Martha C. Moreno-Lafont

Natural Killer T Cells Activated by a Lipopeptidophosphoglycan from Entamoeba histolytica Are Critically Important To Control Amebic Liver Abscess

The innate immune response is supposed to play an essential role in the control of amebic liver abscess (ALA), a severe form of invasive amoebiasis due to infection with the protozoan parasite Entamoeba histolytica. In a mouse model for the disease, we previously demonstrated that Jα18-/- mice, lacking invariant natural killer T (iNKT) cells, suffer from more severe abscess development. Here we show that the specific activation of iNKT cells using α-galactosylceramide (α-GalCer) induces a significant reduction in the sizes of ALA lesions, whereas CD1d−/− mice develop more severe abscesses. We identified a lipopeptidophosphoglycan from E. histolytica membranes (EhLPPG) as a possible natural NKT cell ligand and show that the purified phosphoinositol (PI) moiety of this molecule induces protective IFN-γ but not IL-4 production in NKT cells. The main component of EhLPPG responsible for NKT cell activation is a diacylated PI, (1-O-[(28∶0)-lyso-glycero-3-phosphatidyl-]2-O-(C16:0)-Ins). IFN-γ production by NKT cells requires the presence of CD1d and simultaneously TLR receptor signalling through MyD88 and secretion of IL-12. Similar to α-GalCer application, EhLPPG treatment significantly reduces the severity of ALA in ameba-infected mice. Our results suggest that EhLPPG is an amebic molecule that is important for the limitation of ALA development and may explain why the majority of E. histolytica-infected individuals do not develop amebic liver abscess.

Antigen-specific activation and proliferation of CD4+ and CD8+ T lymphocytes from brucellosis patients

Salt-extractable antigen from Brucella melitensis 16M (RCM-BM) was used to evaluate the immune response from acute and chronic patients suffering from Brucella infections (in Mexico); their responses were compared with those of healthy controls. As a readout we used upregulation of CD69 (a well-established early activation marker for lymphocytes), lymphocyte proliferation by 3[H]thymidine or 5-bromo-2-deoxyuridine (BrdU) incorporation measured by liquid scintillation or flow cytometry, respectively, and production of gamma interferon (IFN gamma). We compared the antigen-specific response with the response induced by phytohaemagglutinin (PHA) as a positive control. There was no difference between acute patients and the healthy controls in the percentages of CD3+, CD4+ or CD8+ lymphocytes. However, we found that chronic patients had a significant (P < 0.05) increase in the CD8+ T cells, in line with previous studies. Antigen-specific responses to RCM-BM showed a significant (P < 0.05) upregulation of CD69 in both CD4+ and CD8+ T lymphocytes in acute brucellosis patients and in CD8+ T lymphocytes in chronic patients, indicating that both populations became activated by this antigen preparation. Moreover, lymphocyte proliferation from both acute and chronic patients in response to RCM-BM was highly significant (P < 0.001) when compared with healthy controls. However, there were no apparent differences between acute and chronic patients. Although the incorporation of BrdU showed similar results it provided additional information, since we demonstrated that both CD4+ and CD8+ T lymphocytes from acute and chronic patients proliferated equally well in response to RCM-BM. Similar results were observed with intracellular IFN gamma determination. As a whole, our data suggest an important role for both CD4+ and CD8+ T lymphocytes in Brucella infection in humans. As has been reported in mice, it is feasible that activated CD8+ T cells participate in protection against Brucella in humans through cytotoxicity or/and by the production of factors such as interferon and granulysin. The role of these cells should be carefully analysed to understand better their participation in human infection by Brucella.

Comparative proteome analysis of Brucella abortus 2308 and its virB type IV secretion system mutant reveals new T4SS-related candidate proteins

Brucella abortus is an alpha-2 proteobacteria with a type IV secretion system (T4SS) known as virB, which is necessary to gain virulence by building up a replicative vacuole associated with the endoplasmic reticulum of the host cell. A virB T4SS mutant of the B. abortus 2308 strain and its wild-type strain were grown in acid medium in order to obtain and analyze their proteomes, looking for putative proteins that may serve as T4SS substrates and those that may be subjected to T4SS regulation. A total of 47 overexpressed and 22 underexpressed proteins from the virB T4SS mutant strain were selected and sequenced. Some of the 69 analyzed proteins have not been described before either as over or under-expressed in relation to a virB T4SS mutation, whereas some of them have been already described by other groups as potentially important secretory proteins in other Brucella species. An important number of the proteins identified are outer membrane and periplasmic space protein, which makes them become particularly important new T4SS-related candidate proteins.

