Maria Carmen Jimenez-Martinez

Intracellular expression of interleukin-4 and interferon-γ by a Mycobacterium tuberculosis antigen-stimulated CD4+ CD57+ T-cell subpopulation with memory phenotype in tuberculosis patients

In some chronic pathological conditions, antigen persistence activates and expands the CD4+ CD57+ T‐cell subset. The host immune response against tuberculosis infection is maintained through the continuous presence of antigen‐stimulated effector/memory helper T cells. To determine whether CD4+ CD57+ T cells were also expanded in human tuberculosis, we analysed (by flow cytometry) the phenotype of peripheral blood CD4+ T cells from 30 tuberculosis patients and 30 healthy controls. We observed a significant increase in the CD4+ CD57+ T‐cell subset in tuberculosis patients in comparison to healthy controls (P < 0·001). Most CD4+ CD57+ T cells exhibited a CD28− CD45RO+ CD62L− phenotype, which is associated with memory cells. In vitro, a higher number of antigen‐stimulated CD4+ CD57+ T cells produced intracellular interferon‐γ and interleukin‐4 compared with antigen‐stimulated CD4+ CD57− T cells (P < 0·001). These findings suggest that the majority of CD4+ CD57+ T cells correspond to a phenotype of activated memory T cells.

Amniotic Membrane is an Immunosuppressor of Peripheral Blood Mononuclear Cells

Amniotic membrane (AM) is the inner layer of the placenta, which is in contact with the fetus; it has been used for transplantation in ocular surface diseases. It has been reported that amniotic membrane promotes epithelialization, inhibits angiogenesis and diminishes ocular inflammation. A persistent epithelial defect is the delay in epithelial wound healing caused by infiltrating inflammatory cells into the cornea and amniotic membrane transplantation has been successfully used in its treatment, however the mechanism of action in inhibiting inflammation it is not well understood. This study was aimed at determining whether denuded amniotic membrane (dAM) induces anti-inflammatory effects on peripheral blood mononuclear cells. Methods: Human peripheral blood mononuclear cells (PBMC) were cultured on dAM. Proliferation and apoptosis assays were performed on PBMC; and synthesis and secretion of pro-inflammatory cytokines by these cells was analyzed. Results: dAM induced apoptosis and inhibited cell proliferation of PBMC; and abolished the synthesis and the secretion of pro-inflammatory cytokines even when they were LPS stimulated. In contrast, when PBMC were cultured on hydrophilic membrane cell culture inserts, apoptosis was not significantly induced, cell proliferation was conserved, and synthesis and secretion of pro-inflammatory cytokines were not affected. Conclusions: Taken together, these results could explain the anti-inflammatory in vivo effects observed when the amniotic membrane is used as a transplant.

Pars planitis is associated with an increased frequency of effector-memory CD57+ T cells

Aim: To evaluate the frequency, phenotype and the potential function of CD57+ T cell subsets in patients with pars planitis. Methods: CD4+CD57+ and CD8+CD57+ T cells were quantitated in peripheral blood from 15 patients with pars planitis and 15 healthy controls. To evaluate the phenotype and potential function of CD57+ T cell subsets CCR7, CD27, CD28, CD45RA, CD45RO, intracellular IFN-&ggr;, IL-4, perforin and granzyme-A expression were assessed by flow cytometry. Results: CD57+ T cells subsets were increased in patients with pars planitis (p = 0.002). The majority of CD4+CD57+ T cells were CCR7−CD27−CD28−CD45RO+, while the most CD8+CD57+ T cells were CCR7−CD27−CD28−CD45RA+. The number of cells positive for intracellular IFN-&ggr; and IL-4 was higher in the CD57+ T cell populations. A greater number of CD8+CD57+ T cells than CD8+CD57− T cells were positive to perforin (p = 0.006) and granzyme-A (p = 0.01). Conclusions: CD57+ T cells had a phenotype associated with peripheral memory (CCR7−CD27−CD28−). Cytokine production by CD57+ T cells suggests that these cells may play a role in helper cell regulation. High expression of intracellular proteins involved in cytotoxicity suggests that CD8+CD57+ T cells may play an effector role. Taken together, this study proposes that CD57+ T cells function as memory-effector T cell subsets during pars planitis pathogenesis.

