Maria Jania Teixeira

In vitro and in vivo Leishmanicidal activity of 2‐hydroxy‐3‐(3‐methyl‐2‐butenyl)‐1,4‐naphthoquinone (lapachol)

This study aims to evaluate the in vitro and in vivo leishmanicidal activity of lapachol, a naphthoquinone found in the seeds and heartwood of certain tropical plants, and to compare its efficacy with a reference drug, sodium stibogluconate (Pentostam®). These compounds (0.0125–4.0 mg/mL) were evaluated in vitro against intracellular amastigotes of Leishmania (Viannia) braziliensis (LVb), then tested in an animal model (hamster) to try to reproduce the leishmanicidal activity. In vitro, lapachol exhibited an anti‐amastigote effect, whereas in vivo it did not prevent the development of LVb‐induced lesions at an oral dose of 300 mg/kg/day for 42 days. Pentostam® demonstrated a significant anti‐amastigote effect in vitro for LVb and apparent clinical cure in vivo (60 mg/kg/day). However, it could not completely eradicate parasites from the tissues of infected animals. The observation that lapachol exerts leishmanicidal activity in vitro without offering significant protection against LVb‐infected lesions in hamsters suggests that lapachol in vivo might possibly inhibit the microbicidal functioning of macrophages. Alternatively, it might be transformed into an inactive metabolite(s) or neutralized, losing its leishmanicidal activity. It is also possible that an optimal and sustained plasma level of the drug could not be achieved at the dose used in this study. Copyright

Controle do calazar canino: comparação dos resultados de um programa de eliminação rápida de cães sororreagentes por ensaio imuno-enzimático com outro de eliminação tardia de cães sororreagentes por teste de imunofluorescência indireta de eluato de papel filtro

