Calophyllum brasiliense Modulates the Immune Response and Promotes Leishmania amazonensis Intracellular Death.

Calophyllum brasiliense is a plant from the Brazilian rain forests and has been used in folk medicine for the treatment of various diseases, including leishmaniasis. This infectious disease depends on the Leishmania sp. and the host immune response. C. brasiliense antileishmanial activity is well known, but the effects on immune response remain to be investigated. This study showed the leishmanicidal and immunomodulatory effects of a 30 µg/mL of hydroalcoholic extract of C. brasiliense in murine macrophages before and after Leishmania (Leishmania) amazonensis infection. The semiquantitative cytokine RNA expression was determined by RT-PCR and the anti-Leishmania activity was measured by infection index (IF). Hydroalcoholic extract of C. brasiliense reduced more than 95% of IF when used before and after Leishmania infection, with 3 and 24 h of treatment (p < 0.05). C. brasiliense inhibited or reduced significantly (p < 0.05) the TNF-α, IL-1ß, IL-18, and IL-10 mRNA expression. The antileishmanial and anti-inflammatory effects showed the potential of C. brasiliense as an alternative therapy for leishmaniasis and it must be investigated.

Association between HLA-C*04 and American cutaneous leishmaniasis in endemic region of southern Brazil.

Leishmaniasis is a parasitic infectious disease with global repercussions. American cutaneous leishmaniasis (ACL) is endemic in southern Brazil and its pathogenesis varies according to parasite species, immune response, and host genetics. In terms of immunogenetics, many host genes, including HLA (human leukocyte antigen), could be involved in susceptibility to and protection against ACL. Accordingly, the aim of this study was to investigate the association between HLA class I genes (HLA-A, -B, and -C) and ACL in an endemic region of southern Brazil. The allele frequencies of 186 patients diagnosed with ACL and 278 healthy individuals were compared. HLA class I (HLA-A, -B, and -C) typing was carried out by PCR-SSO using Luminex technology. The results revealed an association between the HLA-C*04 allele and the patient study group, in which it appeared more frequently than in the control group [21.5 vs 13.49% (P = 0.0016 and Pc = 0.0258; OR = 1.7560; 95%CI = 1.2227-2.5240)], thereby suggesting an increased susceptibility to ACL. Additional allelic groups such as HLA-A*02, HLA-B*35, HLA-B*45, HLA-C*01, and HLA-C*15 were also implicated; however, further investigation is necessary to confirm their association with ACL. Therefore, the results obtained in this study demonstrate the involvement of HLA class I genes in the susceptibility or resistance to ACL, with significant association between HLA-C*04 and ACL susceptibility.

Immune Responses in Broiler Chicks Fed Propolis Extraction Residue-supplemented Diets.

This study was conducted to evaluate the effect of inclusion of propolis extraction residue in the feed of broilers from 1 to 21 d of age on phagocytic activity of macrophages, cutaneous basophil hypersensitivity response to phytohemagglutinin, antibody production against Newcastle disease, lymphoid organ weight and hematological profile and to determine the optimal level of inclusion. 120 chicks, reared in metabolism cages until 21 days of age, were distributed in a completely randomized design, with five treatments (0%, 1%, 2%, 3%, and 4% of propolis residue) and six replications. The relative weight of thymus and monocyte percentage were affected by propolis residue, with a quadratic response (p<0.05) and lowest values estimated at 2.38% and 2.49%, respectively. Changes in relative weight of cloacal bursa and spleen, percentage of lymphocyte, heterophil, basophil, eosinophil, and heterophil:lymphocyte ratio, antibody production against Newcastle disease, phagocytic activity of macrophages and the average number of phagocytosed erythrocytes were not observed. The nitric oxide production with regard to positive control (macrophages+erythrocytes) decreased linearly (p<0.05) with increased doses of propolis residue. The remaining variables of nitric oxide production (negative control - macrophages, and difference between the controls) were not affected by propolis residue. The cutaneous basophil hypersensitivity response to phytohemagglutinin as determined by the increase in interdigital skin thickness exhibited a quadratic response (p<0.05), which predicted a lower reaction response at a dose of 2.60% of propolis residue and highest reaction response after 43.05 hours of phytohemagglutinin injection. The inclusion of 1% to 4% of propolis extraction residue in broiler diets from 1 to 21 days of age was not able to improve the immune parameters, despite the modest changes in the relative weight in thymus, blood monocyte percentage, nitric oxide concentration, and interdigital reaction to phytohemagglutinin.

