RESUMO
Trachyspermum ammi (L.) Sprague (Apiaceae), commonly known as "Ajwain" is distributed throughout India. Ajwain fruits contain fiber, carbohydrates, phenolic acids, flavonoids, and tannins. The fruits also yield a small amount of essential oil, with Thymol as the principal constituent. Ajwain has various pharmacological activities like anti-leishmanial, antimicrobial, cytotoxic, antispasmodic, nematocidal, and anthelmintic. The fruits are of high therapeutic value; thus, it becomes quite essential to evaluate the quality of Trachyspermum ammi (L.) Sprague to authenticate and ensure its therapeutic and nutritional properties. The ethyl acetate fraction of Trachyspermum ammi (L.) Sprague fruits exhibited the highest total phenolic and flavonoid content values of 149.55 ± 1.19 mg rutin equivalent and 682.85 ± 3.68 mg gallic acid equivalent, respectively. Metabolite profiling of the ethyl acetate fraction using ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry analysis resulted in identifying 19 phytomolecules. A validated high-performance thin-layer chromatography method was developed to quantify standard phytomolecules in the ethyl acetate fraction. The highest and lowest percentages of phytomarker were found to be caffeic acid (5.51% ± 0.16% w/w) and gallic acid (1.29% ± 0.09% w/w), respectively. This validated rapid, accurate, and precise method for standardization of Trachyspermum ammi (L.) Sprague will be beneficial for its quality evaluation as well as the derived products.
Assuntos
Ammi , Apiaceae , Espectrometria de Massas em Tandem , Cromatografia em Camada Fina , Apiaceae/química , Cromatografia Líquida , Cromatografia Líquida de Alta PressãoRESUMO
Here, we report a simple, economic and autoclavable monophasic LGPY medium supplemented with 10% fetal bovine serum (FBS), for routine maintenance of Leishmania donovani promastigotes for laboratory use. In comparison to commercially available M199 and RPMI-1640 media, LGPY has shown approximately seven fold more cell growth. The parasite has been observed to survive in the medium for at least 15 days post-inoculation. The medium also supports long-term sub-passaging of the promastigotes and can also be stored at 4 °C or room temperature for 14 months and 45 days, respectively.
Assuntos
Meios de Cultura , Leishmania donovani , Leishmania donovani/crescimento & desenvolvimentoRESUMO
Visceral leishmaniasis (VL) is a disease caused by protozoan species of the genus Leishmania and is transmitted through bites from the Phlebotomus sand fly; it is associated with considerable morbidity and mortality in many parts of world, including India. Reports on the protective role played by saliva proteins of Lutozomyia longipalpis, Phlebotomus papatasi and Phlebotomus duboscqi. are available. However, no studies have explored the salivary proteins of P. argentipes, which is the known proven vector for the transmission of VL in the Indian sub-continent. Herein we revealed the presence of two proteins of 14.2 and one protein of 13.6â¯kDa in Indian strain P. argentipes which is absolute identical to previously reported protein of SP15 family (PagSP01, PagSP02 and PagSP07) of P. argentipes of NIH colony, USA. In an experimental study on P. argentipes from Bihar, India, we demonstrated that a strong humoral and cellular immune response was triggered to reduce the concomitant Leishmania load in groups of immunized mice. The immunized group produced a considerable amount of IgG antibodies, and their splenocytes generated TH1 cytokines (IL-12, IFN-γ) with the support of delayed-type hypersensitivity (DTH) reactivity in such mice at the challenged site. We summarize from our data that some identical proteins to previous from SP15 family protein of 14.2 and 13.6â¯kDa molecular size, derived from Indian P. argentipes and reported its first time, can also be significant in resolution of VL infection after modulation of host protective T cell response in VL.