The kala-azar control program, adopted by the Fundação Nacional de SaúdeFNS (National Health Foundation) has not been able to reduce to an acceptable level the incidence of human cases. The diagnostic method utilized is a blood eluate immunofluorescence. A dogs diagnosed as infected is eliminated a mean of eighty days after the blood collection. The low sensitivity of the test used and the continuing residence of the infected dog in the region due to the elimination delay may be critical in the lack of success of this program. In this study, the FNS standard canine control method is compared to a strategy based on ELISA identification of infected dog and elimination within 7 days. In both study areas the canine seroprevalence was noted ten months before and ten months after the intervention. In the routine FNS area a 9% decrease in seroprevalence was noted, compared to statistically significant greater 27%, reduction (p = 0.0015) in the ELISA intervention area. Key-words: Visceral leishmaniasis. Kala-azar. Epidemiologic control. Reservoir. Canine kala-azar control: aftermath comparison of a fast deletion program of serum-reactive dogs by immuno-enzimatic essay with another of late deletion program of serum-reactive dogs by indirect immunofluorescence of filter paper eluate Marcus Davis Machado Braga, Ivo Castelo Branco Coêlho, Margarida Maria Lima Pompeu, Thomas G. Evans, Isabel Tavares MacAullife, Maria Jania Teixeira e José Wellington de Oliveira Lima Revista da Sociedade Brasileira de Medicina Tropical 31:419-424, set-out, 1998. 420 No Brasil a leishmaniose visceral é causada pela Leishmania chagasi 6 22, e transmitida ao homem através da picada de um psicodídeo, a Lutzomya longipalpis 6 9 13 14. Duas espécies de mamíferos já foram incriminadas como reservatório deste parasita, uma silvestre, a raposa12, e outra doméstica, o cão1 2 3 10 11. A leishmaniose visceral humana e canina são endêmicas no Brasil, principalmente no Nordeste6 18. Nesta região, e mais especificamente, no Estado do Ceará, em humanos a doença apresenta uma prevalência de 10,08/100.000 habitantes17. O Ministério da Saúde do Brasil, desde o início da década de 60, através da então Superintendência de Campanhas de Saúde Pública (SUCAM), e mais recentemente, da Fundação Nacional de Saúde (FNS), vem desenvolvendo atividades de controle da infecção canina, em vários estados da Federação, incluindo o Ceará2 20. Este controle inclui medidas para diminuir a densidade populacional do vetor, e a identificação e eliminação de cães infectados6 20 22. Ao longo destes anos, em função da necessidade de se utilizar técnicas cada vez mais sensíveis, diferentes métodos de identificação de cães infectados têm sido propostos. Inicialmente, foi utilizado o exame parasitológico da pele e/ou vísceras3, outra técnica utilizada foi a detecção de anticorpos antileishmania, por reação de fixação do complemento8. A partir de 1982, vêm-se usando a técnica de imunofluorescência realizada em eluato de sangue colhido em papel filtro6 22. Embora a introdução da imunofluorescência ainda represente um grande avanço, esta técnica, quando realizada em eluato de papel filtro, apresenta uma sensibilidade muito baixa, quando comparada com ELISA realizada no soro4 15. Após 5 anos de atividades de controle do reservatório canino, utilizando a imunofluorescência no eluato para detectar cães infectados, observou-se que é possível reduzir a prevalência da infecção no cão até certo limite, em torno de 0,5 a 1%. Esta redução, no entanto, não se acompanha necessariamente de uma interrupção da transmissão ao homem21. Acreditamos que, entre outros fatores, a baixa sensibilidade do teste de IFI no eluato, e o tempo decorrido entre a coleta de sangue e a eliminação do cão infectado, sejam responsáveis pela permanência de cães infectados e pela manutenção da transmissão da infecção. Neste trabalho, avalia-se o impacto que a eliminação precoce de cães infectados, detectados por uma técnica de diagnóstico mais sensível, possa ter na prevalência da infecção do cão pela L. chagasi, comparando esta estratégia com aquela adotada pelo programa de controle da leishmaniose visceral, na qual cães infectados são detectados pela IFI no eluato e eliminados, em média, 80 dias depois.The kala-azar control program, adopted by the Fundacao Nacional de Saude- FNS (National Health Foundation) has not been able to reduce to an acceptable level the incidence of human cases. The diagnostic method utilized is a blood eluate immunofluorescence. A dogs diagnosed as infected is eliminated a mean of eighty days after the blood collection. The low sensitivity of the test used and the continuing residence of the infected dog in the region due to the elimination delay may be critical in the lack of success of this program. In this study, the FNS standard canine control method is compared to a strategy based on ELISA identification of infected dog and elimination within 7 days. In both study areas the canine seroprevalence was noted ten months before and ten months after the intervention. In the routine FNS area a 9% decrease in seroprevalence was noted, compared to statistically significant greater 27%, reduction (p = 0.0015) in the ELISA intervention area.The kala-azar control program, adopted by the Fundação Nacional de Saúde-FNS (National Health Foundation) has not been able to reduce to an acceptable level the incidence of human cases. The diagnostic method utilized is a blood eluate immunofluorescence. A dogs diagnosed as infected is eliminated a mean of eighty days after the blood collection. The low sensitivity of the test used and the continuing residence of the infected dog in the region due to the elimination delay may be critical in the lack of success of this program. In this study, the FNS standard canine control method is compared to a strategy based on ELISA identification of infected dog and elimination within 7 days. In both study areas the canine seroprevalence was noted ten months before and ten months after the intervention. In the routine FNS area a 9% decrease in seroprevalence was noted, compared to statistically significant greater 27%, reduction (p = 0.0015) in the ELISA intervention area.

Saliva from Lutzomyia longipalpis Induces CC Chemokine Ligand 2/Monocyte Chemoattractant Protein-1 Expression and Macrophage Recruitment

Saliva of bloodfeeding arthropods has been incriminated in facilitating the establishment of parasite in their host. We report on the leukocyte chemoattractive effect of salivary gland homogenate (SGH) from Lutzomyia longipalpis on saliva-induced inflammation in an air pouch model. SGH (0.5 pair/animal) was inoculated in the air pouch formed in the back of BALB/c or C57BL/6 mice. L. longipalpis SGH induced a significant influx of macrophages in BALB/c but not in C57BL/6 mice. SGH-induced cell recruitment reached a peak at 12 h after inoculation and was higher than that induced by the LPS control. This differential cell recruitment in BALB/c mice was directly correlated to an increase in CCL2/MCP-1 expression in the air pouch lining tissue. In fact, treatment with bindarit, an inhibitor of CCL2/MCP-1 synthesis, and also with a specific anti-MCP-1 mAb resulted in drastic reduction of macrophage recruitment and inhibition of CCL2/MCP-1 expression in the lining tissue. CCL2/MCP-1 production was also seen in vitro when J774 murine macrophages were exposed to L. longipalpis SGH. The SGH effect was abrogated by preincubation with serum containing anti-SGH IgG Abs as well as in mice previously sensitized with L. longipalpis bites. Interestingly, the combination of SGH with Leishmania chagasi induced an increased recruitment of neutrophils and macrophages when compared with L. chagasi alone. Taken together these results suggest that SGH not only induces the recruitment of a greater number of macrophages by enhancing CCL2/MCP-1 production but also synergizes with L. chagasi to recruit more inflammatory cells to the site of inoculation.

