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1.
PLoS Negl Trop Dis ; 14(12): e0008921, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33370302

RESUMO

BACKGROUND: Alveolar (AE) and cystic echinococcosis (CE) in humans are caused by the metacestode of the tapeworms Echinococcus multilocularis and Echinococcus granulosus sensu lato (s.l.). Immunohistochemistry with the monoclonal antibodies (mAb) Em2G11, specific for AE, and the mAb EmG3, specific for AE and CE, is an important pillar of the histological diagnosis of these two infections. Our aim was to further evaluate mAb EmG3 in a diagnostic setting and to analyze in detail the localization, distribution, and impact of small particles of Echinococcus multilocularis (spems) and small particles of Echinococcus granulosus s.l. (spegs) on lymph nodes. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the mAb EmG3 in a cohort of formalin-fixed, paraffin embedded (FFPE) specimens of AE (n = 360) and CE (n = 178). These samples originated from 156 AE-patients and 77 CE-patients. mAb EmG3 showed a specific staining of the metacestode stadium of E. multilocularis and E. granulosus s.l. and had a higher sensitivity for spems than mAb Em2G11. Furthermore, we detected spegs in the surrounding host tissue and in almost all tested lymph nodes (39/41) of infected patients. 38/47 lymph nodes of AE showed a positive reaction for spems with mAb EmG3, whereas 29/47 tested positive when stained with mAb Em2G11. Spegs were detected in the germinal centers, co-located with CD23-positive follicular dendritic cells, and were present in the sinuses. Likewise, lymph nodes with spems and spegs in AE and CE were significantly enlarged in size in comparison to the control group. CONCLUSIONS/SIGNIFICANCE: mAb EmG3 is specific for AE and CE and is a valuable tool in the histological diagnosis of echinococcosis. Based on the observed staining patterns, we hypothesize that the interaction between parasite and host is not restricted to the main lesion since spegs are detected in lymph nodes. Moreover, in AE the number of spems-affected lymph nodes is higher than previously assumed. The enlargement of lymph nodes with spems and spegs points to an immunological interaction with the small immunogenic particles (spems and spegs) of Echinococcus spp.


Assuntos
Equinococose Hepática/diagnóstico , Equinococose/diagnóstico , Echinococcus granulosus/imunologia , Echinococcus multilocularis/imunologia , Linfadenopatia/parasitologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos Monoclonais/imunologia , Criança , Testes Diagnósticos de Rotina , Equinococose/parasitologia , Equinococose/patologia , Equinococose Hepática/parasitologia , Equinococose Hepática/patologia , Feminino , Humanos , Imuno-Histoquímica/métodos , Linfonodos/parasitologia , Linfonodos/patologia , Masculino , Pessoa de Meia-Idade , Adulto Jovem
2.
PLoS Negl Trop Dis ; 14(12): e0009015, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33370305

RESUMO

Trypanosoma rangeli is a non-pathogenic protozoan parasite that infects mammals, including humans, in Chagas disease-endemic areas of South and Central America. The parasite is transmitted to a mammalian host when an infected triatomine injects metacyclic trypomastigotes into the host's skin during a bloodmeal. Infected mammals behave as parasite reservoirs for several months and despite intensive research, some major aspects of T. rangeli-vertebrate interactions are still poorly understood. In particular, many questions still remain unanswered, e.g. parasite survival and development inside vertebrates, as no parasite multiplication sites have yet been identified. The present study used an insect bite transmission strategy to investigate whether the vector inoculation spot in the skin behave as a parasite-replication site. Histological data from the skin identified extracellular parasites in the dermis and hypodermis of infected mice in the first 24 hours post-infection, as well as the presence of inflammatory infiltrates in a period of up to 7 days. However, qPCR analyses demonstrated that T. rangeli is eliminated from the skin after 7 days of infection despite being still consistently found on circulating blood and secondary lymphoid tissues for up to 30 days post-infection. Interestingly, significant numbers of parasites were found in the spleen and mesenteric lymph nodes of infected mice during different periods of infection and steady basal numbers of flagellates are maintained in the host's bloodstream, which might behave as a transmission source to insect vectors. The presence of parasites in the spleen was confirmed by fluorescent photomicrography of free and cell-associated T. rangeli forms. Altogether our results suggest that this organ could possibly behave as a T. rangeli maintenance hotspot in vertebrates.


