively. Tachypnea, mydriasis, miosis, vomiting and cyanosis were also registered in both groups. The adverse reactions observed in this study were attributed to the activation of the complement system by lipid vesicles in a phenomenon known as Complement Activation-Related Pseudoallergy (CARPA). The influence of the physical-chemical characteristics of liposomal formulation in the triggering of CARPA is discussed.

A presença de anticorpos anti-Neospora caninum e anti-Toxoplasma gondii foi avaliada em cães não infectados e naturalmente infectados com Leishmania chagasi manifestando diferentes formas clínicas da enfermidade. Amostras de sangue foram coletadas de 110 cães sem raça definida. Os soros foram avaliados por meio da reação de imunofluorescência indireta (RIFI) e os animais com leishmaniose visceral (LV) (n=60) foram classificados clinicamente. Dos 110 soros analisados, 5 (4,5%) foram reativos para N. caninum (RIFI>50) e 36 (32,7%) para T. gondii (RIFI>16). Os títulos de anticorpos anti-L. chagasi em cães assintomáticos (n=10) foram significativamente (P<0,05) mais baixos que aqueles verificados em oligossintomáticos (n=22), que por sua vez foram significativamente menores (P<0,05) que em cães sintomáticos (n=28). Não foi observada associação entre infecções por Leishmania e N. caninum. Entre os cães infectados com L. chagasi, verificou-se uma tendência de associação (P=0.053) entre infecção com T. gondii e aparecimento de sinais clínicos da LV, o que sugere que a manifestação clínica da LV em cães pode aumentar sua susceptibilidade ao T. gondii. A provável influência do quadro de imunossupressão em diferentes formas clínicas da leishmaniose canina é abordada.

Uninfected dogs and those naturally infected with Leishmania chagasi exhibiting different clinical forms of disease were evaluated for the presence of anti-Neospora caninum and anti-Toxoplasma gondii antibodies. Blood samples were collected from 110 mongrel dogs. Sera were tested using the indirect fluorescent antibody test (IFAT), and the animals with visceral leishmaniasis (VL) (n=60) were classified clinically. Out of the 110 sera investigated, 5 (4.5%) were positive for N. caninum (IFAT>50) and 36 (32.7%) for T. gondii (IFAT>16). Anti-L. chagasi antibody titers in asymptomatic dogs (n=10) were found to be significantly lower (P<0.05) than those in oligosymptomatic ones (n=22), which were in turn significantly lower (P<0.05) than those in symptomatic ones (n=28). No association between Leishmania and N. caninum infections was observed. Among dogs infected with L. chagasi, a tendency (P=0.053) towards an association between the infection with T. gondii and the appearance of VL symptoms was observed, suggesting that the clinical manifestation of VL in dogs may enhance their susceptibility to T. gondii. The possible influence of the immunosuppressive status of canine leishmaniasis in the different clinical forms of the disease is discussed.

Complexos de platina(II) derivados de N-alquil-propanodiamina com cadeia carbônica variável (C8, C10, C12 ou C14) foram incorporados em lipossomas e a atividade citotóxica dessas formulações foi avaliada em linhagens tumorais (A549, MDA-MB-231, B16-F1 and B16-F10) e não-tumorais (BHK-21 and CHO). Suspensões de lipossomas estáveis e monodispersas incorporando os complexos de platina foram obtidas com uma composição lipídica de diestearoil-sn-glicero-3-fosfocolina, colesterol, e 1,2-diestearoil-sn-glicero-3-fosfoetanolamina-N-(metoxi(polietilenoglicol)-2000) na razão molar 5:3:0,3. A eficiência da incorporação dos complexos de platina em lipossomas aumentou com o tamanho da cadeia carbônica e foi maior que 80% com os derivados C12 e C14. O efeito da encapsulação em lipossomas na atividade citotóxica dos complexos mostrou-se dependente do tamanho da cadeia carbônica. Os dados indicam que a biodisponibilidade da platina a partir das formulações de lipossomas foi maior para o complexo apresentando uma cadeia carbônica (C12) e uma partição entre a membrana e a fase aquosa intermediárias.

Antitumor platinum(II) complexes derived from N-alkyl-propanediamine differing in the length of their carbon chain (C8, C10, C12 and C14) were incorporated in liposomes and the cytotoxic activity of these formulations was evaluated against tumor (A549, MDA-MB-231, B16-F1 and B16-F10) and non-tumor (BHK-21 and CHO) cell lines. Stable and monodisperse liposome suspensions incorporating the platinum complexes were obtained from the lipid composition consisting of distearoyl-sn-glycero-3-phosphocholine, cholesterol and 1,2-distearoyl-sn-glycero-3-phophoethanolamine-N-(methoxy(polyethylene glycol)-2000) at 5:3:0.3 molar ratio. The entrapment efficiency (EE%) of the platinum complexes in liposomes increased with the carbon chain length. EE% was higher than 80% in C12- and C14-derivatives. The effect of liposome encapsulation on the cytotoxic activity of the complexes was found to depend on the carbon chain length. These data indicate that the highest drug bioavailability from liposome formulations was achieved with the complex showing intermediate carbon chain length and partition between the liposome membrane and aqueous phase.