Assuntos
Leishmania/imunologia , Leishmaniose Visceral/imunologia , Phlebotomus/imunologia , Psychodidae/imunologia , Saliva/imunologia , Proteínas e Peptídeos Salivares/imunologia , Animais , Citocinas/imunologia , Feminino , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Células Th1/imunologiaRESUMO
Sterile cure from visceralized Leishmania donovani (L. donovani) needs Th1 cell support along with the assistance from innate immune cells, NK cells and NKT cells. NKT cells play as a connecting link between innate and adaptive immune cell and support T helper cell function. Earlier, a categorical function of CD56 positive CD4+ or CD8+ NKT cells was reported in visceral leishmaniasis (VL). It was observed in in vitro that CD4+CD56+NKT cells, but not CD8+CD56+NKT cells, were accumulated at the L. donovani infection site. Therefore, in vitro experiments have been carried out to decipher the mechanism behind preferential accumulation of CD4+CD56+NKT cells at infection site. In this study, 1.89 fold higher expression of CCL4/MIP-1ß was noticed in infected macrophages. The higher expression of CCL4 was correlated with preferential accumulation of CCR5+CD4+CD56+NKT cells and apoptosis of CD8+CD56+NKT cells at in vitro infection site. The CD4+CD56+NKT cells were also observed expressing TGF-ß dominantly. Interaction of CCL4 chemotaxis was interrupted by blocking, which led to drift back the TGF-ß producing CD4+CD56+NKT cells and promoted CD8+CD56+NKT cells recruitment in in vitro infection site. CCR5 blockade also reduced CD25 and FoxP3 positive CD4+CD56+NKT cells in in vitro infection site. Therefore, it was concluded that Leishmania promotes strategic expression of CCL4, which alternately attracts CCR5+ cells, mostly expressing regulatory cytokines, at infection site. This reduces the CD8+CD56+NKT cells at infection site through Smad4 mediated TGF-ß expression and activation of caspases. Data indicates that L. donovani induces higher expression of CCL4 in host cell to attract CCR5+ cells under its strategic plan to downregulate host immune response.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Antígeno CD56/imunologia , Linfócitos T CD8-Positivos/imunologia , Quimiocina CCL4/imunologia , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Células T Matadoras Naturais/imunologia , Adolescente , Adulto , Apoptose/imunologia , Caspases/imunologia , Criança , Feminino , Fatores de Transcrição Forkhead/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Smad4/imunologia , Fator de Crescimento Transformador beta/imunologia , Adulto JovemRESUMO
Visceral leishmaniasis (VL) is a disease that is associated with compromised immunity and drug un-responsiveness as well as with the emergence of drug resistance in Leishmania donovani (Ld). Ld down-modulates cellular immunity by manipulating signaling agents, including a higher expression of the adhesion molecule CD58. The expression of CD58 and CD2 on natural killer (NK) cells facilitates intercellular adhesion and signaling. The influence of drug-resistant Ld on the expression of CD58 and CD2 was addressed in this study. The mean florescence intensity (MFI) of CD58 but not of CD2 was twofold higher on CD56+ cells during VL, but was down-regulated after treatment. In addition, MFI of CD58 on CD56+ cells was further exacerbated in VL subjects who had relapsed after Ambisome or Miltefosine treatment. The same pattern of CD58 expression was also obtained upon stimulation of healthy peripheral blood mononuclear cells with Miltefosine- or Ambisome-resistant Ld. The ratio of CD56+CD58+IFN-γ+/CD56+CD58+IL-10+ cells was reduced by 6.98-fold after stimulation with Ld. Further, an antagonist to CD58 or its counter-receptor CD2 down-regulated CD56+ NK cell recruitment across a polycarbonate trans-membrane at Ld infection sites. This study reports that factors associated with drug resistance in Ld probably promote higher expression of CD58 on CD56+ cells and their migration to the infection site in association with CD2.