Assuntos
Doença de Chagas/transmissão , Linfonodos/parasitologia , Pele/parasitologia , Baço/parasitologia , Trypanosoma rangeli/isolamento & purificação , Animais , América Central/epidemiologia , Doença de Chagas/epidemiologia , Modelos Animais de Doenças , Interações Hospedeiro-Parasita , Humanos , Mordeduras e Picadas de Insetos/parasitologia , Insetos Vetores/parasitologia , Camundongos , Rhodnius/parasitologia , Sepse/parasitologia , América do Sul/epidemiologia
3.
PLoS Negl Trop Dis ; 14(8): e0008482, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776923

RESUMO

Current drug therapies for cutaneous leishmaniasis are often difficult to administer and treatment failure is an increasingly common occurrence. The efficacy of anti-leishmanial therapy relies on a combination of anti-parasite activity of drugs and the patient's immune response. Previous studies have reported in vitro antimicrobial activity of histamine 1-receptor antagonists (H1RAs) against different pathogens. We used an ex vivo explant culture of lymph nodes from mice infected with Leishmania major to screen H1RAs compounds. Azelastine (AZ) and Fexofenadine (FX) showed remarkable ex vivo efficacy (EC50 = 0.05 and 1.50 µM respectively) and low in vitro cytotoxicity yielding a high therapeutic index. AZ significantly decreased the expression of H1R and the proinflammatory cytokine IL-1ẞ in the ex vivo system, which were shown to be augmented by histamine addition. The anti-leishmanial efficacy of AZ was enhanced in the presence of T cells from infected mice suggesting an immune-modulatory mechanism of parasite suppression. L. major infected BALB/c mice treated per os with FX or intralesionally with AZ showed a significant reduction of lesion size (FX = 69%; AZ = 52%). Furthermore, there was significant parasite suppression in the lesion (FX = 82%; AZ = 87%) and lymph nodes (FX = 81%; AZ = 36%) with no observable side effects. AZ and FX and potentially other H1RAs are good candidates for assessing efficacy in larger studies as monotherapies or in combination with current anti-leishmanial drugs to treat cutaneous leishmaniasis.


Assuntos
Antagonistas dos Receptores Histamínicos H1 não Sedativos/uso terapêutico , Leishmaniose Cutânea/tratamento farmacológico , Ftalazinas/uso terapêutico , Terfenadina/análogos & derivados , Animais , Leishmania major , Linfonodos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Ftalazinas/química , Terfenadina/química , Terfenadina/uso terapêutico , Técnicas de Cultura de Tecidos
4.
Exp Parasitol ; 217: 107934, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32698075

RESUMO

The inadequacy of available treatments for leishmaniasis has presented up to 40% therapeutic failure. This fact suggests an urgency in the discovery of new drugs or alternative approaches for treating this disease. The objective of this study was to evaluate the antileishmanial activity of combined therapy between crotamine (CTA) from Crotalus durissus terrificus and the pentavalent antimonial Glucantime® (GLU). The assays were in vitro performed measuring the inhibition of Leishmania amazonensis amastigotes, followed by the evaluation of cellular production of cytokines and nitrites. After that, analytical methods were performed in order to characterize the molecules involved in the study by Mass Spectrometry, molecular affinity through an in silico assay and Surface Plasmon Resonance. In vivo experiments with BALB/c mice were performed by analyzing parasitemia, lesion size and immunological mediators. In the in vitro experiments, the pharmacological association improved the inhibition of the amastigotes, modulated the production of cytokines and nitric oxide. The therapy improved the effectiveness of the GLU, demonstrating a decreased parasitemia in the infected tissues. Altogether, the results suggest that the combined approach with CTA and GLU may be a promising alternative for the treatment of cutaneous leishmaniasis.


Assuntos
Antiprotozoários/uso terapêutico , Venenos de Crotalídeos/uso terapêutico , Crotalus , Leishmania mexicana/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Antimoniato de Meglumina/uso terapêutico , Animais , Antiprotozoários/farmacologia , Venenos de Crotalídeos/farmacologia , Combinação de Medicamentos , Interleucina-12/sangue , Interleucina-12/metabolismo , Leishmania mexicana/isolamento & purificação , Linfonodos/parasitologia , Macrófagos Peritoneais , Espectrometria de Massas , Antimoniato de Meglumina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Óxido Nítrico/metabolismo , Nitritos/análise , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/metabolismo
5.
Parasitol Res ; 119(7): 2245-2255, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32447515

RESUMO

This is the first study showing an in vivo microautophagy upregulation by Leishmania infantum in dogs. Both Leishmania amastigotes and promastigotes were detected in the cytoplasm of many professional and nonprofessional phagocytic cells of popliteal lymph node of three dogs suffering from chronic cutaneous leishmaniasis. Ultrastructurally, parasites appeared to be wrapped by lysosomes and/or multivesicular bodies. Neither phagophores nor double-membraned vacuoles consistent with autophagosomes were observed. Transcription factor EB (TFEB), a key factor involved in lysosome biogenesis, showed a statistically significant increase in the total component when examined by western blot in samples from leishmaniotic dogs compared with samples from healthy dogs. Instead, phosphorylated TFEB showed unmodified expression levels both in leishmaniotic and healthy dogs. Furthermore, Hsc70 and endosomal sorting complex required for transport (ESCRT)-I, which are known to play a role in microautophagy, showed no variation in expression levels both in diseased and healthy animals. Vps4A/B, an evolutionary conserved ATPase responsible for ESCRT-I complex disassembly and MVB maturation, was statistically significantly overexpressed in lymph nodal samples from leishmaniotic dogs. Bag3 was downregulated in diseased dogs whereas CHIP, p62, and LC3-II did not show any variation in expression levels. The altered expression profile of Bag3 suggested an altered interaction of Bag3 with Hsc70 and CHIP, which usually form a molecular complex involved in autophagosome-lysosome pathways. Ultrastructural and molecular findings suggested that the microautophagy pathway is upregulated in lymph nodes of dogs suffering from a chronic natural infection by Leishmania infantum.