Differences in gamma interferon production in vitro predict the pace of the in vivo response to Leishmania amazonensis in healthy volunteers

ABSTRACT The initial encounter of Leishmania cells and cells from the immune system is fundamentally important in the outcome of infection and determines disease development or resistance. We evaluated the anti-Leishmania amazonensis response of naive volunteers by using an in vitro priming (IVP) system and comparing the responses following in vivo vaccination against the same parasite. In vitro stimulation allowed us to distinguish two groups of individuals, those who produced small amounts of gamma interferon (IFN-γ) (n = 16) (low producers) and those who produced large amounts of this cytokine (n = 16) (high producers). IFN-γ production was proportional to tumor necrosis factor alpha and interleukin 10 (IL-10) levels but did not correlate with IL-5 production. Volunteers who produced small amounts of IFN-γ in vitro remained low producers 40 days after vaccination, whereas high producers exhibited increased IFN-γ production. However, 6 months after vaccination, all individuals tested produced similarly high levels of IFN-γ upon stimulation of their peripheral blood mononuclear cells with Leishmania promastigotes, indicating that low in vitro producers respond slowly in vivo to vaccination. In high IFN-γ producers there was an increased frequency of activated CD8+ T cells both in vitro and in vivo compared to the frequency in low producers, and such cells were positive for IFN-γ as determined by intracellular staining. Such findings suggest that IVP responses can be used to predict the pace of postvaccination responses of test volunteers. Although all vaccinated individuals eventually have a potent anti-Leishmania cell-mediated immunity (CMI) response, a delay in mounting the CMI response may influence resistance against leishmaniasis.

Distinct Leishmania braziliensis Isolates Induce Different Paces of Chemokine Expression Patterns

ABSTRACT Inflammatory events during Leishmania braziliensis infection in mice were investigated. Large lesions were directly correlated with the inflammatory reaction but not with parasite burden. Different L. braziliensis strains induce different paces of chemokine expression patterns, leading to diverse cell recruitment and differential inflammatory responses.

1,8‐Cineole protects against liver failure in an in‐vivo murine model of endotoxemic shock

The effects of 1,8‐cineole on d‐galactosamine/lipopolysaccharide (GalN/LPS)‐induced shock model of liver injury was investigated in mice. The co‐administration of GalN (700 mg kg−1, i.p.) and LPS (5 μg kg−1, i.p.) greatly elevated serum concentrations of tumour necrosis factor‐α (TNF‐α), alanine aminotransferase and aspartate aminotransferase, and induced massive hepatic necrosis and lethality in 100% of control mice. Pretreatment with 1,8‐cineole (400 mg kg−1, p.o.) and dexamethasone (1 mg kg−1, s.c.),60 min before GalN/LPS, offered complete protection (100%) against the lethal shock and acute elevation in serum TNF‐α and serum transaminases. Hepatic necrosis induced by GalN/LPS was also greatly reduced by both 1,8‐cineole and dexamethasone treatment. The results indicate that 1,8‐cineole protects mice against GalN/LPS‐induced liver injury through the inhibition of TNF‐α production, and suggest that 1,8‐cineole may be a promising agent to combat septic‐shock‐associated pathologies.

The safety of oral use of L-glutamine in middle-aged and elderly individuals.