Assuntos
Anfotericina B/farmacologia , Antígenos CD2/genética , Antígenos CD58/genética , Células Matadoras Naturais/imunologia , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/imunologia , Fosforilcolina/análogos & derivados , Antígenos CD2/antagonistas & inibidores , Antígenos CD2/metabolismo , Antígeno CD56/genética , Resistência a Medicamentos , Humanos , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/parasitologia , Ativação Linfocitária/efeitos dos fármacos , Fosforilcolina/farmacologiaRESUMO
BACKGROUND & OBJECTIVES: The existing antileishmanial drugs for complete cure of visceral leishmaniasis (kala-azar) are limited. The available drugs are either toxic or less effective leading to disease relapse or conversion to post-kala-azar dermal leishmaniasis. Several herbal extracts have been shown to have antileishmanial activity, but a herbal drug may not always be safe. In the present study, the extract of Cedrus deodara leaves has been standardized and tested for immunomodulatory antileishmanial activities. METHODS: The extracts of C. deodara leaves with different solvents such as benzene, chloroform, ethyl acetate and methanol were made by soxhlation process. Solvents were removed under reduced pressure and temperature using rotary evaporator. The antileishmanial bioassay test was performed with in vitro maintained parasites. Immunomodulatory activity of different extracts was tested by flow cytometry. Standardization of the effective fraction was performed with Linalool as a marker compound through reverse-phase high-performance liquid chromatography. RESULTS: The extract with the use of benzene solvent showed strong antileishmanial activities within a dose 25-200 µg/ml culture with non-significant haemolytic activities and significant immunomodulant activities against the host cells. Linalool was found to be 1.29 per cent in the effective extract of C. deodara. INTERPRETATION & CONCLUSIONS: The antileishmanial activity of C. deodara, as assessed by bioassay testing on. LEISHMANIA DONOVANI: parasites and immunomodulatory effect of benzene extract of leaves on host cells indicated that it might be a potential new safe therapeutic target to cure the visceral leishmaniasis.
Assuntos
Antiprotozoários/farmacologia , Cedrus/química , Leishmaniose Visceral/tratamento farmacológico , Extratos Vegetais/farmacologia , Animais , Antioxidantes/química , Antioxidantes/farmacologia , Antiprotozoários/química , Humanos , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/patogenicidade , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia , Extratos Vegetais/química , Folhas de Planta/químicaRESUMO
Currently the main concerns regarding control of visceral leishmaniasis (VL) caused by L. donovani are immunosuppression, relating toxicity of anti-leishmanial drug and little development in appropriate vaccine and vector (P. argentipes) control. Reports available from ex-vivo studies reflect significance of vector salivary gland homogenate (SGH) in reverting immunosuppression of infected VL subjects and as such the immunogenic nature of SGH can be a strategy to modulate immune system and anti-leishmanial function to enable immune response to control the disease. Several related studies also identified a better utility of vector anti-saliva antibodies in achieving such effects by an adoptive transfer approach instead of direct stimulation with SGH protein. However, conclusive evidences on VL cases are far beyond satisfactory to suggest role of SGH into modulation of host immune response in VL subjects in India. This study was under taken to make comparison on change in cytokines (TH1 and TH2) response pattern and anti-leishmanial macrophage (MÏ) function following stimulation of their PBMCS with SGH protein derived from P. argentipes sand fly vector for VL or anti SGH antibodies raised in rabbit. This study reports for the first time that L. donovani sensitized healthy subject demonstrates an up-regulated Interferon-γ (TH1) and down regulate Interleukin-10 (TH2) production following stimulation of their PBMCs by P. argentipes anti-saliva antibodies accompanied with an improvement in anti-leishmanial MÏ function for nitric oxide (NO) production. Subsequent experiments suggest that P. argentipes based anti-SGH antibodies when used to stimulate LD infected PBMCs in healthy subjects resulted in better clearance of Leishmania amastigotes load compare to SGH protein. Possibly the immunogenic components of anti-saliva an antibody maintains the level of protective cytokine (INF-γ) and seems to restrict the infection by host protection by vector saliva.