Assuntos
Leishmania infantum/fisiologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/veterinária , Linfonodos/parasitologia , Microautofagia/imunologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Western Blotting , Doenças do Cão/parasitologia , Cães , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Leishmaniose Visceral/parasitologia , Pele/parasitologia , Ativação Transcricional , Regulação para Cima/imunologia , ATPases Vacuolares Próton-Translocadoras/metabolismo
6.
Parasitol Res ; 119(5): 1683-1690, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32285265

RESUMO

The diagnosis of canine visceral leishmaniasis (CVL) has been a problem for public health services due to the variety of clinical signs similar to other diseases and low sensitivity and specificity of available tests. In this sense, our main objective was to develop a simple, rapid, and accurate quantitative real-time PCR (qPCR) diagnosis for CVL. Thus, low-invasive samples from bone marrow (BM), popliteal lymph nodes (PLN), and conjunctival swabs (CS) were selected from negative and VL-positive dogs, using as gold standard, immunological and parasitological tests performed with different tissues. Oligonucleotides for Leishmania infantum kDNA were designed and the limit of quantification and amplification efficiency of the qPCR were determined using tissue-specific standards produced with DNA from those different tissues, mixed with DNA from a known amount of L. infantum promastigotes. Endogenous control was used to validate a comparative Ct method, and tissue parasite concentrations were estimated by comparison with tissue-specific reference standard samples. The overall analysis of the qPCR data suggests the following ranking for tissue choice: PLN > BM > CS. Finally, we have concluded that this molecular approach simplifies and accelerates the quantitative diagnostic process because it is easy to perform, requiring no DNA dosing or standard curve application, and it shows good diagnostic parameters, especially when using popliteal lymph node samples.


Assuntos
Doenças do Cão/diagnóstico , Leishmania infantum/genética , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/veterinária , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Medula Óssea/parasitologia , DNA de Cinetoplasto/genética , Doenças do Cão/parasitologia , Cães , Leishmaniose Visceral/parasitologia , Linfonodos/parasitologia , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Sensibilidade e Especificidade , Baço/parasitologia
7.
Immunity ; 52(4): 606-619.e6, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32160524

RESUMO

Group 2 innate lymphoid cells (ILC2s) regulate immunity, inflammation, and tissue homeostasis. Two distinct subsets of ILC2s have been described: steady-state natural ILC2s and inflammatory ILC2s, which are elicited following helminth infection. However, how tissue-specific cues regulate these two subsets of ILC2s and their effector functions remains elusive. Here, we report that interleukin-33 (IL-33) promotes the generation of inflammatory ILC2s (ILC2INFLAM) via induction of the enzyme tryptophan hydroxylase 1 (Tph1). Tph1 expression was upregulated in ILC2s upon activation with IL-33 or following helminth infection in an IL-33-dependent manner. Conditional deletion of Tph1 in lymphocytes resulted in selective impairment of ILC2INFLAM responses and increased susceptibility to helminth infection. Further, RNA sequencing analysis revealed altered gene expression in Tph1 deficient ILC2s including inducible T cell co-stimulator (Icos). Collectively, these data reveal a previously unrecognized function for IL-33, Tph1, and ICOS in promoting inflammatory ILC2 responses and type 2 immunity at mucosal barriers.


Assuntos
Imunidade Celular , Proteína Coestimuladora de Linfócitos T Induzíveis/imunologia , Interleucina-33/imunologia , Nippostrongylus/imunologia , Infecções por Strongylida/imunologia , Subpopulações de Linfócitos T/imunologia , Triptofano Hidroxilase/imunologia , Animais , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Suscetibilidade a Doenças , Regulação da Expressão Gênica/imunologia , Imunidade Inata , Imunidade nas Mucosas , Proteína Coestimuladora de Linfócitos T Induzíveis/genética , Interleucina-33/genética , Larva/crescimento & desenvolvimento , Larva/imunologia , Larva/patogenicidade , Linfonodos/imunologia , Linfonodos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nippostrongylus/crescimento & desenvolvimento , Nippostrongylus/patogenicidade , Cultura Primária de Células , Transdução de Sinais , Infecções por Strongylida/genética , Infecções por Strongylida/parasitologia , Infecções por Strongylida/patologia , Subpopulações de Linfócitos T/classificação , Subpopulações de Linfócitos T/parasitologia , Triptofano Hidroxilase/genética
8.
Sci Rep ; 10(1): 3865, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32123265