Granulocyte colony-stimulating factor produces a decrease in IFNγ and increase in IL-4 when administrated to healthy donors

Hematopoietic stem cells transplantation (HSCT) is the leading curative therapy for a variety of hematological and hereditary diseases; however, graft versus host disease (GVHD), an immunologic phenomenon that is favored by Th1 cytokines and cytotoxic cells from donors, is present frequently and is one of the most important causes of transplant related mortality. Peripheral blood HSCT is the preferred source of stem cells in almost 100% of the cases of autologous HSCT and in 70% of allogeneic transplants. The best mobilizing agent to get the stem cells out from the bone marrow is the Granulocyte‐Colony Stimulating Factor (G‐CSF). In this work, our main objective was to study a possible correlation between the graft cell dose and the patients clinical outcome. We evaluated the immunologic changes produced by G‐CSF in the lymphocyte and cytokine profiles in allogeneic HSC donors. HSC from twelve donors were mobilized with G‐CSF at 16 μg/kg/day, for 5 days. Basal Peripheral Blood (BPB), Mobilized Peripheral Blood (MPB), and aphaeresis mononuclear cells (G‐MNC) samples were taken from all donors. Using flow cytometry, we quantified CD19+, CD3+, CD3+CD4+, CD3+CD8+, NK, NKT, DC1, and DC2 cells. Cytokines were determined by ELISA in culture supernatants. CD19+ (p = 0.001), DC1 (p < 0.002) and DC2 (p < 0.001) cells were increased in MPB with respect to BPB. An increase in Th2 cytokines such as (IL‐4) and a decrease in Th1 cytokines (IFNγ, IL‐2) were also found in MPB samples. In conclusion, Th1 and Th2 cytokines are relevant in predicting the clinical outcome after allogeneic peripheral blood HSCT. J. Clin. Apheresis 25:181–187, 2010.

Role of CD8 Regulatory T Cells versus Tc1 and Tc17 Cells in the Development of Human Graft-versus-Host Disease

CD8+ T cells that secrete proinflammatory cytokines play a central role in exacerbation of inflammation; however, a new subpopulation of CD8 regulatory T cells has recently been characterized. This study analyzes the prominent role of these different subpopulations in the development of graft-versus-host disease (GVHD). Samples from 8 healthy donors mobilized with Filgrastim® (G-CSF) and 18 patients who underwent allogeneic hematopoietic stem cell transplantation (HSCT) were evaluated by flow cytometry. Mobilization induced an increase in Tc1 (p < 0.01), Th1 (p < 0.001), Tc17 (p < 0.05), and CD8+IL-10+ cells (p < 0.05), showing that G-CSF induces both pro- and anti-inflammatory profiles. Donor-patient correlation revealed a trend (p = 0.06) toward the development of GVHD in patients who receive a high percentage of Tc1 cells. Patients with acute GVHD (aGVHD), either active or controlled, and patients without GVHD were evaluated; patients with active aGVHD had a higher percentage of Tc1 (p < 0.01) and Tc17 (p < 0.05) cells, as opposed to patients without GVHD in whom a higher percentage of CD8 Treg cells (p < 0.01) was found. These findings indicate that the increase in Tc1 and Tc17 cells is associated with GVHD development, while regulatory CD8 T cells might have a protective role in this disease. These tests can be used to monitor and control GVHD.

Determination of Th1/Th2/Th17 Cytokines in Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic cell transplantation emerges as a therapy option to treat the sequels of exposure to radiation, a great concern at the beginning of the atomic age and cold war (Welniak et al., 2007). Hematopoietic cell transplantation emerged as a rescue strategy since there were already antecedents, like the study of Lorenz et al. in 1952 (as cited by Welniak et al., 2007), who showed that infusion of the bone marrow after lethal irradiation healed radiation disease in mice. This lay the foundations for the current consideration of allogeneic hematopoietic cell transplantation as the first-line therapy for many lifethreatening oncological and hematological diseases. Today, it is primarily used to treat patients with hereditary anemias or immunological deficiencies through replacement of the hematopoietic system with cells from a healthy individual. It also allows cancer patients to be treated with myeloablative radiation and/or chemotherapy (known as myeloablative conditioning) in an attempt to eliminate tumoral cells, and although this strategy brings loss of bone marrow function, the latter can be recovered with infusion of normal hematopoietic cells (Jenq & Van den Brink, 2010).

Description of CD8

Gershon and Kondo described CD8+ Treg lymphocytes as the first ones with regulating activity due to their tolerance ability to foreign antigens and their capacity to inhibit the proliferation of other lymphocytes. Regardless, CD8+ Treg lymphocytes have not been fully described—unlike CD4+ Treg lymphocytes—because of their low numbers in blood and the lack of specific and accurate population markers. Still, these lymphocytes have been studied for the past 30 years, even after finding difficulties during investigations. As a result, studies have identified markers that define their subpopulations. This review is focused on the expression of cell membrane markers as CD25, CD122, CD103, CTLA-4, CD39, CD73, LAG-3, and FasL as well as soluble molecules such as FoxP3, IFN-γ, IL-10, TGF-β, IL-34, and IL-35, in addition to the lack of expression of cell activation markers such as CD28, CD127 CD45RC, and CD49d. This work also underlines the importance of identifying some of these markers in infections with several pathogens, autoimmunity, cancer, and graft-versus-host disease as a strategy in their prevention, monitoring, and cure.