A rapid method to verify single-cell deposition setup for cell sorters.

Studying single cells reveals biology that cannot be explored using bulk techniques. Cell sorters provide the opportunity of separating single cells either for cell culture or for downstream molecular applications such as qPCR to study specific gene expression and single cell mRNA sequencing. Some of these molecular studies can be expensive so the investigator will often want reassurance that the cell sorter can reliably deposit a single droplet into each well of a 96-well or 384-well plate. Such plates may contain very small volumes of fluid as reducing the volume of fluid used can reduce the cost of the assay. To miss some of the wells could leave the data set incomplete requiring costly repetition. To verify this by microscopy is at best very time consuming and at worst impossible. Here, an inexpensive colorimetric method is described for verifying whether a well, in either a 96- or 384-well plate, did receive a single sorted droplet from a cell sorter into the fluid at the bottom of the well. The droplet consists of particles suspended in an enzymatic solution, horseradish peroxidase, which is deposited into microtiter plate wells containing a substrate, 3,3',5,5'-tetramethylbenzidine. This method requires no special equipment or expertise and is rapid enough to be performed directly prior to the single-cell sorting experiment. © 2016 International Society for Advancement of Cytometry.

New insights into neutrophil and Leishmania infantum in vitro immune interactions.

The interaction between polymorphonuclear leukocytes (PMN) or neutrophils and Leishmania became an interesting focus of research, since PMN turn out to be essential cells in transiently hosting the parasites. This study aims to evaluate whether L. infantum, the etiological agent of zoonotic visceral leishmaniasis, influences the in vitro functional activity of murine neutrophils. Phagocytosis, chemotaxis, oxidative burst, degranulation and apoptosis assays were performed. Cytokines, chemokines and toll-like receptors gene expression were evaluated by Real-time PCR. Results indicate that some of the innate features of PMN immunity were activated when in contact with L. infantum. However, parasites might negatively interfere with PMN defense mechanisms compromising the link between innate and acquired immunity. This work provides additional insights on the inflammatory immune interactions between neutrophils and L. infantum highlighting the role of PMN in Leishmania infection.

Canine leishmaniasis. Immunophenotypic profile of leukocytes in different compartments of symptomatic, asymptomatic and treated dogs.

Canine visceral leishmaniasis (CanL) is an emerging disease, expanding in various parts of the world. The infection caused by Leishmania, an intracellular protozoan parasite, can show different clinical manifestations, from asymptomatic or subclinical to symptomatic dogs, in which a wide spectrum of clinical signs is evident. The fact that the parasite replicates in different organs raises the hypothesis that each organ may have a specific immune response. The local immune responses should be evaluated and taken into consideration when developing prophylactic tools. Therefore, phenotypic characterization of peripheral blood, lymph node and bone marrow lymphocyte populations and the expression of class II molecules of major histocompatibility complex (MHCII) were performed in asymptomatic and symptomatic dogs and in dogs that had been diagnosed and treated for leishmaniasis. Our findings showed that blood and bone marrow lymphocytes from symptomatic dogs were highly activated. In bone marrow of asymptomatic and treated dogs, a high frequency of MHCII(+) lymphocytes was observed, as well as MHCII(+) monocytes in the treated group. These results show increased expression of MHCII molecules giving evidence for antigenic presentation mainly by lymphocytes. The symptomatic and treated dogs showed an expansion of CD4(+) T cells subpopulations in lymph nodes, revealing an important contribution of these cells in controlling local parasite replication. This study also underlines the eventual importance of CD3(+)CD4(-)CD8(-) (double negative) and CD3(+)CD4(+)CD8(+) (double positive) T cell subsets in sensing and controlling latent infections and their possible function in the immune dynamics during CanL. The specific cellular immune responses raised in different compartments where the parasite replicates seem to have variable effects on local parasite control, highlighting the complexity of the cellular immune response developed by the dog infected by Leishmania infantum.

Influence of dyslipidemia and smoking on redox markers in humans--a critical study.