OBJECTIVE To evaluate the safety of nutraceutical oral administration of L-glutamine (L-Gln) in middle-aged and elderly individuals. METHODS In this randomized, crossover, double-blind clinical study, 30 residents of a long-term-care institution, selected according to a modified SENIEUR protocol (Working Party of the EURAGE Concerted Action Programme on Ageing of the European Community), were studied. Fourteen subjects received orally 0.5 g kg(-1) d(-1) of L-Gln and 16 received calcium caseinate for 14 d, followed by a 5-d washout. Supplements were switched for the second 14-d trial. Laboratory tests for hepatic and renal functions and ammonemia were performed and the estimated glomerular filtration rate (eGFR) was calculated. RESULTS Of the 30 subjects, 16 were men, mean age was 69+/-8.8 y, average weight was 61.8+/-14.2 kg, and mean serum albumin was 4.0+/-0.3g/dL. Neither adverse clinical effects nor clinically significant laboratory changes were noted during L-Gln supplementation. There was no difference in ammonemia between the groups. There were statistically but not clinically significant increases in plasma urea nitrogen and creatinine concentrations. There was no significant decrease in eGFR during calcium caseinate supplementation (-2.9%). The eGFR decreased significantly after L-Gln supplementation (-13.3%) but well below the 25% limit for biologic significance. CONCLUSION Increases in serum urea nitrogen and creatinine and decrease in eGFR are probably due to difficulties by older kidneys in metabolizing the supplemented protein sources. Although not clinically significant, those alterations impose a rigorous control on the evaluation parameters of renal function during oral L-Gln supplementation, with doses of 0.5 g kg(-1) d(-1) in middle-aged and elderly individuals.

Effect of Lutzomyia whitmani (Diptera: Psychodidae) salivary gland lysates on Leishmania (Viannia) braziliensis infection in BALB/c mice

Previous reports showed that Lutzomyia longipalpis saliva exacerbate Leishmania braziliensis infection in mice. The sand fly Lu. whitmani is one of the vectors of L. (Viannia) braziliensis (LVb), a causative agent of cutaneous leishmaniasis in the State of Ceará, Brazil. To determine whether saliva of Lu. whitmani could increase the infectivity of LVb in mice, we inoculated groups of BALB/c mice with LVb promastigotes in the presence or absence of the salivary glands lysate from Lu. whitmani. We found that coinjection with Lu. whitmani saliva increased size but not longevity of cutaneous LVb lesions in BALB/c mice, since the formed lesions gradually resolved. The mechanism(s) by which Lu. whitmani saliva might exacerbate LVb infection in BALB/c mice is speculated.