Assuntos
Anticorpos/imunologia , Leishmaniose Visceral/imunologia , Macrófagos/imunologia , Phlebotomus/imunologia , Glândulas Salivares/imunologia , Células Th1/imunologia , Animais , Citocinas/biossíntese , Citocinas/imunologia , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Leishmania donovani/imunologia , Leishmaniose Visceral/prevenção & controle , Leucócitos Mononucleares/imunologia , Macrófagos/metabolismo , Óxido Nítrico/biossíntese , Carga Parasitária , Phlebotomus/química , Coelhos , Glândulas Salivares/química , Proteínas e Peptídeos Salivares/imunologiaRESUMO
In Leishmania species, protein disulfide isomerase (PDI) - a redox chaperone is primarily associated with virulence and survival. The precise mechanism, especially in relation to redox changes and its effects on immunological responses in visceral leishmaniasis (VL) is not completely understood as yet. Therefore, we purified a recombinant PDI from Leishmania donovani (r-LdPDI) which was of â¼15 kDa molecular size and examined its effects on immunological responses in peripheral blood (PBMC) of human VL cases. For these studies, alanine was tested as an inhibitor and was used in parallel to all experiments. This protein was identified to have a direct correlation with parasite growth which significantly increased number of promastigotes as well as axenic amastigotes after 96 h of culture. Our experiments examining the immunological response against r-LdPDI also indicate the activation of pro-L. donovani dictated immunological responses in VL. The stimulation of PBMC with r-LdPDI induced lactate dehydrogenase (LDH) activities and up regulated interleukin-10 (IL-10) production but not the HLA-DR expression, Nitric oxide (NO) release and IFN-γ production indicating a pivoted role for r-LdPDI in causing a strong immunosuppression in a susceptible host. Further, we observed that an addition of alanine in L. donovani culture offers a significant inhibition in growth of parasite and helps in reconstitution of protective immune response in VL cases. Therefore, we demonstrate a future cross talk on use of alanine which can reduce the activities of PDI of L. donovani, eliminating the parasite induced immunosuppression and inducing collateral host protective response in VL.
Assuntos
Alanina/farmacologia , Leishmania donovani/enzimologia , Leishmaniose Visceral/imunologia , Isomerases de Dissulfetos de Proteínas/imunologia , Adolescente , Adulto , Feminino , Humanos , Imunidade Celular/imunologia , Fatores Imunológicos/imunologia , Terapia de Imunossupressão , Interferon gama/biossíntese , Interleucina-10/biossíntese , L-Lactato Desidrogenase , Macrófagos/imunologia , Macrófagos/parasitologia , Masculino , Óxido Nítrico/metabolismo , Adulto JovemRESUMO
Anemia in kala-azar patients is a serious problem. The present study has addressed this problem with the hypothesis that as the Leishmania parasite is completely devoid of heme biosynthetic pathway, therefore the excessive use of heme by the parasites in the human patients can be one of the possible reason of anemia. We investigated that whether, the inability of Leishmania donovani to synthesize heme, can enforce Leishmania parasite to utilize heme derived from host sources in Indian KA patients. Patients had higher tendency of their macrophages to bind with Hb which was pronounced after sensitization with drug resistant Leishmania strain compared to sensitive.
Assuntos
Anemia/parasitologia , Heme/metabolismo , Leishmania donovani/metabolismo , Leishmaniose Visceral/complicações , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Feminino , Hemoglobinas/metabolismo , Humanos , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/parasitologia , Macrófagos/metabolismo , Masculino , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/metabolismo , Índice de Gravidade de DoençaRESUMO
A large body of evidence has shown a direct link between arsenic exposure and drug resistance to Leishmania parasites against antimonial preparations in visceral leishmaniasis (VL) hyper-endemic regions, especially in India and its sub-continent. However, the implicated roles of arsenic on the VL host, pathophysiological changes, and immune function have not yet been clarified, particularly at the reported concentration of arsenic in the VL hyper-endemic area of Bihar, India. Herein, we exposed the mouse VL model to arsenic (0.5 mg/L to 2 mg/L) through their drinking water and analyzed its effect on T cells proliferation, Th1/Th2-mediators, MAPK signaling cascade, and parasite load in preclinical models. Coherently, the parasite count in Giemsa stained spleen imprint has been investigated and found significant positive associations with levels of arsenic exposure. The liver and kidney function tests (AST, ALT, ALP, BUN, Creatinine, Urea, etc.) are apparent to hepatonephric toxicity in arsenic exposed VL mice compared to unexposed. This observation appears to be consistent with the up-regulated expression of immune regulatory Th2 mediators (IL-4, IL-10, TGF-ß) and down-regulated expression of Th1 mediators (IL-12, IFN-γ, TNF-α) with a suppressed leishmanicidal function of macrophage (ROS, NO, iNOS). We also established that arsenic exposure modulated the host ERK-1/2 and p38 MAPK signaling cascade, limited T lymphocyte proliferation, and a lower IgG2a/IgG1 ratio to favor the Leishmania parasite survival inside the host. This study suggests that the contorted Th1-subtype and exacerbated Th2-subtype immune responses are involved in the increased susceptibility and pathogenesis of Leishmania parasite among subjects/individuals regularly exposed to arsenic.