UNLABELLED: Experimental research indicates that oxidative processes play a role in susceptibility to a large number of diseases. A better understanding of the parameters affecting redox balance could delay and even prevent such processes. OBJECTIVE: The present study aims to investigate blood parameters associated with antioxidant systems in a Portuguese population for the first time, taking into consideration gender, age range, lipid profile and smoking habits as influencing factors. DESIGN AND PARTICIPANTS: One hundred and eighty-three healthy Portuguese subjects of both genders were recruited from the metropolitan area of Lisbon. The group consisted of individuals aged from 20 to 70 years, who gave their informed consent before participating in the study. All subjects were screened to determine eligibility, which was based on a clinical report. Subjects were considered eligible if they had no acute or chronic illness and were not taking any drugs or dietary supplements that could compromise the values of the studied parameters. The subjects were then divided into different subgroups according to gender, age range, lipid profile and smoking habits. METHODS: Whole blood glutathione peroxidase activity and serum albumin, transferrin and uric acid were determined using commercially available kits. Superoxide dismutase activity in erythrocytes and thiobarbituric acid reactive substances in serum were measured using methods published elsewhere. RESULTS: Glutathione peroxidase activity was not affected by any of the studied variables, but superoxide dismutase activity decreased with smoking. Albumin levels remained unchanged under all conditions. Hyperlipidemia was associated with higher lipid peroxidation as well as higher uric acid levels. Gender was the strongest predictor for transferrin, total iron binding capacity and uric acid variations. Finally, a multivariate statistical model clearly separated genders and lipid profile and genders and smoking. CONCLUSIONS: The present study suggests that hyperlipidemia and smoking should be considered important selection criteria in epidemiological studies focusing on oxidative stress and on the atherosclerotic process.

Identification of regulatory T cells during experimental Leishmania infantum infection.

Leishmania infantum is the causative agent of zoonotic visceral leishmaniasis (ZVL), a disease frequently characterized by specific impairment of cell-mediated immune responses and uncontrolled parasitization. Regulatory T cells (Treg) have been shown to be involved in the direct induction of immunosuppression of effector immune response during chronic Leishmania infections. The present study aims to investigate the possible involvement of Treg cells during L. infantum infection. Results indicate that CD4(+)CD25(+) regulatory T cells are present in L. infantum-infected BALB/c mice and exhibit phenotypic and functional characteristics of Treg. The presence of high levels of Foxp3 gene expression and surface expression of alpha(E)beta(7) integrin (CD103) suggest a predisposition for Treg retention within sites of L. infantum infection, as is the case of the spleen and draining lymph nodes, consequently influencing local immune response. Th1 and Th2 effector immune responses seem inadequate, due to Treg expansion. Foxp3 expressing CD4(+)CD25(+) T cells are capable of producing TGF-beta and may contribute to immunosuppression and better control of parasite-mediated-immunopathology during infection. Surprisingly, IL-10 producing-CD4(+)CD25(-)Foxp3(-) T cells were also identified as an additional source of IL-10 and may represent a type 1 regulatory T (Tr1) cell subset that is being induced by L. infantum parasites. These findings suggest that distinct regulatory T cells develop in response to L. infantum and may play a possible role in promoting parasite persistence and the establishment of chronic infection.

Immunization with Leishmania infantum released proteins confers partial protection against parasite infection with a predominant Th1 specific immune response.

In this study, protective effect and immune response elicited by protein fractions LiRic1 (>75 kDa) and LiRic2 (<37 kDa) released by Leishmania infantum promastigotes were analysed in challenged BALB/c mice. Viable parasites were quantified in spleen and isolated CD4(+) and CD8(+) T cells were stimulated for evaluation of proliferative response and cytokine production. Immunization triggered 50.4-66.9% of parasite reduction. Stimulated CD4(+) T cells from challenged animals revealed high proliferation. IL-12 and IFN-gamma were released by CD4(+) T cells whereas IL-4 and IL-10 were impaired. LiRic1 and LiRic2 immunization gave partial protection and a CD4(+) Th1 response. LiRic2 generated IL-12 by CD8(+) T cells pointing to its participation in protective response. These results encourage further research on the development of a vaccine that provides long-lasting protection against zoonotic visceral leishmaniasis.