In vitro effects of lapachol and ß-lapachone against Leishmania amazonensis.

Leishmaniasis is a neglected disease that affects millions of people worldwide, and special attention should be given to treatment because the available drugs have limitations, which can lead to low therapeutic adherence and parasitic resistance. This study evaluated the activity of the bioactive naphthoquinones, lapachol and ß-lapachone, against Leishmania amazonensis. The cell alterations were evaluated in vitro on promastigote and amastigote forms. The lethal dose (LD50) at 24, 48, and 72 h on the promastigote's forms using lapachol was 75.60, 72.82, and 58.85 µg/mL and for ß-lapachone was 0.65, 1.24, and 0.71 µg/mL, respectively. The naphthoquinones significantly inhibited the survival rate of L. amazonensis amastigotes at 83.11, 57.59, and 34.95% for lapachol (82.28, 41.14, and 20.57 µg/mL), and 78.49, 83.25, and 80.22% for ß-lapachone (3.26, 1.63, and 0.815 µg/mL). The compounds on the promastigote's forms led to the loss of mitochondrial membrane potential, induced changes in the integrity of the membrane, caused damage to cells suggestive of the apoptotic process, and showed inhibition of tumor necrosis factor (TNF)-α and interleukin (IL)-6 production. The results showed that these naphthoquinones are promising candidates for research on new drugs with anti-Leishmania activity derived from natural products.

Leishmania (Viannia) braziliensis: New primers for identification using polymerase chain reaction.

The objective of this study was to develop specific primers for Leishmania (Viannia) braziliensis species identification using PCR. The designed primers (LBF1 and LBR1) were evaluated for sensitivity and specificity using various L. (V.) braziliensis serodemes and various Leishmania species and also using Trypanosoma cruzi. A specific fragment of 536bp was detected from 50ng of DNA in a crude extract derived from L. (V.) braziliensis. The DNA fragment was not detected when DNA from other Leishmania species or from T. cruzi was used as template in the PCR. Furthermore, when tested with DNA from cutaneous leishmaniasis the designed primers and reaction gave positive results. Taking into consideration that the primers LBF1 and LBR1 could specifically identify L. (V.) braziliensis, they could be considered for use in L. (V.) braziliensis diagnosis and epidemiological studies.

Predominance of Nyssomyia neivai (Pinto) on the Islands of the Paraná River, Southern Brazil.

Leishmaniasis is a vector-borne disease caused by the protozoan Leishmania and it is one of the six most important infectious diseases in the world. The aim of this study was to conduct a research on the fauna and most frequented ecotopes by sandflies on islands of the Paraná River. The sandflies were collected with Falcão light traps in 10 islands in the municipalities of São Pedro do Paraná, Porto Rico, and Querência do Norte, in Paraná State, Brazil, from November 2012 to November 2014. In total, 76,145 specimens of Nyssomyia neivai, the only species found, were collected, of which 63,015 were female and 13,130, male. The largest number of sandflies was collected mainly in domiciles and peridomiciles. The islands of the Paraná River showed predominance of Ny. neivai, which is largely involved in the transmission of Leishmania and a risk of human leishmaniasis to the island inhabitants and tourists.

Phototoxic effect of aluminium-chlorine and aluminium-hydroxide phthalocyanines on Leishmania (l.) amazonensis.

