RESUMO
Cutaneous leishmaniasis (CL) treatment is based on therapy with Glucantime® , yet, there are few laboratory methods to monitor its success. In this study, ex vivo and in vitro evaluations of peripheral blood monocytes were performed in a longitudinal study to characterize the impact of Glucantime® on overall phenotypic/functional features of these cells from CL patients to identify predictive biomarkers for post-therapeutic monitoring by flow cytometry. The ex vivo evaluation from CL patients demonstrated a modulatory profile before treatment, with a decrease in TLR-2, FcγRII, HLA-DR, CD86, IFN-γR, TNF, IL-12, NO, and an increase in FcγRIII and IL-10R. Conversely, treatment changes some of these biomarker expressions by decreasing FcγRIII and IL-10R and increasing IFN-γR, IL-12 and NO. Moreover, an in vitro analysis of these patients showed a reduced phagocytic capacity of Leishmania braziliensis and higher levels of IL-10 and TGF-ß modulating functional profile. Regardless of the compromised L. braziliensis phagocytic capacity, treatment re-established the production of IL-12, IL-10, TGF-ß and NO at the basal level. Notably, monocytes from patients with early cicatrization showed enhanced FcγRI and FcγRII expressions and reduced IL-10, which was further corroborated by a baseline fold change analysis. Finally, the logistic regression model emphasized the performance of FcγRI, FcγRII and IL-10 as robust predictive biomarkers for post-therapeutic cicatrization during cutaneous leishmaniasis.
Assuntos
Biomarcadores/análise , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/imunologia , Receptores de IgG/análise , Adulto , Cicatriz , Citocinas/análise , Feminino , Citometria de Fluxo , Humanos , Interleucina-10/análise , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Modelos Logísticos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Adulto JovemRESUMO
During cryopreservation, sperm was submitted to an increase in reactive oxygen species generation. This work aimed to improve the quality of frozen equine sperm after the addition of antioxidants lactoferrin (Lf) and catalase (Cat) to a freezing extender. Semen from six stallions was frozen with the extenders: F1) control, INRA 82 freezing extender, F2) F1 + 500 µg/ml Lf and F3) F1 + 200 IU/ml Cat. After thawing, sperm motility parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome-reacted sperm were evaluated with a computer-assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite, hydroperoxide and iron concentrations of frozen semen were measured with spectrophotometry. The percentage of functional membrane sperm treated with Lf was higher (50.7% ± 11.6%) compared to that of the control (37.6% ± 15.6%), while the iron (61.4 ± 11.6 vs 73.3 ± 13.8 mg/dl) and nitrite concentrations (16.3 ± 7.1 vs 25.9 ± 4.2 µM/µg protein) were lower, respectively (p < .05). Thus, it can be suggested that Lf protect stallion spermatozoon during freezing as it has increased the percentage of sperm with functional membrane and decreased the lipid oxidant agents.
Assuntos
Criopreservação/veterinária , Crioprotetores/farmacologia , Cavalos , Lactoferrina/farmacologia , Espermatozoides/efeitos dos fármacos , Reação Acrossômica , Animais , Antioxidantes , Catalase/farmacologia , Membrana Celular/fisiologia , Criopreservação/métodos , Masculino , Análise do Sêmen/veterinária , Preservação do Sêmen/veterinária , Motilidade dos Espermatozoides , Espermatozoides/citologiaRESUMO
BACKGROUND: Frozen equine semen has lower fertility compared to cooled semen. Due to the difficulty to obtain equine oocytes, a heterologous zona pellucida binding assay (ZBA) is an alternative method to predict the fertilizing capability of equine frozen sperm. The rate of capacitated and hyperactivated sperm according to their motility characteristics were analyzed with a Computer Assisted Sperm Analyzer. We believe this report describes for the first time the in vitro hyperactivation induction and the heterologous ZBA to predict the fertilizing ability of frozen equine sperm. OBJECTIVE: This work aimed at developing an assay to evaluate the fertilizing ability of frozen equine sperm using a bovine ZBA with the use of an in vitro capacitation and hyperactivation media with procaine and calcium ionophore A23187, respectively. MATERIALS AND METHODS: The sperm motility characteristics, intact and acrosome reacted sperm rates, and number of stallion sperm bound to the bovine ZP were calculated. RESULTS: The procaine group showed a hyperactivation motility pattern, although it improved ZP sperm binding similarly to the capacitation group. CONCLUSION: The capacitation medium improved the IVF capability of frozen equine sperm, allowing the highest possibility of sperm-oocyte interaction.