Leishmania infantum released proteins specifically regulate cytokine expression and production patterns by CD4+ and CD8+ T cells.

Specific immune responses by CD4+ and CD8+ T cells, from two infected mice strains (BALB/c and C57BL/6), induced by High, Inter and Low protein fractions released by Leishmania infantum, were assessed through the evaluation of IL-12, IFN-gamma and IL-10 mRNA by real-time PCR and respective protein production by ELISA. During infection establishment, High and Inter fractions directed both mice strains T cells subsets to increase the production of IFN-gamma, associated to IL-12 release. Later on, parasite replication augmented in BALB/c and stabilised in C57BL/6 mice. Inter fraction induced CD4+ T cells to maintain IFN-gamma production, with the simultaneous release of IL-12 by both cell subsets in BALB/c mice and by CD8+ T cells in C57BL/6 mice. These observations suggested a prophylactic potential for Inter fraction which was able to induce Th1 response with IL-12 involvement, required for the maintenance of memory cells, in mice strains with different parasitic evolution.

H-2 complex influences cytokine gene expression in Leishmania infantum-infected macrophages.

This work aims to study the influence of H-2 locus in the control of Leishmania infantum infection by evaluating whether cytokine responses by host macrophages of different H-2 haplotype are differentially regulated, either induced or actively impaired during parasite growth and replication. This study shows that macrophages of "non-cure" phenotype (H-2(d)) are more susceptible to infection with virulent L. infantum promastigotes. Virulent parasites lead to impaired IL-12 and inhibited TNF-alpha expression. The degree of parasite virulence is an important contributing factor to differences detected in cytokine expression. Virulent parasites also induced TGF-beta, a deactivating cytokine that is known to suppress Th-1 type responses, thus allowing the parasite to subvert antimicrobial activity and increase its chances of survival. Depending on specific host haplotype, cells differentially respond to infection since TNF-alpha expression is inhibited and TGF-beta is enhanced by macrophages of "non-cure" phenotype, thus perhaps determining their degree of susceptibility in this strain of mice.

Dynamics of CD62L/CD45RB CD4+ and CD8+ lymphocyte subsets in hepatic and splenic tissues during murine visceral leishmaniasis.

This study aimed to characterise, for the first time, the dynamics of CD4+ and CD8+ lymphocyte CD62L/CD45RB subsets, during visceral leishmaniasis. Memory/activated status of hepatic and splenic T cells was compared in mice strains with "cure" and "non-cure" phenotypes to Leishmania infantum infection. In both mice strains, a correlation between the dynamics of the memory CD4+ and CD8+ T cells (CD62Llow/CD45RBlow) subsets in the liver and the pre-activated phenotype of lymphocytes (CD62Llow/CD45RBhigh) from the spleen was detected suggesting that this organ is the source of Leishmania-specific T lymphocytes that migrate to the liver, where parasite replication is highly active. In the liver, these pre-activated cells become effector T lymphocytes, however, a strong regulation of CD8+ T cell effector function was observed, probably preventing hepatic tissue damage. Comparing mice strains with "cure" and "non-cure" phenotype, an imbalance between "protective" CD45RBhigh and "pathogenic" CD45RBlow CD4+ subsets in B10.D2/n animals might be involved in the evolution of a non-healing infection.

Hepatic cellular immune responses in mice with "cure" and "non-cure" phenotype to Leishmania infantum infection: importance of CD8+ T cells and TGF-beta production.

The objective of this study was to analyse hepatic cellular immune response of mice with "cure" and "non-cure" phenotypes to Leishmania infantum infection. During infection establishment, elevated TGF-beta levels and absence of a Th1 response may have contributed to parasite multiplication and to similar hepatic parasitic loads. Later in infection, an increase in the number and activation levels of CD8+ cells was observed simultaneously with parasite elimination, but only significant in "cure" strain. During this recovering phase, "non-cure" animals showed low Th2 cytokine levels, while TGF-beta production was higher than in "cure" mice. These results point out to a role for CD8+ T cells in liver acquired immune response and to TGF-beta regulation of "cure" and "non-cure" phenotype to L. infantum infection.