This study investigated the activity of photosensitive phthalocyanines on promastigotes and amastigotes of Leishmania (L.) amazonensis. Aluminum phthalocyanine chloride (AlPcCl), Aluminum phthalocyanine hydroxide (AlPcOH) and zinc phthalocyanine (PcZn) were tested in the presence (matte red LED, potency of 2.5-2.3 µW for 30 min) and absence of light against L. amazonensis promastigotes and the parasite viability was evaluated after 24, 48 and 72 h. The amastigote forms were treated with AlPcCl and AlPcOH, following the same lighting protocols described for the promastigote forms, being evaluated after 24 h. Cytotoxicity to human erythrocytes and peritoneal macrophages was also evaluated. The results showed that AlPcCl and AlPcOH in the presence of light have antileishmania activity, with leishmanistatic effects on promastigotes and amastigotes of L. amazonensis, without causing cytotoxicity to peritoneal macrophages and erythrocytes. The concentrations that inhibited 50% of the promastigote forms after 24 h of light exposure were 0.21 ±â€¯0.08 µM for AlPcCl and 0.23 ±â€¯0.06 µM for AlPcOH. In 48 h and 72 h after the treatment, the IC50 of AlPcCl was 0.13 ±â€¯0.02 and 0.12 ±â€¯0.03 µM and for AlPcOH was 0.14 ±â€¯0.01 µM and 0.11 ±â€¯0.01 µM, respectively. PcZn showed no activity on promastigotes of L. amazonensis. This study showed a substantial photodynamic activity of the phthalocyanines AlPcCl and AlPcOH against intracellular amastigotes forms of L. amazonensis after irradiation, presenting IC50 values of 0.62 ±â€¯0.06 µM and 0.92 ±â€¯0.12 µM, respectively. These results support the possibility of using photodynamic therapy for the treatment of cutaneous leishmaniasis.

In vitro effects of lapachol and β-lapachone against Leishmania amazonensis

Leishmaniasis is a neglected disease that affects millions of people worldwide, and special attention should be given to treatment because the available drugs have limitations, which can lead to low therapeutic adherence and parasitic resistance. This study evaluated the activity of the bioactive naphthoquinones, lapachol and β-lapachone, against Leishmania amazonensis. The cell alterations were evaluated in vitro on promastigote and amastigote forms. The lethal dose (LD50) at 24, 48, and 72 h on the promastigote's forms using lapachol was 75.60, 72.82, and 58.85 μg/mL and for β-lapachone was 0.65, 1.24, and 0.71 μg/mL, respectively. The naphthoquinones significantly inhibited the survival rate of L. amazonensis amastigotes at 83.11, 57.59, and 34.95% for lapachol (82.28, 41.14, and 20.57 µg/mL), and 78.49, 83.25, and 80.22% for β-lapachone (3.26, 1.63, and 0.815 µg/mL). The compounds on the promastigote's forms led to the loss of mitochondrial membrane potential, induced changes in the integrity of the membrane, caused damage to cells suggestive of the apoptotic process, and showed inhibition of tumor necrosis factor (TNF)-α and interleukin (IL)-6 production. The results showed that these naphthoquinones are promising candidates for research on new drugs with anti-Leishmania activity derived from natural products.

Inhibition of macrophage invasion by monoclonal antibodies specific to Leishmania (Viannia) braziliensis promastigotes and characterisation of their antigens.

Monoclonal antibodies that specifically recognise Leishmania (Viannia) braziliensis promastigotes were produced and termed SST-2, SST-3 and SST-4. SST-2 recognises a conformational epitope present in a 24-28 kDa doublet and in a 72 kDa component, as verified by Western blotting. Indirect immunofluorescence showed that the antigen recognised by SST-2 is distributed homogeneously on the parasite surface. SST-3 recognises a flagellar glycoprotein of approximately 180 kDa. The reactivity of this mAb was abolished by sodium m-periodate treatment, indicating that SST-3 reacts with a carbohydrate epitope of the 180 kDa antigen. SST-4 recognises a conformational epitope of a 98 kDa antigen. SST-2, SST-3 and SST-4 were specific to L. (V.) braziliensis promastigote forms. Indirect immunofluorescence did not show reactivity of SST-2 or SST-3 with amastigotes of L. (V.) braziliensis, or with promastigotes of Leishmania (Viannia) panamensis, Leishmania (Viannia) guyanensis, Leishmania (Viannia) naiffi, Leishmania (Viannia) lainsoni, Leishmania (Leishmania) amazonensis, Leishmania (Leishmania) major, or Leishmania (Leishmania) chagasi. We also evaluated the involvement of SST-2, SST-3 and SST-4 antigens in parasite-macrophage interaction. Fab fragments of SST-3 and SST-4 significantly inhibited the infectivity of L. (V.) braziliensis promastigotes to mouse peritoneal macrophages.

