Macrophages play important roles in the innate and acquired immune responses against Leishmania p... more Macrophages play important roles in the innate and acquired immune responses against Leishmania parasites. Depending on the subset and activation status, macrophages may eliminate intracellular parasites; however, these host cells also can offer a safe environment for Leishmania replication. In this sense, the fate of the parasite may be influenced by the phenotype of the infected macrophage, linked to the subtype of classically activated (M1) or alternatively activated (M2) macrophages. In the present study, M1 and M2 macrophage subsets were analyzed by double-staining immunohistochemistry in skin biopsies from patients with American cutaneous leishmaniasis (ACL) caused by L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis ,and L. (L.) infantum chagasi. High number of M1 macrophages was detected in nonulcerated cutaneous leishmaniasis (NUCL) caused by L. (L.) infantum chagasi (M1 = 112 ± 12, M2 = 43 ± 12 cells/mm2). On the other side, high density of M2 macrophages was o...
A genome-wide association study (GWAS) could unravel the complexity of the cell-mediated immunity... more A genome-wide association study (GWAS) could unravel the complexity of the cell-mediated immunity (CMI) to canine leish-maniasis (CanL). Therefore, we scanned 110,165 single-nucleotide polymorphisms (SNPs), aiming to identify chromosomal regions associated with the leishmanin skin test (LST), lymphocyte proliferation assay (LPA), and cytokine responses to further understand the role played by CMI in the outcome of natural Leishmania infantum infection in 189 dogs. Based on LST and LPA, four CMI profiles were identified (LST /LPA , LST /LPA , LST /LPA , and LST /LPA), which were not associated with subclinically infected or diseased dogs. LST /LPA dogs showed increased interferon gamma (IFN-) and tumor necrosis factor alpha (TNF-) levels and mild parasitism in the lymph nodes, whereas LST /LPA dogs, in spite of increased IFN-, also showed increased interleukin-10 (IL-10) and transforming growth factor (TGF-) levels and the highest parasite load in lymph nodes. Low T cell proliferation under low parasite load suggested that L. infantum was not able to induce effective CMI in the early stage of infection. Altogether, genetic markers explained 87%, 16%, 15%, 11%, 0%, and 0% of phenotypic variance in TNF-, TGF-, LST, IL-10, IFN-, and LPA, respectively. GWAS showed that regions associated with TNF-include the following genes: IL12RB1, JAK3, CCRL2, CCR2, CCR3, and CXCR6, involved in cytokine and chemokine signaling; regions associated with LST, including COMMD5 and SHARPIN, involved in regulation of NF-B signaling; and regions associated with IL-10, including LTBP1 and RASGRP3, involved in T regulatory lymphocytes differentiation. These findings pinpoint chromosomic regions related to the cell-mediated response that potentially affect the clinical complexity and the parasite replication in canine L. infantum infection.
International journal of experimental pathology, 2014
During the natural transmission of Leishmania parasites, the infected sand fly female regurgitate... more During the natural transmission of Leishmania parasites, the infected sand fly female regurgitates promastigotes into the host's skin together with its saliva. It has been reported that vector saliva contains immunomodulatory molecules that facilitate the establishment of infection. Thus, the main objective of this study was to evaluate the specificity of Lutzomyia (Lu.) flaviscutellata and Lu. (Psychodopygus) complexus salivas on the infectivity of Leishmania (L.) (Leishmania) amazonensis and L. (Viannia) braziliensis, respectively. BALB/c mice were inoculated into the skin of hind footpad with L. (L.) amazonensis and L. (V.) braziliensis promastigotes in the absence or presence of Lu. flaviscutellata and Lu. (P.) complexus salivary gland homogenates (SGHs). The evolution of the infection was evaluated by lesion size, histopathological analysis and determination of the parasite load in the skin biopsies collected from the site of infection at 4 and 8 weeks PI. The lesion size a...
