Platelet-Derived Microvesicles Contribute to the Pathophysiogenesis of Human Cutaneous Leishmaniasis: A Nano-Flow Cytometric Approach in Plasma Samples from Patients before and under Antimonial Treatment.

Cutaneous leishmaniasis is a neglected tropical disease caused, in Brazil, mainly by Leishmania braziliensis, which is a protozoan transmitted during the blood feeding of infected female sandflies. To control leishmaniasis, the participation of CD4+ Th1 cells together with macrophages, neutrophils, and other peripheral blood cells, including platelets, is necessary. These anuclear fragments, when activated, produce microvesicles (MVs) that can reach locations outside the blood, carrying molecules responsible for activating pro-inflammatory responses and antigen presentation. Using flow cytometry, this current study evaluated the frequency and concentration of platelet-derived MVs (pMVs) in plasma samples obtained from patients in the acute phase and undergoing treatment, as well as from healthy volunteers. Our results revealed a higher frequency and concentration of pMVs in the plasma of patients with acute CL when compared to all other groups studied. These results highlight the impact of pMVs in modulating the immune response of CL patients, correlating their higher concentrations and frequencies in CL-patient plasmas, with the acute inflammatory status of the disease and their reduction with beneficial results of systemic treatment with antimony. This knowledge is essential to define potential treatment protocols, as well as highlight pMVs as biomarkers for the different clinical stages of CL.

Successful editing and maintenance of lactogenic gene expression in primary bovine mammary epithelial cells.

In vitro investigation of bovine lactation processes is limited by a lack of physiologically representative cell models. This deficiency is most evident through the minimal or absent expression of lactation-specific genes in cultured bovine mammary tissues. Primary bovine mammary epithelial cells (pbMECs) extracted from lactating mammary tissue and grown in culture initially express milk protein transcripts at relatively representative levels. However, expression drops dramatically after only three or four passages, which greatly reduces the utility of primary cells to model and further examine lactogenesis. To investigate the effects of alternate alleles in pbMECs including effects on transcription, we have developed methods to deliver CRISPR-Cas9 gene editing reagents to primary mammary cells, resulting in very high editing efficiencies. We have also found that culturing the cells on an imitation basement membrane composed of Matrigel, results in the restoration of a more representative lactogenic gene expression profile and the cells forming three-dimensional structures in vitro. Here, we present data from four pbMEC lines recovered from pregnant cows and detail the expression profile of five key milk synthesis genes in these MECs grown on Matrigel. Additionally, we describe an optimised method for preferentially selecting CRISPR-Cas9-edited cells conferring a knock-out of DGAT1, using fluorescence-activated cell sorting (FACS). The combination of these techniques facilitates the use of pbMECs as a model to investigate the effects of gene introgressions and genetic variation in lactating mammary tissue.

Flow-Cytometric Method for Viability Analysis of Mycoplasma gallisepticum and Other Cell-Culture-Contaminant Mollicutes.

Mycoplasma is the smallest self-replicating bacteria, figuring as common contaminant of eukaryotic cell cultures. Production inputs and operator's manipulation seem to be the main sources of such contamination. Many analytical approaches have been applied for mycoplasma detection in cell cultures and also in biological products. However, unless they were validated, only indicator cell culture and bacteriological culture are considered as compendial methods for quality control of biological products. Nano-flow cytometry has been pointed out as an alternative technique for addressing prokaryotic and eukaryotic cell viability being a substantial tool for reference material production. In this study, a viability-flow-cytometry assay was standardized for M. gallisepticum and then applied to other cell-culture-contaminant mycoplasmas. For this, M. galliseticum's growth rate was observed and different treatments were evaluated to establish low viability cultures (cell death-induced control). Distinct viability markers and their ideal concentrations (titration) were appraised. Ethanol treatment showed to be the best death-inducing control. CFDA and TOPRO markers revealed to be the best choice for detecting live and dead mycoplasma frequencies, respectively. The standardized methodology was applied to Mycoplasma arginini, M. hyorhinis, M. orale, Spiroplasma citri and Acholeplasma laidlawii. Significant statistical difference was observed in the percentage of viable cells in comparison to ethanol treatment for A. laidlawii in CFDA and in both markers for M. gallisepticum, M. hyorhinis and S. citri. In summary, we standardized a flow cytometry assay for assessing M. gallisepticum - and potentially other species - viability and ultimately applied for reference material production improving the quality control of biological products.