Assuntos
Arsênio , Água Potável , Leishmania donovani , Leishmaniose Visceral , Humanos , Animais , Camundongos , Leishmaniose Visceral/parasitologia , Arsênio/toxicidade , Progressão da DoençaRESUMO
We have evaluated the effect of combining CD2 with conventional antimonial (sb) therapy in protection in BALB/c mice infected with either drug sensitive or resistant strain of Leishmania donovani with 3×10(7) parasites via-intra-cardiac route. Mice were treated with anti CD2 adjunct SAG sub-cutaneously twice a week for 4 weeks. Assessment for measurement of weight, spleen size, anti-Leishmania antibody titer, T cell and anti-leishmanial macrophage function was carried out day 0, 10, 22 and 34 post treatments. The combination therapy was shown boosting significant proportion of T cells to express CD25 compared to SAG monotherapy. Although, the level of IFN-γ was not statistically different between combination vs monotherapy (p=0.298) but CD2 treatment even alone significantly influenced IFN-γ production than either SAG treatment (p=0.045) or with CD2 adjunct SAG treatment (p=0.005) in Ld-S strain as well as in Ld-R strain. The influence of CD2 adjunct treatment was also documented in anti-leishmanial functions in macrophages. As shown, the super-oxide generation began enhancing very early on day 10 after SAG treatment with CD2 during which SAG action was at minimum. Interestingly, the super-oxide generation ability remained intact in macrophage after treatment with immuno-chemotherapy even in mice infected with Leishmania resistant strain. Unlike SAG treatment, treatment of SAG with CD2 also led to production of nitric oxide and TNF-α, resulting in resulting in most effective clearance of L. donovani from infected macrophages. Our results indicate that CD2, which can boost up a protective Th1 response, might also be beneficial to enable SAG to induce Macrophages to produce Leishmanicidal molecules and hence control the infection in clinical situation like Kala-azar. Drug resistance is the major impedance for disease control but the encouraging results obtained after infecting mice with resistant strain of the parasite strongly imply that this drug can be effective even in treating resistant cases of Kala-azar.
Assuntos
Gluconato de Antimônio e Sódio/uso terapêutico , Antiprotozoários/uso terapêutico , Antígenos CD2/imunologia , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Células Th1/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Resistência a Medicamentos , Quimioterapia Combinada , Humanos , Fatores Imunológicos/imunologia , Fatores Imunológicos/uso terapêutico , Interferon-alfa/metabolismo , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/prevenção & controle , Leucócitos Mononucleares/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico/metabolismo , Estudos Prospectivos , Explosão Respiratória , Baço/citologia , Baço/imunologia , Superóxidos/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
One obstacle faced in the effective control of visceral leishmaniasis (VL) is the limited number of available treatment options. Furthermore, control efforts have been hindered further by the emergence of Leishmania resistance to many of the available drugs. In this study, we investigated the anti-leishmanial properties of 30 medicinally important plants from the VL endemic area of Bihar, India and compared them to two available anti-leishmanial drugs (sodium antimony gluconate and amphotericin B) and two plant lectins (phytohemagglutinin and concanavalin A) on Leishmania donovani promastigotes in vitro at 24 and 48 h after initiation of culture. We identified eight plant extracts in addition to phytohemagglutinin and amphotericin B that significantly inhibited the growth of promastigotes (p < 0.03). We further studied the minimum effective concentrations as well as the effect on axenic amastigotes viability and the cell cytotoxicity on human peripheral blood of four (Agave americana, Azadirachta indica, Eclipta alba and Piper longum) of the eight plant extracts that induced significant promastigotes killing (p = 0.00098). Effect-based dose finding analysis revealed that the threshold concentration of A. americana required to eliminate L. donovani after 24h was 0.05 mg/ml. A. indica and P. longum plant extracts eliminated L. donovani promastigotes after 48 h at concentrations of 0.1 and 0.5mg/ml, respectively. E. alba eliminated the promastigotes at a concentration of 0.5mg/ml within 24h. The axenic amastigote killing response was 1.90-, 2.52- and 1.3-fold higher than the promastigote killing response with A. indica, A. americana and E. alba plant extracts, respectively. A. americana and A. indica, respectively, led to approximate 2.5- and 1.3-fold declines in mitochondrial dehydrogenase activity compared with control. E. alba stimulation resulted in an up-regulation of dehydrogenase activity (p = 0.00329). The CSA from P. longum was found to be least cytotoxic; the observed difference in mitochondrial activity was insignificant (p = 0.16314). Further studies may reveal the pharmacological significance of many of the plants with anti-leishmanial properties identified in the present study.