A influência da nucleobase (uracila, U; citosina, C; adenina, A; guanina, G) nas propriedades físico-químicas e atividades biológicas in vitro dos complexos SbV-ribonucleosídeo foi investigada. Os complexos 1:1 Sb-U e Sb-C foram obtidos no estado sólido e caracterizados por RMN, ESI-MS e análise elementar. As constantes de estabilidade e as constantes aparentes de velocidade de formação e dissociação dos complexos 1:1 Sb-U, Sb-C e Sb-A foram determinadas. Embora o SbV se ligue através dos mesmos átomos de oxigênio das posições 2' e 3' nos diferentes nucleosídeos, as mudanças conformacionais na ribose e as propriedades físico-químicas do complexo dependem da nucleobase. A nucleobase influenciou fortemente as atividades citotóxica e leishmanicida dos complexos SbV-ribonucleosídeo. Os complexos SbV-nucleosídeos purínicos foram mais citotóxicos e eficazes contra a Leishmania chagasi do que os pirimidínicos, o que reforça o modelo de que a interação do SbV com os nucleosídeos purínicos pode mediar a atividade dos medicamentos antimoniais.

The influence of the nucleobase (uracyl, U; cytosine, C; adenine, A; guanine, G) on the physicochemical characteristics and in vitro biological activities of SbV-ribonucleoside complexes has been investigated. The 1:1 Sb-U and Sb-C complexes were characterized by NMR and ESI-MS spectroscopies and elemental analysis. The stability constant and the apparent association and dissociation rate constants of 1:1 SbV-U, SbV-C and SbV-A complexes were determined. Although SbV most probably binds via oxygen atoms to the same 2' and 3' positions in the different nucleosides, the ribose conformational changes and the physicochemical characteristics of the complex depend on the nucleobase. The nucleobase had a strong influence on the cytotoxicity against macrophages and the antileishmanial activity of the SbV-ribonucleoside complexes. The SbV-purine complexes were more cytotoxic and more effective against Leishmania chagasi than the SbV-pyrimidine complexes, supporting the model that the interaction of SbV with purine nucleosides may mediate the antileishmanial activity of pentavalent antimonial drugs.

Como o antimônio é a espécies ativa no medicamento leishmanicida, antimoniato de meglumina (AM), o conhecimento da concentração exata do metal é crítico para o seu uso experimental e clínico. Por outro lado, o papel da cisteína no metabolismo dos antimoniais deve ser esclarecido. No presente trabalho, a reação de redução do SbV pela cisteína foi caracterizada cineticamente. As ordens de reação com relação ao Sb e à cisteína foram iguais a 1,0 e 3,3, respectivamente. A constante de velocidade da reação de redução foi dependente do pH. Essa reação foi usada para reduzir o SbV no AM, para subseqüente determinação fotométrica do SbIII com o cromóforo vermelho de bromopirogalol. Eficiências de redução de 100% foram alcançadas em pH 3. Soluções apresentando concentrações de Sb baixas como 0,5 mmol L-1 foram dosadas com sucesso. O método foi também aplicado para a determinação de Sb em formulações lipossomais do AM.

Since antimony is the active species in the antileishmanial drug meglumine antimoniate (MA), the knowledge of the exact metal concentration is critical for its experimental and clinical use. On the other hand, the involvement of thiols, such as cysteine (Cys), in the metabolism of this drug remains to be clarified. In the present work, the reduction reaction of SbV by Cys was kinetically characterized. The reaction orders with respect to Sb and Cys were equal to 1.0 and 3.3, respectively. The rate constant for the reduction reaction was pH-dependent. This reaction was exploited to effectively reduce SbV in MA, for the subsequent photometric determination of SbIII using the chromogen bromopyrogallol red. Reduction efficiencies of 100% were achieved at pH 3. Solutions with Sb concentration as low as 0.5 mmol L-1 were successfully assayed. The method was also applied to the determination of Sb in liposomal formulations of MA.

The use of organoantimonial complexes in the therapeutic of leishmaniasis and schistosomiasis has been limited mainly by the need for daily parenteral administration, their adverse side-effects and the appearance of drug resistance. Liposome encapsulation has been so far the most effective means to improve the efficacy of pentavalent antimonials against visceral leishmaniasis. Pharmacologically- and pharmaceutically-acceptable liposomal compositions are still being investigated through manipulation of preparation method, lipid composition and vesicle size. Recently, the encapsulation of a trivalent antimonial within "stealth" liposomes was found to reduce its acute toxicity and effectively deliver this compound to the parasite in experimental schistosomiasis.