Genetic variation in populations of Leishmania species in Brazil.

The population structure of strains of Leishmania (Viannia) braziliensis sensu lato from Pará State and Paraná State in Brazil, of L. (V.) shawi and L. (Leishmania) amazonensis from Pará State, and the relationships of type strains of the subgenera L. (Viannia) and L. (Leishmania) were examined by the random-amplified polymorphic deoxyribonucleic acid (RAPD) technique. Four different primers (M13-40, QG1, L15996 and delta gt11R) were used. The bands were analysed using the neighbor-joining (NJ) and unweighted pair-group method with arithmetic averages (UPGMA) algorithms of the MEGA package. The topology of the NJ and UPGMA trees was very similar but they were not always identical. Both trees differentiated the standard strains of the different species. Strains from the same location were grouped together only in the UPGMA phenogram of the M13-40 primer. L. (V.) braziliensis isolates from Paraná State were genetically closer to those from Paragominas, Pará State than to those from the Amazonian regions of Carajás in Pará State and Peru. The relationship was not dependent on geographical distance. It is postulated that the groups arose from different origins, in which the Amazonian stocks were related to Psychodopygus sand flies while the Paraná strains originated from a gene pool transmitted by Lutzomyia sand flies such as Lutzomyia (Nyssomyia) whitmani. Transmission by Ps. complexus in Paragominas is considered to be a secondary adaptation from the Lutzomyia leishmanial gene pool. Although the vectors of L. (V.) braziliensis are poorly known in the Amazon region, there is strong evidence that the major vectors are all Psychodopygus spp. There was a high degree of genetic variability amongst the L. (V.) shawi strains and there was no clear grouping according to the strains' origins. The genetic variability amongst L. (L.) amazonensis strains from the same locations was much lower but they formed 2 groups which coincided with their origin. Our results support the clonal population structure of Leishmania isolates and suggest that their distribution is related to the origin of the gene pool as well as to present vector and reservoir movements.

Leishmania braziliensis: isolation of carbohydrate-containing antigen and possibility of its use in the immunodiagnosis of American cutaneous leishmaniasis.

Leishmania braziliensis is a causative agent of American Cutaneous Leishmaniasis (ACL). The 034-JCG strain, isolated from a patient from the northern region of Paraná State, Brazil, was cultivated in Blood Agar Base medium, lyophilized and submitted to phenol-water extraction. The extract was treated with RNase I. The carbohydrate containing-antigen (Ag-CHO) was immunogenic to rabbits and showed at least a fraction with some negative charge at pH 8.2. This antigen showed cross-reactivity with the phenol-water extract of the growth medium used for the culture of promastigotes and with the surface antigens of promastigotes. Its composition is: 24.3% of total sugars, from which 11.2% of galactose, 7.5% of mannose and 5.6% of ribose. Protein content was 5.4% and phosphate 18.5%. The antigenic activity was maintained after: repeated freezing-thawing; lyophilization; heating at 100 degrees C for 30 minutes; treatment with RNase, trichloroacetic acid and sodium metaperiodate. The precipitin line obtained is Periodic Acid Schiff positive. The application of the Ag-CHO in counterimmunoelectrophoresis reaction for the immunodiagnosis of ACL showed 60% sensitivity, and no cross-reaction with the five sera of Chagas' disease patients tested. The use of this antigen in a more sensitive technique, with more samples of sera, may improve these results.

[The laboratory diagnosis and epidemiology of cutaneous leishmaniasis in Paraná State, southern Brazil]. / Observações sobre o diagnóstico laboratorial e a epidemiologia da leishmaniose tegumentar no Estado do Paraná, sul do Brasil.