Assuntos
Antiprotozoários/farmacologia , Leishmania donovani/efeitos dos fármacos , Extratos Vegetais/farmacologia , Anfotericina B/farmacologia , Gluconato de Antimônio e Sódio/farmacologia , Antiprotozoários/toxicidade , Concanavalina A/farmacologia , Humanos , Índia/epidemiologia , Leishmania donovani/crescimento & desenvolvimento , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/epidemiologia , Leucócitos Mononucleares/efeitos dos fármacos , Fito-Hemaglutininas/farmacologia , Extratos Vegetais/toxicidadeRESUMO
The arsenic contamination of ground water in visceral leishmaniasis (VL) endemic areas in Bihar, India leads to human exposure through drinking water. Possibly, the consumed arsenic (As) accumulates in the tissues of VL patients, who subsequently internalize intracellular amastigotes to confer resistance against chemotherapy to the parasite, leading to modulation in the host's immune response. This hypothesis appears to be consistent with the in vitro findings that in arsenic-exposed parasites, the mitochondrial membrane potential became depolarized, whereas the reduced thiol and lactate production was overexpressed with enhanced glucose consumption; therefore, the reduced thiol possibly supports an immunosuppressive state in the host cells. This observation was well supported by the down-regulated expression of pro-inflammatory cytokines (IL-2, IL-12, IFN-γ, and TNF-α) with a suppressed anti-leishmanial function of macrophage (NO, ROS). In contrast, the pathophysiological mechanism of VL has received ample support by the promotion of Th2 cytokines (IL-4 and IL-10) in the presence of arsenic-exposed Leishmania parasites (LdAS). Dysfunction of mitochondria and the overexpression of lactate production raise the possibility of the Warburg effect being operative through the up-regulation of glucose consumption by parasites to enhance the energy production, possibly augmenting virulence. Therefore, we surmise from our data that arsenic exposure to Leishmania donovani modulates the immune response and infection pattern by impairing parasite function, which may affect the anti-leishmanial effect in VL.
Assuntos
Arsênio/farmacologia , Leishmania donovani/imunologia , Leishmaniose Visceral , Macrófagos Peritoneais , Animais , Citocinas/imunologia , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/patologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/parasitologia , Macrófagos Peritoneais/patologia , Camundongos , Óxido Nítrico/imunologia , Espécies Reativas de Oxigênio/imunologiaRESUMO
The communication presents clinical response of cases of visceral leishmaniasis to treatment by two different brands of Amphotericin B. Fungizone was found to be slightly better than Amphotericin B, however, the difference is not statistically significant.
Assuntos
Anfotericina B , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Adolescente , Adulto , Anfotericina B/farmacologia , Anfotericina B/uso terapêutico , Animais , Criança , Humanos , Leishmania donovani/isolamento & purificação , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/patologia , Camundongos , Baço/efeitos dos fármacos , Baço/patologia , Resultado do TratamentoRESUMO
This study reports a structural and functional heterogeneity of CD8+CD56+NKT cells, which usually decrease quantitatively during visceral leishmaniasis. Based on fluorescence intensity of CD8 receptors on CD56+NKT cells, two populations of CD8+CD56+NKT cells have been identified. These cells were recognized as CD8dimCD56+NKT and CD8brightCD56+NKT cells. We further analyzed the functional nature of CD8dim and CD8bright positive CD56+NKT cells. In comparison to CD8brightCD56+NKT cells, a significantly higher percentage of CD8dimCD56+NKT cells expressed KIR during VL. The percentage of CD8dimCD56+NKT cells expressing KIR was found 4 fold higher in VL as compared to healthy subjects. But, the difference was insignificant in case of CD8brightCD56+NKT cells. CD8+CD56+NKT cells release granzyme B to kill the infected cells. A categorical difference was also observed in the function of CD8dimCD56+NKT and CD8brightCD56+NKT cells during visceral leishmaniasis. The percentage of granzyme B expressing CD8dimCD56+NKT cells was 2.83 fold higher in VL compared to healthy subjects. But, there was no significant difference in granzyme B expressing CD8brightCD56+NKT cells in samples from healthy and VL subjects. However, within VL subject, the percentage of granzyme B expressing CD8dimCD56+NKT cells was 5.7 fold higher in comparison to CD8brightCD56+NKT cells. This study concludes that CD8dimCD56+NKT cells are more cytotoxic than CD8brightCD56+NKT cells during VL.