Magnetic liposomes are spherical vesicles containing magnetic particles. Their practical interest relies in their potential use as "intelligent" drug delivery systems, with magnetic driving or targeting possibilities. In the presence of a field, the vesicles become magnetized and interact. A strong interaction leads to aggregation and could in extreme lead to undesirable clogging in physiological conditions. We investigated the formation of chain aggregates in liposome dispersions in the presence of magnetic fields by light scattering measurements. Data analysis showed that aggregation number increased linearly with elapsed time, as for ordinary isotropic colloid diffusion limited aggregation. The rate of aggregation increased with the field also linearly for low field strengths, and saturates at higher fields. These results are in contrast with measurements for the pure magnetic fluid, i.e., non-encapsulated magnetic particle dispersions.

Distinct Toxoplasma gondii antigens were entrapped within liposomes and evaluated for their ability to protect Balb/c mice against congenital transmission: soluble tachyzoite antigen (L/STAg), soluble tissue cyst antigen (L/SCAg), soluble tachyzoite plus tissue cyst (L/STCAg) or purified 32kDa antigen of tachyzoite (L/pTAg). Soluble tachyzoite antigen alone in PBS (STAg) or emulsified in Freund's Complete Adjuvant (FCA/STAg) was also evaluated. Dams were inoculated subcutaneously with these antigens 6, 4 and 2 weeks prior to a challenge with four tissue cysts of the P strain of T. gondii orally between 10 and 14 days of pregnancy. Significant diminution differences were observed between the frequency of infected pups born of the dams immunized with the antigens incorporated into liposomes and that of pups born of the dams immunized with antigen emulsified in FCA or non immunized group (p<0.05). There was a significant decrease in the number of pups born dead in the groups L/STAg, L/SCAg and L/pTAg when compared with pups from all other groups (p <0.05). All dams immunized with or without adjuvant showed an antibody response and a proliferation of T-cells. However, no correlation was found between immune response and protection against the challenge.

Avaliação da eficácia esquistossomicida da oxamniquina encapsulada em liposomas. Oxamniquina (OXA) foi encapsulada em vesículas unilamelares pequenas de distearoilfosfatidilcolina usando-se técnica de encapsulação ativa em gradiante de pH. Este procedimento produziu uma alta eficiência de encapsulação (> 85%) com uma razão molar de 1/10, além de reter, eficientemente, a droga encapsulada sob condições de diálise à 37º C. OXA encapsulada (LOXA), OXA livre (OXA), (10 mg/kg respectivamente) ou liposomas vazios foram testados durante o curso da infecção experimental pelo Schistosoma mansoni. Verificou-se uma redução significativa do número de parasitos recuperados no grupo tratado com LOXA, por via subcutânea 1 dia antes ou 3, 7, ou 14 dias após a infecção, quando comparado aos grupos controle, lipossomas vazios e OXA. LOXA não foi efetiva quando administrada 7 dias antes ou 35 dias após a infecção. OXA livre (OXA) apresentou atividade em relação ao grupo controle quando administrada 3 dias após a infecção. O efeito máximo de LOXA, com 97% de redução da carga parasitária, foi observado quando administrada um dia antes da infecção. Por outro lado, não foi observada redução do número de parasitos quando LOXA foi inoculada intraperitonealmente 1 dia antes da infecção. Pode-se concluir que LOXA é mais efetiva que OXA no tratamento da esquistossomose experimental quando administrada subcutaneamente em período próximo à infecção

Oxamniquine (OXA) was sucessfully encapsulated in small unilamellar vesicles using a pH gradient method. This procedure led to a high drug encapsulation efficiency (> 85%) at a drug to lipid molar ratio of 1/10. Moreover, these liposomes were found to retain encapsulated OXA efficciently under dialysis conditions at 37º C. Liposome-entrapped OXA (LOXA), OXA, and empty liposomes were tested against Schistosoma mansoni in a murine model. LOXA produced a significant reduction of the worm burden compared to the other preparations, when inoculated by subcutaneous route (s.c.) with 10 mg OXA/kg animal one day before the infection, and 3, 7, and 14 days after. However, LOXA was not effective when given 7 days before, or 35 days after infections. OXA, in the free form, was effective in relation to the untreated group, only when administered 3 days after the infection. Maximum effect of LOXA, with 97% reduction of the parasite number, was observed when the preparation was given s.c.one day before the infection. On the other hand, LOXA inoculated intraperitoneally one day before the infection didn’t show any reduction of the parasite count. It can be concluded that LOXA is more effective than OXA for the treatment of experimental schistosomiasis, particularly when administered subcutaneously at a time close to the infection