Vernonia brasiliana (L.) Druce induces ultrastructural changes and apoptosis-like death of Leishmania infantum promastigotes.

The present study aimed to evaluate the antileishmanial effect, the mechanisms of action and the association with miltefosine of Vernonia brasiliana essential oil against Leishmania infantum promastigotes. This essential oil was obtained by hydrodistillation and its chemical composition was determined by gas chromatography-mass spectrometry (GC-MS). The antileishmanial activity against L. infantum promastigotes and cytotoxicity on DH82 cells were evaluated by MTT colorimetric assay. Ultrastructural alterations were evaluated by transmission electron microscopy. Changes in mitochondrial membrane potential, in the production of reactive oxygen species, and analysis of apoptotic events were determined by flow cytometry. The association between the essential oil and miltefosine was evaluated using the modified isobologram method. The most abundant component of the essential oil was ß-caryophyllene (21.47 %). Anti-Leishmania assays indicated an IC50 of 39.01 ±â€¯1.080 µg/mL for promastigote forms after 72 h of treatment. The cytotoxic concentration for DH82 cells was 63.13 ±â€¯1.211 µg/mL after 24 h of treatment. The effect against L. infantum was proven through the ultrastructural changes caused by the oil, such as kinetoplast and mitochondrial swelling, vesicles in the flagellar pocket, discontinuity of the nuclear membrane, nuclear fragmentation and condensation, and loss of organelles. It was observed that the oil leads to a decrease in the mitochondrial membrane potential (35.10 %, p = 0.0031), increased reactive oxygen species production, and cell death by late apoptosis (17.60 %, p = 0.020). The combination of the essential oil and miltefosine exhibited an antagonistic effect. This study evidences the antileishmanial action of V. brasiliana essential oil against L. infantum promastigotes.

CD49d Is Upregulated in Circulating T Lymphocytes from HTLV-1-Infected Patients.

INTRODUCTION: HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic progressive myelopathy associated with an inflammation of the central nervous system (CNS), being characterized by perivascular infiltration of inflammatory cells. HTLV-1-infected cells have the capacity to migrate through endothelial layers by enhancing adhesion receptor expression and corresponding ligands. T cells interact with the extracellular matrix via integrin receptors and these interactions affect both cell migration and proliferation. The importance of these interactions in retrovirus-induced diseases, however, remains less clear. METHODS: Herein we studied the expression of 3 integrin alpha chains (CD49d, CD49e, and CD49f) on the membrane of T-cell subsets in patients infected by HTLV-1, both HAM/TSP patients and oligo/asymptomatic subjects who were asymptomatic or presented slight manifestations related to the virus infection. RESULTS: We observed higher peripheral blood frequency of CD49dhiCD4+ and CD49dhiCD8+ T cells in HTLV-1-infected patients. CONCLUSION: Our findings suggest that the increased expression of adhesion molecules, such as CD49d on T lymphocytes from HTLV-1-infected patients may contribute to the pathogenesis of the disease, in both oligo/asymptomatic and HAM/TSP-infected subjects. Accordingly, it is conceivable that there is a potential use of CD49d as target for a therapeutic approach aiming at blocking migration of activated T cells from HTLV-1-infected patients into the CNS, thus avoiding the progression to HAM/TSP.

A protocol for rapid monocyte isolation and generation of singular human monocyte-derived dendritic cells.