Assuntos
Leishmaniose Visceral/imunologia , Subpopulações de Linfócitos/imunologia , Células T Matadoras Naturais/imunologia , Antígeno CD56/metabolismo , Antígenos CD8/metabolismo , Separação Celular , Células Cultivadas , Citotoxicidade Imunológica , Feminino , Citometria de Fluxo , Granzimas/metabolismo , Humanos , Imunofenotipagem , Masculino , Receptores KIR/metabolismoRESUMO
We report here a Leishmania donovani ornithine decarboxylase (Ld-ODC) gene used as a DNA vaccine against visceral leishmaniasis in a murine Balb/c mouse model. This study also evaluated the possible mechanism of action directed by this candidate. We found a Th1 immune response after immunization using an Ld-ODC DNA vaccine, with results based on the rearrangement of TCR-V-α-2, proliferation of Carboxy fluorescein Succinimidyle ester positive T cells, which were able to produce cytokines such as TNF-α, IFN-γ, IL-12 and IL-2, but not IL-4, IL-5, IL-6 and IL-10, and modulations of the STAT-1 and p38 MAP kinase signaling pathways. The results were corroborated with the reduction in the amastigote proliferation and parasite killing in spleens after infection in vitro. We conclude this study suggesting that the Ld-ODC DNA construct could be a new vaccine candidate against visceral leishmaniasis.
Assuntos
Imunomodulação , Leishmania donovani/imunologia , Vacinas contra Leishmaniose/uso terapêutico , Leishmaniose Visceral/prevenção & controle , Ornitina Descarboxilase/imunologia , Vacinas de DNA/uso terapêutico , Imunidade Adaptativa/fisiologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Imunização/métodos , Imunomodulação/genética , Imunomodulação/imunologia , Leishmania donovani/genética , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ornitina Descarboxilase/genética , Vacinas de DNA/imunologiaRESUMO
Protein disulphide isomerase (PDI) is one of the key enzymes essential for the survival of Leishmania donovani in the host. Our study suggested that PDI is associated with the generation of Th1-type of cellular responses in treated Visceral leishmaniasis (VL) subjects. The stimulation of Peripheral blood mononuclear cells (PBMCs) with recombinant Protein Disulphide Isomerase upregulated the reactive oxygen species generation, Nitric oxide release, IL12 and IFN-γ production indicating its pivotal role in protective immune response. Further, a pre-stimulation of PBMCs with Protein disulphide isomerase induced a strong IFN-γ response through CD8+ T cells in treated VL subjects. These findings also supported through the evidence that this antigen was processed and presented by major histocompatibility complex class I (MHC-1) dependent pathway and had an immunoprophylactic potential which can induce CD8+ T cell protective immune response in MHC class I dependent manner against VL. To find out the possible epitopes that might be responsible for CD8+ T cell specific IFN-γ response, computational approach was adopted. Six novel promiscuous epitopes were predicted to be highly immunogenic and can be presented by 32 different HLA allele to CD8+ T cells. Further investigation will explore more about their immunological relevance and usefulness as vaccine candidates.