Assuntos
Criopreservação , Fertilização , Espermatozoides/fisiologia , Zona Pelúcida , Animais , Bovinos , Cavalos , Masculino , Motilidade dos Espermatozoides , Interações Espermatozoide-ÓvuloRESUMO
The immunological biomarkers profiles were evaluated using Luminex as putative measures to monitor canine mammary carcinomas (MCs). Forty female dogs were categorized into benign mixed tumour (MC-BMT = 28) and mammary carcinoma (MC=12). The ascendant biomarker signatures were used to compare the groups. For example, a higher frequency of MC-BMT animals producing IL-6, CXCL-8 and CXCL-10 was observed, whereas for the MC group IL-2 and CXCL-8 were detected. MC-BMT animals without metastasis had an increase in the levels of IL-2, CXCL-8, CXCL-10, IL-6, TNF-α, IL-15 and a decrease in IL-10 and CXCL-8. MC-BMT animals with metastasis showed only an increase in CXCL-10 and a decrease in IL-18. After comparing the ascendant signatures following the presence of metastasis in both groups, a higher frequency of dogs exhibiting IL-10 production was observed. Pearson correlation (P = 0.0273) and receiver operating characteristic (ROC) curve analysis revealed that this pattern was associated with worse outcome and lower survival rates in MC animals.
Assuntos
Biomarcadores Tumorais/sangue , Carcinoma/veterinária , Doenças do Cão/sangue , Neoplasias Mamárias Animais/sangue , Animais , Carcinoma/sangue , Carcinoma/metabolismo , Carcinoma/patologia , Citocinas/sangue , Citocinas/genética , Citocinas/metabolismo , Doenças do Cão/metabolismo , Doenças do Cão/patologia , Cães , Feminino , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologiaRESUMO
In this study, we described, for the first time, specific aspects of an anti-Leishmania immune response in a Brazilian Xakriabá indigenous community. Induction of an intracellular NO pathway, triggered by the binding of IgE to CD23 receptor in IFN-γ/IL-4 cytokines environment, was evaluated in localized cutaneous leishmaniasis (LCL) carriers and positive Montenegro skin test (MST) individuals without skin lesion (MT(+) SL(-)). Our data demonstrated that the higher frequency of CD23(+) CD14(+) monocytes and the increased serum levels of IgE observed in the LCL group were even higher in LCL carriers with late lesions (LCL≥60). Furthermore, patients with LCL presented increased NO production after Leishmania (Viannia) braziliensis stimulation and this NO profile was independent of the time of the lesion (recent LCL<60 or late LCL≥60). We also showed that the increased frequency of IFN-γ(+) and IL-4(+) CD4(+) T cells is related to the MT(+) SL(-) group. The results of biomarker signature curves demonstrated that in the MT(+) SL(-) group, the index signature was characterized by DAF-2T(+) CD14(+)/IL-4(+) CD8(+)/IFN-γ(+) CD4(+)/IL-4(+) CD4(+). On the other hand, the LCL group presented a higher index of DAF-2T(+) CD14(+)/CD23(+) CD14(+)/IL-4(+) CD8(+), associated with a lower index of IFN-γ(+) CD8(+). Considering the time of lesion, data analysis demonstrated that the main differences observed were highlighted in LCL<60 patients, with a higher index of CD23(+) CD14(+), which was also present in LCL≥60 patients. In conclusion, our data suggest that the protective immune response involving CD23-IgE-mediated NO release is a hallmark of patients with LCL. However, in MT(+) SL(-) individuals, another different leishmanicidal mechanism seems to be involved.
Assuntos
Imunoglobulina E/imunologia , Leishmaniose Cutânea/imunologia , Óxido Nítrico/imunologia , Receptores de IgE/imunologia , Adolescente , Adulto , Brasil , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Criança , Feminino , Citometria de Fluxo , Humanos , Imunoglobulina E/sangue , Interferon gama/sangue , Interferon gama/imunologia , Interleucina-4/sangue , Interleucina-4/imunologia , Leishmaniose Cutânea/sangue , Receptores de Lipopolissacarídeos/sangue , Receptores de Lipopolissacarídeos/imunologia , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/metabolismo , Óxido Nítrico/metabolismo , Grupos Populacionais , Receptores de IgE/sangue , Testes Cutâneos/métodos , Adulto JovemRESUMO
Eleven commercially available PE-labeled anti-human (IL-1-α, IL-6, IL-8, TNF-α, IL-17A, IL-5, IL-10, IL-12 and IL-13) and anti-mouse (IL-10, TNF-α) cytokine monoclonal antibodies (mAbs) were tested for cross-reactivity with cattle, goat, and sheep cytokines. Cross-reactivity was assessed by comparative analysis with the standard reactivity of the target species. Our data demonstrated that anti-human IL-1-α, IL-6, IL-8, IL-17A and IL-10 mAbs cross-react with all ruminant species tested. Anti-human IL-5 mAb showed a strong cross-reactivity with cattle and goat IL-5, while anti-human TNF-α mAb showed a selective cross-reactivity with goat TNF-α. No cross-reactivity with the ruminant cytokines was observed for anti-human IL-12 and IL-13 mAbs or for the two anti-mouse cytokine mAbs tested. The present study demonstrated the cross-reactivity of various anti-human cytokine mAbs with cattle, sheep, and goat cytokines, increasing the range of immunological biomarkers for studies in veterinary medicine.