Paraffin-embedded samples commonly stored at
educational and research institutions constitute tis... more Paraffin-embedded samples commonly stored at educational and research institutions constitute tissues banks for follow-up or epidemiological studies; however, the paraffin inclusion process involves the use of substances that can cause DNA degradation. In this study, a PCR protocolwas applied to identify Leishmania strains in 33 paraffinembedded skin samples of patients with American cutaneous leishmaniasis. DNA was obtained by the phenol-chloroform protocol following paraffin removal and then used in PCR or nested PCR based on the nucleotide sequence of the small subunit ribosomal RNA (SSU rDNA). The amplicons obtained were cloned and sequenced to determine the single nucleotide polymorphism that distinguishes between different Leishmania species or groups. This assay allowed to distinguish organisms belonging to the subgenus Viannia and identify L. (Leishmania) amazonensis and L. (L.) chagasi of the Leishmania subgenus. Of the 33 samples, PCR and nested PCR identified 91% of samples. After sequencing the PCR product of 26 samples, 16 were identified as L. (L.) amazonensis, the other 10 contain organisms belonging to the L. (Viannia) sub-genus. These results open a huge opportunity to study stored samples and promote relevant contributions to epidemiological studies.
We investigated the performance of the DPP®canine visceral leishmaniasis (CVL) rapidtest, a novel... more We investigated the performance of the DPP®canine visceral leishmaniasis (CVL) rapidtest, a novel immunochromatographic assay launched by BioManguinhos (Brazil), whichwas recently included in the new Brazilian protocol for screening CVL in serological surveys.The present study compared the DPP®with the ELISA and IFA produced by BioManguinhos(Brazil) both with L. major-like antigens and with in-house tests using Leishmania infantumchagasi (in-house ELISA and in-house IFA). We analyzed the sera from clinically symp-tomatic (n = 47) and asymptomatic (n = 38) infected dogs from an endemic area of CVL, aswell as from healthy (n = 18) dogs, in addition to the sera of dogs (n = 81) infected withother pathogens. The DPP®and the in-house ELISA showed a sensitivity of 90.6% and 94.1%,respectively, and specificity of 95.1% and 97.5%, respectively, and both presented cross-reactivity only with the sera of dogs with babesiosis, 44% for the DPP®and 22% for thein-house ELISA. The clinical groups were detected equally by the two assays. The ELISABioManguinhos, IFA BioManguinhos, and in house-IFA showed a good sensitivity, 90.6%,96.5% and 89.4%, respectively, but very low specificity, 77.8%, 69.1% and 65.8%, respec-tively, due to the high cross-reactivity with the sera from the animals harboring otherpathogens. The in-house ELISA provided the highest accuracy (95.8%), followed by theDPP®(92.7%), ELISA BioManguinhos (84.3%), IFA BioManguinhos (83.1%), and in-house IFA(78.0%). The simultaneous use of the DPP®and ELISA BioManguinhos reached a sensitiv-ity of 99.1% and 82.1% when used sequentially. In conclusion, the DPP®performed well asserological test for CVL, and detected both asymptomatic and symptomatic dogs in equalproportions. Although its sensitivity is not ideal yet, discarding the IFA and including theDPP®improved the accuracy of the new Brazilian CVL diagnostic protocol, particularly ofdetecting truly infected dogs. Moreover, considering the higher specificity of DPP®(95.1%vs 77.8%), positive predictive value (95.1% vs 81.1%) and positive likelihood value (18.3% vs4.1%) in comparison with the ELISA BioManguinhos, the use of DPP®as a confirmatory testinstead of a screening test is suggested.
Macrophages play important roles in the innate and acquired immune responses against Leishmania p... more Macrophages play important roles in the innate and acquired immune responses against Leishmania parasites. Depending on the subset and activation status, macrophages may eliminate intracellular parasites; however, these host cells also can offer a safe environment for Leishmania replication. In this sense, the fate of the parasite may be influenced by the phenotype of the infected macrophage, linked to the subtype of classically activated (M1) or alternatively activated (M2) macrophages. In the present study, M1 and M2 macrophage subsets were analyzed by double-staining immunohistochemistry in skin biopsies from patients with American cutaneous leishmaniasis (ACL) caused by L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis ,and L. (L.) infantum chagasi. High number of M1 macrophages was detected in nonulcerated cutaneous leishmaniasis (NUCL) caused by L. (L.) infantum chagasi (M1 = 112 ± 12, M2 = 43 ± 12 cells/mm2). On the other side, high density of M2 macrophages was o...