Leishmania infantum Infection in Blood Donors, Northeastern Brazil

To the Editor: Leishmania infantum is endemic to northeastern Brazil. It is responsible for visceral leishmaniasis (VL), a major emerging health problem in urban areas. Transmission occurs predominantly by the Lutzomyia longipalpis sand fly, but transfusion-associated VL, caused by L. infantum, has been reported from southern Europe and, by L. donovani, on the Indian subcontinent (1,2). Most L. infantum infections are asymptomatic (3), raising concern that the parasite could be present in donated blood from otherwise healthy residents in areas to which it is endemic (4). VL caused by L. infantum is endemic to 20 of Brazil’s 27 states; an annual average of 3,553 cases occur nationwide, with 54% of all cases reported from Brazil’s northeastern region. The state of Ceara historically ranks first or second in number of cases; an annual average of 467 cases were reported during the last decade (5). Thirty-eight percent of cases were reported from Fortaleza, the capital, where 28.4% of the state’s population resides. Over a 10-year-period, 277 (7.8%) persons with VL have died, and 109 (39%) of VL-related deaths have occurred in Fortaleza. Sixty-nine percent of blood donors for Ceara reside in Fortaleza. To determine the prevalence of Leishmania infection among healthy blood donors, we tested blood donated to the State of Ceara Public Blood Bank. Compulsory serologic testing was also done for Trypanosoma cruzi (Chagas disease), hepatitis B and C, Treponema pallidum (syphilis), human T-cell lymphotropic virus types 1 and 2, and HIV-1 and -2. In the blood bank, 60% of units are centrifuged to separate the buffy coat in preparation for platelet separation. During May–November 2011, we randomly selected 431 buffy coats and tested them for Leishmania spp. by ELISA and PCR. To separate plasma from cells, 10 mL of buffy coat was centrifuged in Ficoll-Hypaque (Histopaque −1077, Sigma-Aldrich, Sao Paulo, Brazil). We tested plasma for leishmanial IgG by ELISA using a modified protocol of Evans et al. (6). IOC/L2906 L. infantum strain (MHOM/BR/2002/LPC-RPV) was used as the source of promastigote antigens. In addition, DNA was extracted from the mononuclear cell preparation, and PCR was performed with primers 150 (5′-GGG[G/T]AGGGGCGTTCT[G/C]CGAA-3′) and 152 (5′-[G/C][G/C][G/C][A/T]CTAT[A/T]TTACACCAACCCC-3′) that target the 120-bp conserved region of the Leishmania kDNA minicircle present in all Leishmania spp (7). As a positive control, kDNA was extracted from L. amazonensis promastigotes, strain BA-125 (MHOM/BR/87), characterized by PCR and isoenzymes (8). All PCR-positive samples were purified and sent to Ludwig Biotec (Alvorada, Brazil) for sequencing by ACTGENE-Molecular Analysis. The Federal University of Ceara Ethics Committee approved this study. Buffy coats from 57 (13.2%) serum samples from 431 donors were positive for leishmanial IgG, and 20 (4.6%) were positive for Leishmania spp. DNA. Sequencing of all PCR-positive samples confirmed the Leishmania genus. Three donors tested positive by both ELISA and PCR. Overall, the prevalence of leishmanial infection was 17.1% of blood donors. Eighty of the 431 units tested positive for >1 of compulsorily screened infections and were rejected. Of the remaining 351 that were negative for co-infection, 43 (12.2%) were positive for leishmanial IgG and 15 (4.3%) for Leishmania spp. DNA. Two donors were positive for both by ELISA and PCR. The prevalence of Leishmania infection among blood units accepted for transfusion was 16%. The results demonstrate a surprisingly high prevalence of Leishmania infection in blood donors in Fortaleza, several times higher than that other diseases for which blood is screened (Figure). In a recent study in Salvador, Brazil (9), 5.4% of blood donors had leishmanial antibodies, of which 68% were positive by its PCR targeting kDNA amplification. Figure Comparison of the prevalence of Leishmania infantum as tested by PCR and ELISA and of other infections compulsorily tested in 431 blood donors in Fortaleza, state of Ceara, northeastern Brazil. HBV, hepatitis B virus; HCV, hepatitis C virus; HTLV, ... The percentages of antibody- or PCR-positive units capable of transmitting Leishmania and the outcomes are unknown. Viable Leishmania might not be in the blood of all PCR-positive donors, and even when present, the inoculum might be reduced by removal of infected circulating mononuclear phagocytes in the buffy coat, or parasites might be affected by steps involved in preparation or storage. However, if we consider units that test positive by PCR as being potentially infectious, the number of recipients at risk is of substantial concern. For example, in 2011, there were 99,933 blood donations to the State of Ceara Public Blood Bank. After compulsory screening for the other bloodborne pathogens, 93,238 units were accepted for transfusion. Extrapolating from the PCR-positive rate of 4.3%, a total of 4,009 recipients possibly were exposed to infection. Further studies are needed to determine whether recipients of blood from donors who are PCR positive and/or leishmanial antibody positive become infected with L. infantum. Persons with advanced AIDS or other immunosuppressive conditions seemingly would be at greatest risk for VL. In Brazil, legislation requires that all blood for transfusion be tested for T. cruzi ., hepatitis B and C, T. pallidum, human T-cell lymphotropic virus types 1 and 2, and HIV-1 and -2. As additional information becomes available, screening for L. infantum also might be advisable to reduce the possibility of the recipient becoming infected, developing VL, and possibly being a reservoir of infection in the community (10), particularly in Ceara and other regions where the prevalence of L. infantum infection is high.

DISTINCT CELLULAR MIGRATION INDUCED BY Leishmania infantum chagasi AND SALIVA FROM Lutzomyia longipalpis IN A HEMORRHAGIC POOL MODEL

Recruitment of a specific cell population after Leishmania infection can influence the outcome of the disease. Cellular migration in response to Leishmania or vector saliva has been reported in air pouch model, however, cellular migration induced by Leishmania associated with hosts blood and vector saliva in this model has not been described. Herein we investigated cellular migration into air pouch of hamster after stimulation with combination of L. chagasi and hosts blood and Lutzomyia longipalpis saliva. Migration induced by saliva was 3-fold more than those induced by L. chagasi alone. Additionally, L. chagasi associated with blood and saliva induced significantly even more leukocytes into air pouch than Leishmania alone. L. chagasi recruited a diverse cell population; however, most of these cells seem to have not migrated to the inflammatory exudate, remaining in the pouch lining tissue. These results indicate that L. chagasi can reduce leukocyte accumulation to the initial site of infection, and when associated with vector saliva in the presence of blood components, increase the influx of more neutrophils than macrophages, suggesting that the parasite has developed a strategy to minimize the initial inflammatory response, allowing an unlimited progression within the host. This work reinforces the importance of studies on the salivary components of sand fly vectors of leishmaniasis in the transmission process and the establishment of the infection.