Assuntos
Anticorpos Monoclonais/imunologia , Biomarcadores/sangue , Reações Cruzadas/imunologia , Citocinas/imunologia , Animais , Bovinos , Reações Cruzadas/genética , Citocinas/genética , Cabras/imunologia , Humanos , Camundongos , Ovinos/imunologiaRESUMO
In the present study, we characterized the phagocytic capacity, cytokine profile along with the FCγ-R and TLR expression in leukocytes from Chagas disease patients (indeterminate-IND and cardiac-CARD) before and one-year after Bz-treatment (INDT and CARDT). A down-regulation of IL-17, IFN-γ and IL-10 synthesis by neutrophils was observed in CARDT. The Bz-treatment did not impact on the expression of phagocytosis-related surface molecules or monocyte-derived cytokine profile in INDT. Although CARDT showed unaltered monocyte-phagocytic capacity, up-regulated expression of Fcγ-RI/III and TLR-4 may be related to their ability to produce IL-10 and TGF-ß. Down-regulation of lymphocyte-derived cytokine was observed in INDT whereas up-regulated cytokine profile was observed for lymphocytes in CARDT. Analysis of cytokine network revealed that IND displayed a multifaceted cytokine response characterized by strong connecting axes involving pro-inflammatory/regulatory phagocytes and lymphocytes. On the other hand, CARD presented a modest cytokine network. The Bz-treatment leads to distinct cytokine network: decreasing the links in INDT, with a pivotal role of IL-10(+) monocytes and expanding the connections in CARDT. Our findings highlighted that the Bz-treatment contributes to an overall immunomodulation in INDT and induces a broad change of immunological response in CARDT, eliciting an intricate phenotypic/functional network compatible with beneficial and protective immunological events.
Assuntos
Doença de Chagas/tratamento farmacológico , Neutrófilos/efeitos dos fármacos , Nitroimidazóis/administração & dosagem , Tripanossomicidas/administração & dosagem , Adolescente , Adulto , Doença de Chagas/imunologia , Estudos Controlados Antes e Depois , Citocinas/metabolismo , Feminino , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Imunomodulação , Mediadores da Inflamação/metabolismo , Masculino , Pessoa de Meia-Idade , Neutrófilos/imunologia , Receptores de IgG/genética , Receptores de IgG/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Adulto JovemRESUMO
Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.
Assuntos
Humanos , Adulto Jovem , /sangue , Citometria de Fluxo/normas , Leucócitos Mononucleares/metabolismo , Azul Tripano , Contagem de Células , Separação Celular , Sobrevivência Celular , Membrana Celular/fisiologia , Fluorescência , Imunofenotipagem , Indicadores e Reagentes/normas , Complexos Multiproteicos/normas , Competência Profissional , Propídio/normas , Coloração e Rotulagem , Soroalbumina Bovina/normasRESUMO
Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.
Assuntos
Complexo CD3/sangue , Citometria de Fluxo/normas , Leucócitos Mononucleares/metabolismo , Azul Tripano , Contagem de Células , Membrana Celular/fisiologia , Separação Celular , Sobrevivência Celular , Fluoresceína-5-Isotiocianato , Fluorescência , Humanos , Imunofenotipagem , Indicadores e Reagentes/normas , Complexos Multiproteicos/normas , Competência Profissional , Propídio/normas , Soroalbumina Bovina/normas , Coloração e Rotulagem , Adulto JovemRESUMO
Haemophilia A is a hereditary bleeding disorder linked to the X chromosome characterized by a deficiency or defect in the coagulation factor VIII (FVIII). Individuals with this coagulopathy require constant infusions of FVIII to maintain their physical integrity and haemostasis. During treatment, some patients develop an immune response that produces antibodies to FVIII, also called inhibitors, affecting the pro-coagulant activity of this protein. Despite the clinical relevance of FVIII inhibitors, the immune mechanisms that lead to their production are not known. This study investigated the immunological cytokine profile using plasma from HA patients which were either positive or negative for FVIII inhibitors and from healthy individuals. The results showed that healthy individuals and HA patients that do not develop FVIII inhibitors have a mixed immune response profile with high secretion of IFN-γ, TNF-α IL-2 and IL-5. In contrast, HA patients with FVIII inhibitors exhibited an anti-inflammatory/regulatory immune response characterized by low levels of all measured cytokines except for IL-4 and IL-10. This profile may be related to the development and maintenance of the FVIII inhibitors. By comparing the cytokine profiles of the three different groups we have established a model explaining the immune activation resulting in the production of FVIII inhibitors in haemophilia A patients.