A genome-wide association study (GWAS) could unravel the complexity of the cell-mediated immunity... more A genome-wide association study (GWAS) could unravel the complexity of the cell-mediated immunity (CMI) to canine leish-maniasis (CanL). Therefore, we scanned 110,165 single-nucleotide polymorphisms (SNPs), aiming to identify chromosomal regions associated with the leishmanin skin test (LST), lymphocyte proliferation assay (LPA), and cytokine responses to further understand the role played by CMI in the outcome of natural Leishmania infantum infection in 189 dogs. Based on LST and LPA, four CMI profiles were identified (LST /LPA , LST /LPA , LST /LPA , and LST /LPA), which were not associated with subclinically infected or diseased dogs. LST /LPA dogs showed increased interferon gamma (IFN-) and tumor necrosis factor alpha (TNF-) levels and mild parasitism in the lymph nodes, whereas LST /LPA dogs, in spite of increased IFN-, also showed increased interleukin-10 (IL-10) and transforming growth factor (TGF-) levels and the highest parasite load in lymph nodes. Low T cell proliferation under low parasite load suggested that L. infantum was not able to induce effective CMI in the early stage of infection. Altogether, genetic markers explained 87%, 16%, 15%, 11%, 0%, and 0% of phenotypic variance in TNF-, TGF-, LST, IL-10, IFN-, and LPA, respectively. GWAS showed that regions associated with TNF-include the following genes: IL12RB1, JAK3, CCRL2, CCR2, CCR3, and CXCR6, involved in cytokine and chemokine signaling; regions associated with LST, including COMMD5 and SHARPIN, involved in regulation of NF-B signaling; and regions associated with IL-10, including LTBP1 and RASGRP3, involved in T regulatory lymphocytes differentiation. These findings pinpoint chromosomic regions related to the cell-mediated response that potentially affect the clinical complexity and the parasite replication in canine L. infantum infection.
International journal of experimental pathology, 2014
During the natural transmission of Leishmania parasites, the infected sand fly female regurgitate... more During the natural transmission of Leishmania parasites, the infected sand fly female regurgitates promastigotes into the host's skin together with its saliva. It has been reported that vector saliva contains immunomodulatory molecules that facilitate the establishment of infection. Thus, the main objective of this study was to evaluate the specificity of Lutzomyia (Lu.) flaviscutellata and Lu. (Psychodopygus) complexus salivas on the infectivity of Leishmania (L.) (Leishmania) amazonensis and L. (Viannia) braziliensis, respectively. BALB/c mice were inoculated into the skin of hind footpad with L. (L.) amazonensis and L. (V.) braziliensis promastigotes in the absence or presence of Lu. flaviscutellata and Lu. (P.) complexus salivary gland homogenates (SGHs). The evolution of the infection was evaluated by lesion size, histopathological analysis and determination of the parasite load in the skin biopsies collected from the site of infection at 4 and 8 weeks PI. The lesion size a...
Paraffin-embedded samples commonly stored at
educational and research institutions constitute tis... more Paraffin-embedded samples commonly stored at educational and research institutions constitute tissues banks for follow-up or epidemiological studies; however, the paraffin inclusion process involves the use of substances that can cause DNA degradation. In this study, a PCR protocolwas applied to identify Leishmania strains in 33 paraffinembedded skin samples of patients with American cutaneous leishmaniasis. DNA was obtained by the phenol-chloroform protocol following paraffin removal and then used in PCR or nested PCR based on the nucleotide sequence of the small subunit ribosomal RNA (SSU rDNA). The amplicons obtained were cloned and sequenced to determine the single nucleotide polymorphism that distinguishes between different Leishmania species or groups. This assay allowed to distinguish organisms belonging to the subgenus Viannia and identify L. (Leishmania) amazonensis and L. (L.) chagasi of the Leishmania subgenus. Of the 33 samples, PCR and nested PCR identified 91% of samples. After sequencing the PCR product of 26 samples, 16 were identified as L. (L.) amazonensis, the other 10 contain organisms belonging to the L. (Viannia) sub-genus. These results open a huge opportunity to study stored samples and promote relevant contributions to epidemiological studies.