Familial case of Blau syndrome associated with a CARD15/NOD2 mutation

Purpose: Blau syndrome is a rare autosomal dominant disorder characterized by early onset granulomatous arthritis, uveitis, skin rash and camptodactyly. We report a familial case of Blau syndrome associated with a CARD15/NOD2 mutation. Methods: PCR amplification and automated DNA sequencing of the complete CARD15/NOD2 coding sequence was performed. Results: Molecular analysis in affected subjects disclosed a heterozygous c.1147G>C point mutation in CARD15/NOD2 exon 4, that predicts a p.E383K change at the protein level. Conclusions: Blau syndrome should be considered in the differential diagnosis of childhood uveitis and the genetic analysis of the CARD15/NOD2 gene is helpful in the diagnosis.

Differential expression of a 70 kDa O-glycoprotein on T cells: a possible marker for naive and early activated murine T cells.

We purified a 70 kDa O-glycoprotein that binds to the GalNAc specific lectin from Amaranthus leucocarpus (ALLr) and determined its expression pattern on T lymphocytes from different murine lymphoid organs. High level of ALLr expression was demonstrated in 95-98% of both CD4(+)8(+) and CD4(-)8(+) thymocytes, and in 80-95% of CD8(+) T cells from peripheral blood, lymph nodes, and spleen, whereas a minor fraction of CD4(+)8(-) thymocytes (46-67%) and peripheral CD4(+) T cells (9-40%) showed low ALLr expression. Peripheral CD19(+) B cells were ALLr negative and most of the peripheral ALL(+) T cells showed a CD62L(hi)CD45RB(hi)CD44(lo/-) phenotype, indicating features of naive cells. Mitogenic activation of peripheral T cells increased 3-fold the number of ALL(+)CD4(+) T cells 24 h after stimulation, as opposed to a >80% decrease in CD8(+) T cells 72 h after stimulation. Our results suggest that ALL detects a non-described surface O-glycoprotein selectively expressed by naive CD8(+) T cells and by early activated CD4(+) T cells.

An imbalance between frequency of CD4+CD25+FOXP3+ regulatory T cells and CCR4+ and CCR9+ circulating helper T cells is associated with active perennial allergic conjunctivitis.

Allergic conjunctivitis (AC) is one of the most common eye disorders in ophthalmology. In mice models, it has been suggested that control of allergic conjunctivitis is a delicate balance between Tregs and inflammatory migrating effector cells. Our aim was to evaluate the frequency of Tregs and the frequency of homing receptors expressing cells in peripheral blood mononuclear cells (PBMC) from patients with perennial allergic conjunctivitis (PAC). The analyses of phenotypic markers on CD4+ T cells and both soluble or intracellular cytokines were performed by flow cytometry. CD4+CD25+ cells were 15 times more frequent in PBMC from patients than HC; the vast majority of these CD4+CD25+ cells were FOXP3−, and most of CD4+ T cells were CCR4+ and CCR9+ cells. Upon allergen-stimulation, no significant changes were observed in frequency of Treg; however, an increased frequency of CD4+CCR4+CCR9+ cells, CD4+CD103+ cells and CD4+CD108+ cells with increased IL-5, IL-6, and IL-8 production was observed. These findings suggest an immune dysregulation in PAC, characterized by diminished frequency of Tregs and increased frequency of circulating activated CD4+ T cells; upon allergen-stimulation, these cells were expressing cell-surface molecules related to mucosa homing and were able to trigger an inflammatory microenvironment.

Increased expression of CD30 and CD57 molecules on CD4 T cells from children with atopic asthma: A preliminary report

T helper type 2 (Th2) cells play an important role in the onset and persistence of allergic airway inflammation. Consequently, many authors have attempted to identify cell surface markers associated with a Th2 phenotype. This work was aimed at correlating CD30 and CD57 expression on CD4(+) T cells with interleukin (IL)-4 production in peripheral blood mononuclear cells (PBMCs) from allergic patients. PBMCs from 17 children with atopic asthma and 12 nonatopic healthy control children were analyzed. The CD28, CD30, CD40L, CD57, CD62L, CD69, IL-4, and IFN-gamma expressions on CD4(+) T cells were determined by double immunofluorescence and flow cytometry in PBMCs ex vivo and after phorbol-12-myristate-13-acetate plus ionomycin (PMA/I) stimulation. An increased percentage of peripheral CD4(+)CD30(+) T cells was observed in asthmatic patients (p < 0.001). In addition, the percentage of CD4(+) T cells expressing IL-4, IFN-gamma, CD30, CD40L, CD57, or CD69 significantly increased (p < 0.01) after PMA/I stimulation, in asthmatic patients. The CD30 expression on CD4(+) T cells from asthmatic patients, after stimulation, correlated with both IL-4 and IFN-gamma production, whereas CD57 expression only correlated with IL-4 production. These data suggest that the expression of CD30 and CD57 cell markers on T cells could reflect circulating effector T cell early activation in the allergic airway disease.