Between 1986 and 1997 a total of 1418 patients were examined at the Clinical Analysis Teaching and Research Laboratory of Maringá State University (LEPAC/UEM) for cutaneous leishmaniasis by direct examination of stained smears made from the lesions, the Montenegro skin test and the indirect immunofluorescent antibody test. Nine hundred and fifty five patients (67.3%) were positive for at least one of the three tests and of these 804 (84.2%) were considered to have contracted the disease in Paraná State; 665 (69.6%) were between 15 and 49 years old; 658 (68.9%) were males; 523 (54.8%) sought medical advice during the first three months of their infections and 74 (7.7%) had mucosal lesions. Of the 83 counties of Paraná State, where the patients had most probably acquired their infections, 44.7% were from the counties of São Jorge do Ivaí (10.2%), Doutor Camargo (9.8%), Terra Boa (7.3%), Maringá (7.3%), Jussara (6.0%) and Cianorte (4.5%). Seventy seven strains of Leishmania (Viannia) braziliensis were isolated and 63.6% of these strains belong to serodema 1.

[Phlebotomines in an area of transmission of tegumentar leishmaniasis in the northern region of Parana State, Brazil: seasonal variation and nocturnal activity]. / Flebotomíneos em área de transmissão de leishmaniose tegumentar na região norte do Estado do Paraná-Brasil: Variação sazonal e atividade noturna.

In two years of phlebotomine captures in a modified forest an appreciable decrease in the size of this insect population has been observed when compared to previous data from the same locality. The captures with the Shannon light trap were made in the outlying forest of Palmital farm in Terra Boa county, Parana State, Brazil. The predominant species were Lutzomyia withmani, Lutzomyia migonei, Lutzomyia intermedius e Lutzomyia fischeri. These insects were active from 6 p.m. to 6 a.m. and the period of greatest activity varied according to the species. These species' densities were higher in the summer and autumn months. Environmental modifications may have influenced the observed changes in phlebotomine behavior.

[Frequency of anti-Cysticercus cellulosae antibodies in individuals from five counties in the Northern region of Brazil]. / Freqüência de anticorpos anti-Cysticercus cellulosae em indivíduos de cinco municípios da região Norte do Estado do Paraná-Brasil.

An epidemiological and serological study was carried out on a sample of 2,180 individuals, in five counties in the north of Paraná State-Brazil, using the indirect immunofluorescence test to detect anti-Cysticercus cellulosae antibodies. These individuals, 69 (3.2%) showed significant titers of antibodies. No single significant difference between the proportion of reactivity in Sarandi (6.6%) and in Marialva (4.7%) was observed (Z = 1,319, P = 0.0936), but it was significantly higher than that observed in Mandaguaçu, Paiçandu and Maringá (P < 0.01). Of these individuals, 47.9% were within 21-49 years old and 79.4% were of female sex. "Headache" (70.6%), "faintness" (57.4%), and "convulsions" (7.4%) were among the most frequent by reported, moreover, cases of Taenia infections (22.1%) and the custom of eating uncooked beef (41.2%) or pork (27.9%) and meat containing cysticerci (25.0%) were also related.

Efficacy of components from leaves of Calophyllum brasiliense against Leishmania (Leishmania) amazonensis.

Leishmanicide potential of Calophyllum brasiliense leaves on promastigote and amastigote of Leishmania (Leishmania) amazonensis is evaluated. The LD(50) of dichloromethane extract and hexane fraction for promastigotes was respectively 40 microg/ml and 20 microg/ml. In mouse peritoneal macrophages infected with Leishmania amastigotes the Infection Index decreased respectively 100% and 84.2% in 80 microg/ml and 40 microg/ml concentrations of dichloromethane extract. Hexane fraction decreased infection index respectively by 98.7% and 91.3% within the same concentrations. It was found that pretreatment with dichloromethane extract or with hexane fraction of experimentally infected BALB/c mice decrease the volume of the lesions by L. (L.) amazonensis. Moreover, animals treated topically also revealed healing lesions. Besides, the parasite load in the animals' popliteal lymph nodes was significantly reduced in treated animals, showing that plant components actually control infection. Results show that crude extract and hexane fraction of C. brasiliense reveal a significant in vitro and in vivo leishmanicide activity.