Assuntos
Epitopos de Linfócito T/química , Antígenos de Histocompatibilidade Classe I/química , Leishmania donovani/enzimologia , Isomerases de Dissulfetos de Proteínas/química , Adolescente , Adulto , Sequência de Aminoácidos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Citocinas/metabolismo , Epitopos de Linfócito T/imunologia , Feminino , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunomodulação , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Masculino , Isomerases de Dissulfetos de Proteínas/imunologia , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Adulto JovemRESUMO
Diagnosis of visceral leishmaniasis (VL) is often hindered by cross-reactions with antigens from other related parasite infections. This study aimed to develop an immunochromatographic test (ICT) which can detect the antigen present in circulating immune complexes (CICs) of VL patients using B-cell epitope-specific antibodies. MS analysis of six immunoreactive 2DE spots revealed two epitopes i.e. RFFVQGDGIGQHSLQEALERR (P1) and RRVAVLVLLDRL (P2) (From a hypothetical protein [Acc No: XP_003861458.1]). The epitope conservancy analysis suggested that the linear epitope (P1P2) is 97-100% conserved among Leishmania species and diverged from Homo sapiens (61% query coverage and 80% identity). Further, immunoinformatics analysis of hydrophilicity and flexibility confirmed the antigenicity of the peptide fragment. The linear epitope (P1P2) was synthesized (98% purity) and the purity was confirmed by high-performance liquid chromatography and MS. The indirect Enzyme linked immunosorbent assay results confirmed the presence of the corresponding antibody in VL patient's sera but not in those of healthy and other diseases. The result demonstrated a sensitivity 90%; Se Cl95% (82.16-96.27)% and specificity 100%; Sp Cl95% (84.56-100)% which indicated the possibility to be used as a diagnostic tool. Sensitivity, specificity, and diagnostic efficiency of colloidal gold conjugated anti-P1P2 antibody ICT strip was 100, 95.2, and 96.7%, respectively, which is slightly better as compared to other ICT for VL. Though, our result indicated the utility of anti-P1P2 antibody to detect CICs epitopes, a large-scale inspection in endemic and non-endemic area and in different ethnic population is needed for its validation and authentication.
Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/química , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos de Linfócito B/química , Leishmania donovani/imunologia , Leishmaniose Visceral/diagnóstico , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Complexo Antígeno-Anticorpo/sangue , Antígenos de Protozoários/imunologia , Linfócitos B/imunologia , Linfócitos B/parasitologia , Ensaio de Imunoadsorção Enzimática/normas , Epitopos de Linfócito B/imunologia , Humanos , Soros Imunes/química , Leishmania donovani/química , Leishmaniose Visceral/sangue , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , CoelhosRESUMO
The unreliability of most of the existing antibody-based diagnostic kits to discriminate between active and treated VL cases, relapse situation and reinfection are a major hurdle in controlling the cases of Kala-azar in an endemic area. An antigen targeted diagnostic approaches can be an attractive strategy to overcome these problems. Hence, this study was focused on identifying the Leishmania antigens, lies in circulating immune complex (CICs), can be used for diagnostic as well as prognostic purposes. The present study was conducted on peripheral blood samples of 115 human subjects, based on isolation of CICs. The SDS-PAGE patterns showed an up-regulated expression of 55 kDa and 23 kDa fractions in an antigens obtained from CICs of all clinical and parasitologically proven untreated visceral leishmaniasis patients before treatment (VL-BT), which ensured absolute sensitivity. However, light expressions of these bands were observed in some VL treated cases. To ascertain the prognostic value, 2D expression profiles of circulating antigens were carried out, which revealed 3 upregulated and 12 induced immunoreactive spots. Out of these, ten prominent spots were excised and subjected for enzymatic digestion to generate peptides. Mass spectrometry (MS) analysis successfully explored 20 peptides derived from kinase, kinesin, acetyl Co-A carboxylase, dynein heavy chains (cytoplasmic and axonemal/flagellar), 60S ribosomal protein, nucleoporin protein, RNA polymeraseII, protease gp63, tubulin, DNA polymerase epsilon subunit, GTP-binding protein and tyrosyl-methionyl t-RNA synthetase-like protein and 19 hypothetical protein of unknown function. Presence of L. donovani proteins in circulating antigens were further validated using anti-Ld actin and anti-α tubulin antibody. Besides, MS derived peptides confirmed its reactivity with patients' sera. Therefore, these shortlisted potential antigens can be explored as antigen-based diagnostic as well as prognostic kit.