RESUMO

Previous studies have reported activation of the B cell-activating factor (BAFF)/a proliferation-inducing ligand (APRIL) system in T independent immunity against malaria infection. Plasmodium falciparum (P. falciparum) infected animal model is not feasible. Therefore, little is known about the occurrence of BAFF/APRIL system and changes in falciparum lymphoid tissues. This study aimed to investigate the expression of BAFF/APRIL system components in lymphoid tissues from P. falciparum infected patients. Spleen and lymph node samples from 14 patients were collected at autopsy. Normal spleens and bacterially infected tonsils served as controls. The protein and/or mRNA expression of BAFF/APRIL and their cognate receptors, BAFF-R, TACI and BCMA, were determined by immunohistochemistry and RT-qPCR, respectively. The spleens of the patients exhibited significantly higher BAFF-R protein expression than normal spleens. Although without appropriate control, BCMA protein was markedly observed only in the lymph nodes. BAFF and BCMA mRNA levels were also significantly elevated in the spleen tissues of the patients compared with normal spleens. The overall BAFF-R protein levels in the lymphoid tissues of the patients correlated positively with parasitaemia. These findings are the first to confirm that BAFF/APRIL system activation in lymphoid tissues and is positively correlated with the parasitaemia levels in falciparum malaria.


Assuntos
Fator Ativador de Células B/metabolismo , Proteínas de Ligação a DNA/metabolismo , Linfonodos/metabolismo , Malária Falciparum/metabolismo , Plasmodium falciparum/metabolismo , Baço/metabolismo , Fatores de Transcrição/metabolismo , Receptor do Fator Ativador de Células B/biossíntese , Antígeno de Maturação de Linfócitos B/biossíntese , Regulação da Expressão Gênica , Humanos , Linfonodos/parasitologia , Linfonodos/patologia , Malária Falciparum/patologia , Baço/parasitologia , Baço/patologia , Proteína Transmembrana Ativadora e Interagente do CAML/biossíntese
9.
Otolaryngol Head Neck Surg ; 162(1): 50-55, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31661359

RESUMO

OBJECTIVE: To establish the association between lymph node yield and ratio in neck dissection for well-differentiated thyroid cancer and risk for persistent postoperative disease. STUDY DESIGN: Retrospective cohort study of patients undergoing lymphadenectomy for thyroid carcinoma. SETTING: Tertiary referral center. SUBJECTS AND METHODS: Included patients underwent central and/or lateral neck dissection for papillary thyroid carcinoma at our institution between 1994 and 2015. They were divided into a persistent disease group with biochemical and structural disease (49 patients) and a disease-free group with no disease after a minimum 2 years of follow-up (175 patients). Demographic characteristics, adjuvant therapy, tumor, and lymph node features were compared. RESULTS: There were no significant differences in demographic characteristics between the groups. The mean nodal yield of patients with central and lateral neck persistence was significantly lower than that of patients remaining disease free (4.8 vs. 11.9: odds ratio [OR] 0.69; 95% CI, 0.59 to 0.8; P < .001; 14.8 vs. 31.0: OR, 0.89; 95% CI, 0.84-0.94; P < .001, respectively). Nodal ratio was higher in patients with persistence in the central and lateral neck (74.2% vs 29.4%: OR, 1.06; 95% CI, 1.04-1.08; P < .001; 54.2% vs 19.8%: OR, 1.08; 95% CI, 1.04-1.12; P < .001, respectively). CONCLUSIONS: Lower lymph node yield and higher node ratio from cervical lymph node dissections are associated with persistent disease and have potential applications in surgical adequacy.


Assuntos
Carcinoma Papilar/patologia , Carcinoma Papilar/cirurgia , Linfonodos/parasitologia , Recidiva Local de Neoplasia/patologia , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/cirurgia , Adolescente , Adulto , Carcinoma Papilar/mortalidade , Estudos de Coortes , Bases de Dados Factuais , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Excisão de Linfonodo/métodos , Linfonodos/cirurgia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Esvaziamento Cervical/métodos , Esvaziamento Cervical/mortalidade , Invasividade Neoplásica/patologia , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/terapia , Estadiamento de Neoplasias , Estudos Retrospectivos , Medição de Risco , Análise de Sobrevida , Centros de Atenção Terciária , Neoplasias da Glândula Tireoide/mortalidade , Tireoidectomia/métodos , Tireoidectomia/mortalidade , Estados Unidos
10.
Am J Trop Med Hyg ; 102(2): 268-273, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31872796

RESUMO

In immunocompromised patients, visceral leishmaniasis (VL) can present with atypical clinical symptoms that include poor response to treatment. No optimal therapeutic regimen is available for such cases. In a splenectomized male patient, we observed a disseminated form of the disease in the liver, bone marrow, lymph nodes, and gastrointestinal tract. There was an apparent clinical improvement when he was initially treated with liposomal amphotericin B (L-AmB), but this was followed by a relapse involving severe clinical symptoms. He was finally treated successfully with a combination of L-AmB, meglumine antimoniate, and pentamidine isethionate. It is important to include asplenia as an immunosuppressive condition that induces exotic VL pathologies. In such cases, combination anti-Leishmania drug therapy should be considered.