American cutaneous leishmaniasis in horses from endemic areas in the north-central mesoregion of Paraná state, Brazil.

American cutaneous leishmaniasis (ACL) is an endemic disease in the north-west of Paraná state, Brazil, where it occurs in humans and dogs. Few studies have been undertaken on the occurrence of the disease in other domestic animals and horses. In this study we investigated the infection of horses by Leishmania in ACL-endemic rural areas. Direct agglutination test (DAT) and polymerase chain reaction (PCR) were employed. Fifty-five horses from rural areas in the municipalities of Doutor Camargo, Ourizona, São Jorge do Ivaí, Ivatuba and Santa Fé (Agua do O) were analysed. DAT-detected antibody titres varied from 10 to 640, and 42 (76.3%) animals presented titres > or =20. PCR analyses led to the detection of Leishmania (Viannia) DNA in the blood samples of three (7.1%) of 42 animals. The presence of anti-L. (V.) braziliensis antibodies and Leishmania (Viannia) DNA in horses from endemic areas suggests their participation in the ACL transmission cycle.

Detection of Leishmania (Viannia) DNA in blood from patients with American cutaneous leishmaniasis.

The aim of the present study was to investigate the presence of Leishmania (Viannia) subgenus DNA in peripheral blood from patients with cutaneous lesions due to American cutaneous leishmaniasis. The buffy coats from 68 blood samples were analyzed by polymerase chain reaction using the MP1L/MP3H primers. The parasite DNA was detected in 2 (3.4%) out of 59 patients who had amastigotes present in samples taken from lesions. The presence of Leishmania (Viannia) DNA in the blood of these patients indicates hematogeneous parasite dissemination.

Isolation of Leishmania (Viannia) braziliensis glycolipid antigens and their reactivity with mAb SST-1, specific for parasites of Viannia subgenus.

Specific glycolipids (GLs) from Leishmania (Viannia) braziliensis promastigotes were isolated and purified. A monoclonal antibody directed to carbohydrate epitopes of these GLs was produced. mAb SST-1 recognizes a low molecular weight GL as established by solid-phase radioimmunoassay and HPTLC immunostaining, and does not cross-react with lipophosphoglycan isolated from L. (V.) braziliensis promastigotes. An indirect immunofluorescence study indicated that the antigenic GLs are present at the L. (V.) braziliensis promastigote surface. SST-1 reacted with promastigotes of L. (V.) naiffi and L. (V.) guyanensis, but not with species in the L. Leishmania subgenus i.e. L. (L.) amazonensis, L. (L.) chagasi, or L. (L.) major. All L. (V.) braziliensis serodemes tested were reactive with SST-1. These results indicate that SST-1 recognizes specific GLs expressed by species of the Viannia subgenus, and will be particularly useful for identification of L. (V.) braziliensis.

Cell migration induced by Leishmania (Leishmania) amazonensis, Leishmania (Leishmania) major and Leishmania (Viannia) braziliensis into the peritoneal cavity of BALB/c mice

In American cutaneous leishmaniasis, the initial infection phase is characterized by recruitment of neutrophils and monocytes. The migration of these cells in response to the presence of Leishmania in the peritoneum of affected animals remains unclear. The objective of this study was to investigate cell migration to the peritoneum of BALB/c mice after infection with Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis and Leishmania (Leishmania) major. Initially, Leishmania spp. was intraperitoneally inoculated in five groups of six animals each and the cell migration was analyzed 0, 3, 6, 12, 24 and 48 hours after infection. Different cell counts were performed with a staining kit and showed a higher percentage of polymorphonuclear than mononuclear cells in all three species studied. The total cell count revealed peak migration in L. (L.) amazonensis and L. (L.) major at six hours, and in L. (V.) braziliensis at 12 hours. These results suggest that factors released from different cell types probably act by attracting polymorphonuclear cells, with the peak migration most likely depending on the species of Leishmania inoculated into the host.