Overexpression of peroxiredoxin 2 in pterygium. A proteomic approach.

Pterygium is one of the most frequent pathologies in ophthalmology, and is a benign, fibrovascular lesion originating from the bulbar conjunctiva. It is composed of an epithelium and highly vascular, subepithelial, loose connective tissue. The etiology of pterygium is not clearly understood; the most widely recognized originating factor is ultraviolet radiation. It has been proposed that pterygium and neoplasia have common features, raising the possibility that pterygium is a neoplastic-like growth disorder. In this study, proteomic analysis was performed to show that peroxiredoxin 2 is overexpressed in pterygia compared to healthy conjunctivas. Twelve pterygium specimens were obtained together with healthy conjunctival tissue from the same eyes. Total proteins of pterygia and healthy conjunctivas were analyzed in SDS-PAGE. This analysis showed protein bands expressed exclusively in pterygium samples at the range of 20-25 kDa. After this, 2D electrophoresis was performed for the separation of total proteins; differential spots expressed in pterygium were excised and sequenced. Mass spectrometry (MS) data were searched in the NCBInr and EST databases using the MASCOT program. The spot was identified as peroxiredoxin 2. Real-time PCR, western blot and immunohistochemistry showed that peroxiredoxin 2 was increased in pterygium compared to healthy conjunctiva. Although, these results suggest that overexpression of peroxiredoxin 2 in pterygium could protect the cell against oxidative stress-induced apoptosis, further studies are required to establish the functional role of peroxiredoxin 2 in pterygium to determine its role in peroxidation and apoptosis in this pathology.

Immunophenotyping in peripheral blood mononuclear cells, aqueous humour and vitreous in a Blau syndrome patient caused by a novel NOD2 mutation

The genetic and immunophenotypic characteristics of a 3‐year‐old patient with Blau syndrome (BS), an early onset sarcoidosis caused by mutations in NOD2, were investigated. Molecular analysis of NOD2 gene was achieved by PCR and direct nucleotide sequencing. Immunophenotyping included cytometric analysis of memory‐effector markers on T‐cells, and cytokine in serum, aqueous humour and vitreous. A novel M513R mutation in NOD2 was demonstrated. Immunophenotyping revealed higher frequency of CCR4+ cells and CCR9+ cells on CD4+ cells; most CD8+ cells were CCR7− and CCR9+. IL6 and IL‐8 were detected in a gradient manner: vitreous humour > aqueous humour > serum. The immunophenotype in this patient was characterized by a differential expression of chemokine receptors on T cells and by a particular ocular microenvironment enriched in IL‐6 and IL‐8. To our knowledge, this is the first study analysing the immunological features of BS at aqueous humour, vitreous and blood levels. Our results expand the knowledge of the genetic and immunopathological basis of BS.

Expression of IL-8, IL-6 and IL-1β in Tears as a Main Characteristic of the Immune Response in Human Microbial Keratitis

Corneal infections are frequent and potentially vision-threatening diseases, and despite the significance of the immunological response in animal models of microbial keratitis (MK), it remains unclear in humans. The aim of this study was to describe the cytokine profile of tears in patients with MK. Characteristics of ocular lesions such as size of the epithelial defect, stromal infiltration, and hypopyon were analyzed. Immunological evaluation included determination of interleukine (IL)-1β, IL-6, IL-8, IL-10, IL-12 and tumor necrosis factor (TNF)-α in tear samples obtained from infected eyes of 28 patients with MK and compared with their contralateral non-infected eyes. Additionally, frequency of CD4+, CD8+, CD19+ and CD3−CD56+ cells was also determined in peripheral blood mononuclear cells in patients with MK, and compared with 48 healthy controls. Non-significant differences were observed in the size of the epithelial defect, stromal infiltration, and hypopyon. Nevertheless, we found an immunological profile apparently related to MK etiology. IL-8 > IL-6 in patients with bacterial keratitis; IL-8 > IL-6 > IL-1β and increased frequency of circulating CD3−CD56+ NK cells in patients with gram-negative keratitis; and IL-8 = IL-6 > IL-1β in patients with fungal keratitis. Characterization of tear cytokines from patients with MK could aid our understanding of the immune pathophysiological mechanisms underlying corneal damage in humans.