Assuntos
Anfotericina B/uso terapêutico , Leishmaniose Visceral/tratamento farmacológico , Antimoniato de Meglumina/uso terapêutico , Pentamidina/uso terapêutico , Esplenectomia , Anfotericina B/administração & dosagem , Antiprotozoários/administração & dosagem , Antiprotozoários/uso terapêutico , Medula Óssea/parasitologia , Quimioterapia Combinada , Humanos , Hospedeiro Imunocomprometido , Mucosa Intestinal/parasitologia , Leishmaniose Visceral/imunologia , Linfonodos/parasitologia , Masculino , Antimoniato de Meglumina/administração & dosagem , Pessoa de Meia-Idade , Pentamidina/administração & dosagem
11.
Turk Patoloji Derg ; 36(1): 87-89, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-29630086

RESUMO

Filariasis is a major public health problem in tropical countries like India. Despite the large number of people at risk, detection of eggs with or without larva (microfilaria) on fine-needle aspiration cytology is very unusual, especially in an uncommon site or incidentally detected in clinically unsuspected cases of filariasis with the absence of microfilariae in the peripheral blood. A 19-year-old male presented with swelling over medial aspect of left arm (just above the elbow), with no other specific signs and symptoms. Fine needle aspiration cytology revealed an adult gravid female filarial worm in a background of reactive lymphoid cells and lymphohistiocytic clusters. We report a case with elaborate fine needle aspiration cytology findings of filarial worm infestation with unusual presentation of isolated epitrochlear lymph node involvement in a clinically unsuspected case and recommend clinicians and pathologists to consider a high index of suspicion for such infections at uncommon sites especially in endemic territories, as early diagnosis and treatment prevent the more severe manifestations of disease.


Assuntos
Filariose/patologia , Linfonodos/patologia , Linfadenopatia/patologia , Wuchereria bancrofti/isolamento & purificação , Animais , Biópsia por Agulha Fina , Dietilcarbamazina/uso terapêutico , Filariose/tratamento farmacológico , Filariose/parasitologia , Filaricidas/uso terapêutico , Interações Hospedeiro-Parasita , Humanos , Linfonodos/efeitos dos fármacos , Linfonodos/parasitologia , Linfadenopatia/tratamento farmacológico , Linfadenopatia/parasitologia , Masculino , Resultado do Tratamento , Wuchereria bancrofti/efeitos dos fármacos , Adulto Jovem
12.
J Comp Pathol ; 172: 62-71, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31690418

RESUMO

The identification of the parasite in cytological smears of lymph node aspirates is a widely applied technique for the direct diagnosis of Leishmania spp. infection, especially in endemic areas. Although very specific, this method has limited sensitivity, and improving the technique would be highly desirable. This study aimed to evaluate the efficacy of conventional smear cytology (SC), liquid-based cytology (LBC), cell block (CB) stained with haematoxylin and eosin (HE) and immunocytochemistry (ICC), and formalin-fixed paraffin wax-embedded tissue immunohistochemistry (FFPE-IHC) compared with serology and polymerase chain reaction for the diagnosis of canine visceral leishmaniosis (CVL) in lymphoid tissue. The use of a preservative medium and centrifugation for cytological samples reduced the number of unsatisfactory artefacts/background. Moreover, LBC allowed excellent cellular preservation and the application of ancillary techniques, such as CB and ICC. SC was the most accurate morphological diagnostic method (45.0%). CB-ICC alone or associated with SC demonstrated significantly higher sensitivity (70.0% and 72.0%, respectively) when compared with SC alone (34.00%). CB-ICC was found to be more effective in the detection of infected animals with mild clinical signs, similar to FFPE-IHC. The specificity and positive predictive value were similar between all methods. Finally, the detection limit for CB-ICC and SC + CB-ICC was identical (18.46 amastigotes/mm2). Our study suggests that CB-ICC is a promising tool for improvement of the cytopathological diagnosis of CVL and may be applied in routine epidemiological screening.


Assuntos
Citodiagnóstico/métodos , Doenças do Cão/diagnóstico , Leishmania/isolamento & purificação , Leishmaniose Visceral/veterinária , Linfonodos/parasitologia , Animais , Doenças do Cão/parasitologia , Cães , Imuno-Histoquímica , Leishmania/parasitologia , Leishmaniose Visceral/diagnóstico , Linfonodos/patologia , Reação em Cadeia da Polimerase , Testes Sorológicos
13.
Comp Immunol Microbiol Infect Dis ; 67: 101374, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31707163

RESUMO

Leishmania infantum infection in wildlife is increasingly reported in Europe, but scant data are available in Italy so far. This study aimed to investigate the circulation of L. infantum among sylvatic hosts in Sicily (southern Italy), a highly endemic area for canine leishmaniosis, through serological and molecular tools. Target tissues (skin, spleen, lymph nodes) collected from 71 European rabbits, 2 European hares, 7 red foxes, 11 European wildcats and 1 pine marten, were qPCR analysed for the detection of L. infantum DNA. Additionally, 40 rabbits, older than one year, were serologically screened for specific anti-Leishmania antibodies. Leishmania infantum was molecularly diagnosed in 5.4% (n = 5) of the examined animals (3/71 European rabbits, 2/7 red foxes). In many of the qPCR positive animals (4/5), the parasite DNA was more prevalent in visceral than cutaneous tissues. None of the positive animal showed signs of disease and/or macroscopic alterations of organs; low parasitic burden in all positive tissue samples was also recorded. Only one rabbit serum (i.e., 2.5%) tested positive for anti-Leishmania antibodies. The seropositive rabbit was in good health status and no amastigotes were observed in lymph-node aspirate and blood smears. This study provides first evidence of L. infantum infection in wild animals from Sicily (southern Italy). Despite the low prevalence of infection here reported, the circulation of the Leishmania in wild reservoirs in Sicily remains worthy of future investigations for a better understanding of their role in the epidemiology of the disease as well as to fine-tune control strategies in the area.