The monocyte-derived dendritic cells (moDCs) are a subset of dendritic cells widely used in immunological studies as a convenient and easy approach after isolation of mononuclear cells directly from peripheral blood mononuclear cells (PBMC). Both the purification and cell culture of monocytes impact on the differentiation of monocytes into moDCs. The methodology to isolate and differentiate monocytes into moDCs is still controversial. We aimed to compare three different protocols for monocyte isolation from PBMC: 1) Cold-aggregation; 2) Percoll gradient; and 3) Magnetic beads cell-enrichment. Additionally we also compared four different monocyte differentiation and culture techniques: 1) Cell culture media; 2) Serum sources; 3) required GM-CSF and IL-4 concentrations; 4) Cell culture systems. We used flow cytometry analysis of light scattering and/or expression of pan surface markers, such as CD3, CD14 and CD209 to determine isolation/differentiation degree. Purified PBMC followed by two steps of cold aggregation, yielded cell viability around 95% with poor monocyte enrichment (monocytes increase vs. lymphocytes reduction was not statistically significant, p>0.05). Conversely, monocyte isolation from PBMC with discontinuous Percoll gradient generated around 50% cell viability. Albeit, we observed a significant reduction (p≤0.05) of lymphocytes contaminants. The magnetic beads cell-enrichment yield cell viability higher than 95%, as high as a significant lymphocyte depletion (p≤0.005) when compared to all other techniques employed. The moDCs showed better differentiation based on increased CD209 expression, but lower CD14 levels, when cells were cultured in RPMI medium plus 500IU/mL of both GM-CSF and IL-4 in a semi-adherent fashion. Serum sources showed no influence on the culture performance. In conclusion, the magnetic beads cell-enrichment showed superior cell viability, indicating that this approach is a better choice to isolate monocytes, and moDCs cultured afterwards in appropriate medium, serum, cytokines and culture system might influence the monocytes differentiation into moDC.

Contribution of Leishmania braziliensis antigen-specific CD4+ T, CD8+ T, NK and CD3+CD56+NKT cells in the immunopathogenesis of cutaneous leishmaniasis patients: Cytotoxic, activation and exhaustion profiles.

The pathogenesis of cutaneous leishmaniasis (CL) caused by Leishmania (Viannia) braziliensis is dictated mainly by the immune-mediated-tissue inflammation developed. The understanding of the immunological mechanisms that generate tissue damage or resolution of lesions is the key to the development of effective vaccine protocols and proper therapeutic schemes. It is clear that the specific immune response mediated by T cells is responsible for the beneficial outcome of the disease, however, the roles of CD4+ T, CD8+ T, NK and NKT cell subpopulations in immunopathogenesis of CL need to be elucidated. Peripheral blood cells from patients before, during and after the antimonial therapy, as well as healthy individuals (HI) were cultured with (LbAgS) or without (NS) L. braziliensis antigens (LbAg). Afterwards, the frequencies of LbAg-specific-cytotoxic CD8+ T, CD4+ T, NK and CD3+CD56+ NKT cells, as well as their activation and exhaustion profiles, were defined by flow cytometry. We observed higher frequencies of CD8+ T, NK and CD3+CD56+ NKT cells and lower frequencies of CD4+ T lymphocytes in LbAgS cell cultures from patients before treatment. The specific response to LbAg resulted in an expansion of cytotoxic-activated CD4+ T, CD8+ T, and NK cells, before and during treatment, indicating specificity in the response by these cells against L. braziliensis. Furthermore, comparing the differences of frequencies of cytotoxic-activated CD4+T, CD8+T, and NK cells, among before and during treatment patients and HI groups, we conclude that these cell populations are in charge of immune response elicited by antimonial therapy. Interestingly, we also observed that NK cells were induced by LbAg to an exhaustion profile during all clinical stages of the disease. The increased antigen-specific activation and cytotoxic activity are in line with the strong inflammatory response described in this disease, a likely cause of tissue damage. These findings reinforce the involvement of these distinct cytotoxic-activated cell populations in the immunopathogenesis of CL, showing a character of specificity in this immune response.

Changes in hemocytes of Biomphalaria glabrata infected with Echinostoma paraensei and exposed to glyphosate-based herbicide.

The immune system of snails is highly sensitive to pollutants, which can suppress its immune response. We investigated the effects of exposure to the glyphosate-based herbicide Roundup® Original on the snail Biomphalaria glabrata infected by the platyhelminth Echinostoma paraensei by evaluating changes in the snail's internal defense system. Four cohorts were studied: control group, infected snails, snails treated with Roundup®, and snails infected and treated with Roundup®. The hemocyte viability was assessed, morphological differentiation of cells was observed and flow cytometry was performed to determine the morphology, viability and the lectin expression profiles. The frequencies of dead hemocytes were lower in the infected group and higher in both pesticide treated groups. Three cell types were identified: blast-like cells, hyalinocytes and granulocytes. The highest number of all types of hemocytes, as well as the highest number of dead cells, were observed in the infected, pesticide-treated group. The association between infection and herbicide exposure greatly increased the frequency of dead hemocytes, suggesting that this condition impairs the internal defense system of B. glabrata making the snails more vulnerable to parasitic infections.