We investigated the performance of the DPP®canine visceral leishmaniasis (CVL) rapidtest, a novel... more We investigated the performance of the DPP®canine visceral leishmaniasis (CVL) rapidtest, a novel immunochromatographic assay launched by BioManguinhos (Brazil), whichwas recently included in the new Brazilian protocol for screening CVL in serological surveys.The present study compared the DPP®with the ELISA and IFA produced by BioManguinhos(Brazil) both with L. major-like antigens and with in-house tests using Leishmania infantumchagasi (in-house ELISA and in-house IFA). We analyzed the sera from clinically symp-tomatic (n = 47) and asymptomatic (n = 38) infected dogs from an endemic area of CVL, aswell as from healthy (n = 18) dogs, in addition to the sera of dogs (n = 81) infected withother pathogens. The DPP®and the in-house ELISA showed a sensitivity of 90.6% and 94.1%,respectively, and specificity of 95.1% and 97.5%, respectively, and both presented cross-reactivity only with the sera of dogs with babesiosis, 44% for the DPP®and 22% for thein-house ELISA. The clinical groups were detected equally by the two assays. The ELISABioManguinhos, IFA BioManguinhos, and in house-IFA showed a good sensitivity, 90.6%,96.5% and 89.4%, respectively, but very low specificity, 77.8%, 69.1% and 65.8%, respec-tively, due to the high cross-reactivity with the sera from the animals harboring otherpathogens. The in-house ELISA provided the highest accuracy (95.8%), followed by theDPP®(92.7%), ELISA BioManguinhos (84.3%), IFA BioManguinhos (83.1%), and in-house IFA(78.0%). The simultaneous use of the DPP®and ELISA BioManguinhos reached a sensitiv-ity of 99.1% and 82.1% when used sequentially. In conclusion, the DPP®performed well asserological test for CVL, and detected both asymptomatic and symptomatic dogs in equalproportions. Although its sensitivity is not ideal yet, discarding the IFA and including theDPP®improved the accuracy of the new Brazilian CVL diagnostic protocol, particularly ofdetecting truly infected dogs. Moreover, considering the higher specificity of DPP®(95.1%vs 77.8%), positive predictive value (95.1% vs 81.1%) and positive likelihood value (18.3% vs4.1%) in comparison with the ELISA BioManguinhos, the use of DPP®as a confirmatory testinstead of a screening test is suggested.
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Papers by Thaise Y Tomokane
educational and research institutions constitute tissues banks
for follow-up or epidemiological studies; however, the
paraffin inclusion process involves the use of substances that
can cause DNA degradation. In this study, a PCR protocolwas
applied to identify Leishmania strains in 33 paraffinembedded
skin samples of patients with American cutaneous
leishmaniasis. DNA was obtained by the phenol-chloroform
protocol following paraffin removal and then used in PCR or
nested PCR based on the nucleotide sequence of the small
subunit ribosomal RNA (SSU rDNA). The amplicons
obtained were cloned and sequenced to determine the single
nucleotide polymorphism that distinguishes between different
Leishmania species or groups. This assay allowed to
distinguish organisms belonging to the subgenus Viannia and
identify L. (Leishmania) amazonensis and L. (L.) chagasi of
the Leishmania subgenus. Of the 33 samples, PCR and
nested PCR identified 91% of samples. After sequencing the
PCR product of 26 samples, 16 were identified as L. (L.)
amazonensis, the other 10 contain organisms belonging to
the L. (Viannia) sub-genus. These results open a huge
opportunity to study stored samples and promote relevant
contributions to epidemiological studies.
educational and research institutions constitute tissues banks
for follow-up or epidemiological studies; however, the
paraffin inclusion process involves the use of substances that
can cause DNA degradation. In this study, a PCR protocolwas
applied to identify Leishmania strains in 33 paraffinembedded
skin samples of patients with American cutaneous
leishmaniasis. DNA was obtained by the phenol-chloroform
protocol following paraffin removal and then used in PCR or
nested PCR based on the nucleotide sequence of the small
subunit ribosomal RNA (SSU rDNA). The amplicons
obtained were cloned and sequenced to determine the single
nucleotide polymorphism that distinguishes between different
Leishmania species or groups. This assay allowed to
distinguish organisms belonging to the subgenus Viannia and
identify L. (Leishmania) amazonensis and L. (L.) chagasi of
the Leishmania subgenus. Of the 33 samples, PCR and
nested PCR identified 91% of samples. After sequencing the
PCR product of 26 samples, 16 were identified as L. (L.)
amazonensis, the other 10 contain organisms belonging to
the L. (Viannia) sub-genus. These results open a huge
opportunity to study stored samples and promote relevant
contributions to epidemiological studies.