Assuntos
Felidae/parasitologia , Raposas/parasitologia , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/veterinária , Mustelidae/parasitologia , Animais , Anticorpos Antiprotozoários/sangue , DNA de Protozoário/genética , Itália/epidemiologia , Leishmania infantum/genética , Leishmania infantum/imunologia , Linfonodos/parasitologia , Coelhos , Pele/parasitologia , Baço/parasitologia
14.
Curr Protoc Cytom ; 91(1): e64, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31763771

RESUMO

Confocal microscopy has been an important imaging tool for life scientists for over 20 years. Early techniques focused on indirect staining processes that involved staining with an unconjugated primary antibody, followed by incubation with a secondary fluorescent antibody that would reveal and amplify the signal of the primary antibody. With more and more directly conjugated fluorescent primary antibodies becoming commercially available, staining with multiple fluorescent primary antibodies is now more frequent. To date, staining with up to three primary antibodies and a nuclear dye is widely practiced. Here, we describe an important improvement to the standard polychromatic immunofluorescent staining protocol that allows the simultaneous detection of seven fluorescent parameters using a standard confocal laser scanning microscope with four laser lines and four photomultiplier tubes. By incorporating recently available tandem dyes that emit in the blue and violet regions of the visible light spectrum (Brilliant Blue and Brilliant Violet), we were able to differentiate several additional fluorochromes simultaneously. Due to the added complexity of 7-color immunofluorescent imaging, we developed a clear methodology to optimize antibody concentrations and simple guidelines on how to identify and correct non-specific signals. These are detailed in the following protocol. © 2019 by John Wiley & Sons, Inc. Basic Protocol: 7-Color immunofluorescent staining protocol using directly conjugated antibodies Support Protocol 1: Antibody titration protocol Support Protocol 2: Spillover optimization protocol.


Assuntos
Imunofluorescência/métodos , Microtomia , Coloração e Rotulagem/métodos , Animais , Crioultramicrotomia/métodos , Crioultramicrotomia/normas , Imunofluorescência/normas , Linfonodos/parasitologia , Linfonodos/patologia , Camundongos , Microscopia Confocal/métodos , Microscopia Confocal/normas , Microscopia de Fluorescência/métodos , Microscopia de Fluorescência/normas , Nippostrongylus/fisiologia , Coloração e Rotulagem/normas , Infecções por Strongylida/patologia
15.
Vet Parasitol ; 276: 108976, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31739256

RESUMO

Cutaneous leishmaniosis (CL) is a parasitic disease in animals and human with no satisfactory treatments and vaccination. Rapamycin is a potent inhibitor of mammalian target of rapamycin (mTOR) with various applications. Here, the effect of rapamycin alone or in combination with two other drugs, namely amphotericin B (AmB) and glucantime, was investigated against Leishmania tropica infection. In vitro viability and electron microscopy evaluation of the parasites showed detrimental changes in their appearance and viability. Treatment with clinically relevant dose of rapamycin (10.2 µg/dose) is able to control the parasite load in BALB/c mice infected with L. tropica. Furthermore, the cytokine profiles showed significant polarization towards Th1 immune response. Surprisingly, combination therapy with either AmB or glucantime was not efficient. Rapamycin is showed an effective alternative therapy against leishmaniosis caused by L. tropica.


Assuntos
Antiprotozoários/uso terapêutico , Leishmania tropica/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Sirolimo/uso terapêutico , Anfotericina B/farmacologia , Anfotericina B/uso terapêutico , Animais , Antiprotozoários/farmacologia , Linhagem Celular Tumoral , Citocinas/análise , Feminino , Humanos , Concentração Inibidora 50 , Leishmania tropica/crescimento & desenvolvimento , Leishmania tropica/ultraestrutura , Leishmaniose Cutânea/prevenção & controle , Linfonodos/parasitologia , Antimoniato de Meglumina/farmacologia , Antimoniato de Meglumina/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Carga Parasitária , Distribuição Aleatória , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/efeitos dos fármacos
16.
Exp Parasitol ; 207: 107781, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31626796

RESUMO

The paradigm that Toxoplasma gondii infection generates sterilizing protective immunity was broken by case studies in which reinfections were observed in immunocompetent pregnant women in the chronic phase of toxoplasmosis. Since then, several murine models have suggested that immunoprotection against a previous T. gondii infection may be violated after reinfection with strains of different genotypes. This study aimed to evaluate the dissemination of the parasite after reinfection with the virulent TgCTBr9 and EGS strains in BALB/c mice chronically infected with the avirulent TgCTBr5 strain. Three mice were euthanized at 2, 4, 8, 12, 24 and 48 h post challenge (p.c.) and at 7, 14 and 30 days p.c. Intestines, mesenteric lymph nodes, lungs and brains were collected for PCR-RFLP. Blood samples were collected to measure total IgG, IgG1 and IgG2a by ELISA. The reinfected animals survived and presented reduced morbidity after challenge with the virulent strains. Mice challenged with the TgCTBr9 strain showed a slight increase in anti-T. gondii IgG1. The spread of the TgCTBr5 strain was observed to occur earlier than the dissemination of the virulent TgCTBr9 or EGS strains. The TgCTBr9 strain was observed in the mesenteric lymph node at 7 days post challenge (d.p.c.); in the intestine and lungs at 14 d.p.c.; and in the brain at 30 d.p.c. EGS strain was demonstrated in the mesenteric lymph node and lung at 7 d.p.c and in the intestine and brain at a later time point. The immune response promoted by the primary infection with the avirulent strain (TgCTBr5) protected the animals from death after challenge with the virulent strains (TgCTBr9 or EGS).


Assuntos
Anticorpos Antiprotozoários/sangue , Toxoplasma/fisiologia , Toxoplasmose Congênita/parasitologia , Animais , Peso Corporal , Encéfalo/parasitologia , Brasil , Feminino , Genótipo , Humanos , Imunoglobulina G/sangue , Intestinos/parasitologia , Pulmão/parasitologia , Linfonodos/parasitologia , Mesentério , Camundongos , Camundongos Endogâmicos BALB C , Morbidade , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Recidiva , Toxoplasma/genética , Toxoplasma/imunologia , Toxoplasma/patogenicidade , Toxoplasmose Congênita/imunologia , Virulência
17.
Immunol Lett ; 216: 97-105, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31622634

RESUMO

Mesenchymal stem cell (MSCs) therapy are among new strategies that are used to combat infections through immunomodulation. Cell number, route and frequency of injection and the duration of exposure to the infectious agent are of the main factors to determine the effectiveness of cell therapy. The current study was aimed to assess the effect of multiple intravenous (i.v.) injection of adipose tissue derived (AD)-MSCs on immune response of Leishmania (L.) major-infected BALB/c mice. Therefore, infected mice received AD-MSCs four times during the early phase of infection through i.v. route. They were then monitored weekly for footpad swelling and lesion development. Parasite burden, nitric oxide (NO) and cytokine production were measured in the spleen and lymph node 90 days post-infection. Delayed lesion development, significant reduction in footpad swelling and lower parasite burden in the spleen of AD-MSCs-treated mice showed the relative effect of AD-MSCs therapy in the control of L. major dissemination. In addition, MSCs were able to manage direct cytokine responses toward T-helper 1 (Th1). Although the level of interleukin (IL)-10 was still higher than the associated level of tumor necrosis factor (TNF)-α, a shift towards higher level of TNF-α was also observed.


Assuntos
Gordura Abdominal/citologia , Leishmania major/imunologia , Leishmaniose Cutânea/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Células Th1/imunologia , Animais , Células Cultivadas/transplante , Modelos Animais de Doenças , Feminino , Humanos , Injeções Intravenosas , Leishmania major/isolamento & purificação , Leishmaniose Cutânea/sangue , Leishmaniose Cutânea/imunologia , Linfonodos/imunologia , Linfonodos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Carga Parasitária , Cultura Primária de Células , Pele/imunologia , Pele/parasitologia , Baço/imunologia , Baço/parasitologia
18.
Front Immunol ; 10: 2154, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31572373

RESUMO

CD4+ T follicular helper (Tfh) cells, a new subset of immune cells, have been demonstrated to be involved in granulomatous responses to Schistosoma japonicum (S. japonicum) infection. However, the role and underlying mechanisms of Tfh cell aggregation in S. japonicum infection remain incompletely understood. In this study, we provide evidence that S. japonicum infection enhances the accumulation of Tfh cells in the spleen, lymph nodes, and peripheral blood of C57BL/6 mice. Infection-induced Tfh cells exhibited more potent effects directly on B cell responses than the control Tfh cells (P < 0.05). Furthermore, reduced apoptosis of Tfh cells was found both in S. japonicum infected mice and in soluble egg antigen (SEA) treated Tfh cells (P < 0.05). Mechanistic studies reveal that caspase-3 is the primary drivers of down-regulated apoptotic Tfh cell death in S. japonicum infection. In summary, this study demonstrates that Tfh cell accumulation might have an impact on the generation of immune responses in S. japonicum infection, and caspase-3 signaling mediated apoptosis down-regulation might responsible for the accumulation of Tfh cell in this course.


Assuntos
Apoptose/imunologia , Caspase 3/imunologia , Schistosoma japonicum/imunologia , Esquistossomose Japônica/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Caspase 3/metabolismo , Regulação para Baixo/imunologia , Feminino , Linfonodos/imunologia , Linfonodos/metabolismo , Linfonodos/parasitologia , Camundongos Endogâmicos C57BL , Schistosoma japonicum/fisiologia , Esquistossomose Japônica/metabolismo , Esquistossomose Japônica/parasitologia , Baço/imunologia , Baço/metabolismo , Baço/parasitologia , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Auxiliares-Indutores/parasitologia
19.
Prev Vet Med ; 172: 104781, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31586717

RESUMO

Linguatula serrata is a cosmopolitan zoonotic parasite in which carnivores and herbivores serve as final and intermediate hosts, respectively. The aim of this study was to compare the L. serrata nymphal infection rate and intensity of infection (mean number of nymphs ±â€¯standard error) to the appearance and pathological changes of mesenteric lymph nodes (MLNs) infected with L. serrata using sheep slaughtered in Tabriz, Iran. In addition, the effect of age, sex, and season on the prevalence of L. serrata infection was evaluated. For this purpose, over a four-year period, 31,078 MLNs from 3199 sheep were examined, with 4972 (15.99%) MLNs infected representing 518 (16.20%) sheep. Collected MLNs were categorized by color as normal, red or black and by consistency as normal, soft or hard. L. serrata were found in 8.88% of normal-colored MLNs, 14.45% of red (hemorrhagic) MLNs and 44.57% of black-colored MLNs, with the difference being significant for infection and infection intensity (P < 0.0001). In regards to MLN consistency, 7.98% of normal, 31.52% of soft and 5.42% of hard lymph nodes were found to be infected with the infection rate and intensity in soft nodes being significantly different (P < 0.0001). Pathological changes in MLNs infected with L. serrata nymph with normal color and consistency had calcification of the L. serrata nymph, granulomatous inflammation around the nymph and some neutrophils. Granulomatous inflammation around the L. serrata nymph, haemosiderophage, macrophage and lymph node depletion from lymphocytes were observed in MLNs infected with L. serrata nymph with soft consistency. In addition, MLNs infected with L. serrata nymph with hard consistency and black color contained neutrophils in the capsule's wall, caseous necrotic mass and L. serrata surrounded by a thick capsule. In regards to prevalence, age, sex and season (autumn) were significant (P < 0.0001, P < 0.01, respectively). These study results suggest that targeted meat inspection and targeted animal interventions could be used to decrease human exposure to L. serrata and animal infection.


Assuntos
Linfonodos/parasitologia , Doenças Linfáticas/veterinária , Doenças Parasitárias em Animais/epidemiologia , Pentastomídeos/fisiologia , Doenças dos Ovinos/epidemiologia , Fatores Etários , Animais , Feminino , Irã (Geográfico)/epidemiologia , Linfonodos/patologia , Doenças Linfáticas/epidemiologia , Doenças Linfáticas/parasitologia , Masculino , Ninfa/crescimento & desenvolvimento , Ninfa/fisiologia , Doenças Parasitárias em Animais/parasitologia , Pentastomídeos/crescimento & desenvolvimento , Prevalência , Fatores Sexuais , Ovinos , Doenças dos Ovinos/parasitologia
20.
Exp Parasitol ; 205: 107738, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31442456

RESUMO

Leishmaniases, caused by Leishmania spp., are among the most prevalent infectious diseases in the world and their treatment may present high toxicity and side/adverse effects. This study evaluated the antileishmanial activity of the Hexanic Eluate subfraction from Maytenus guianensis bark (HEMg) incorporated in microparticles of PLGA. One batch of microparticles produced contained HEMg (HEMgP) and another contained the PLGA polymer alone (PCTE). The microparticles were characterized in regards to diameter, Zeta potential, encapsulation rate and morphology and their cytotoxicity was evaluated against J774 macrophages. The infection assay employing peritoneal macrophages witth L. amazonensis and cytokine dosages were performed on the cell supernatants. The groups of infected BALB/C mice were treated, euthanized and the parasite load and cytokine production were evaluated. The diameters and zeta potential were: 4 µm and -11.6 mV (PCTE) and 7.8 µm and -26.7 mV (HEMgP). The encapsulation rate was ≅ 15% and the morphology of the particles was spherical and homogeneous. In the infection assay, HEMgP inhibited the amastigotes by 70% (24 h) and 59% (48 h) and induced IL-12 and TNF-α production. HEMg in solution reduced the number of parasites in the lymph nodes by 50% and HEMgP administration increased the levels of IL-12 and TNF-α cytokines in lymph nodes and in the lesion site. When encapsulated, HEMg maintained its antileishmanial activity, but in a more attenuated and sustained form over time, showing promise as complementary/alternative therapy against cutaneous leishmaniasis.


Assuntos
Antiprotozoários/farmacologia , Leishmania mexicana/efeitos dos fármacos , Maytenus/química , Casca de Planta/química , Extratos Vegetais/farmacologia , Animais , Biodegradação Ambiental , Linhagem Celular , Micropartículas Derivadas de Células/química , Micropartículas Derivadas de Células/ultraestrutura , Concentração Inibidora 50 , Linfonodos/parasitologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Varredura
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