Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation

Flow cytometric detection of antigen-specific CD8+ T cells has previously been limited to MHC-class I tetramer staining or intracellular cytokine production, neither of which measure the cytolytic potential of these cells. Here we present a novel technique to enumerate antigen-specific CD8+ T cells using a marker expressed on the cell surface following activation induced degranulation, a necessary precursor of cytolysis. This assay measures the exposure of CD107a and b, present in the membrane of cytotoxic granules, onto the cell surface as a result of degranulation. Acquisition of cell surface CD107a and b is associated with loss of intracellular perforin and is inhibited by colchicine, indicating that exposure of CD107a and b to the cell surface is dependent on degranulation. CD107a and b are expressed on the cell surface of CD8+ T cells following activation with cognate peptide, concordant with production of intracellular IFNgamma. Finally, CD107-expressing CD8+ T cells are shown to mediate cytolytic activity in an antigen-specific manner. Measurement of CD107a and b expression can also be combined with MHC-class I tetramer labeling and intracellular cytokine staining to provide a more complete assessment of the functionality of CD8+T cells expressing cognate T cell receptors (TCR).     

Microvasculature of the buffalo epididymis

The microvasculature of the water buffalo (Bubalus bubalis) epididymis was investigated using light (LM), scanning electron (SEM), and transmission electron (TEM) microscopy techniques. SEM analysis of the buffalo epididymis showed fenestrations that occupied ovoid inside the endothelium of the postcapillary venules located in the caput, corpus, and cauda. They varied in shape and dimension, but more importantly, they connected the venules of the blood vascular system to the capillaries of the peripheral lymphatic vascular system. Morphofunctional analysis of these connections suggests that the microvasculature of the buffalo epididymis plays a role in facilitating the circulation of biologically active substances, and the absorption and secretion processes necessary for the survival and maturation of spermatozoa. The lymphatic capillaries at the connection points formed a network of variously sized polygonal links. These capillaries then converged to form the precollector lymphatic vessels, which in turn converged with the larger vessels originating from the testis. It was further noted that in the capillary endothelium there were no fenestrations, and in the large veins there were many diverticula. These diverticula appear to play a role in the regulation of the seasonal variations of the blood reflux. In general, the microvascular architecture of the buffalo epididymis, particularly its connection to the lymphatic vascular system, appears to play an important role in the absorption and secretion processes of the epididymal epithelium. Anat Rec 266:58–68, 2002. © 2002 Wiley‐Liss, Inc.     

Candida albicans expresses a focal adhesion kinase-like protein that undergoes increased tyrosine phosphorylation upon yeast cell adhesion to vitronectin and the EA.hy 926 human endothelial cell line

The signaling pathways triggered by adherence of Candida albicans to the host cells or extracellular matrix are poorly understood. We provide here evidence in C. albicans yeasts of a p105 focal adhesion kinase (Fak)-like protein (that we termed CaFak), antigenically related to the vertebrate p125Fak, and its involvement in integrin-like-mediated fungus adhesion to vitronectin (VN) and EA.hy 926 human endothelial cell line. Biochemical analysis with different anti-chicken Fak antibodies identified CaFak as a 105-kDa protein and immunofluorescence and cytofluorimetric analysis on permeabilized cells specifically stain C. albicans yeasts; moreover, confocal microscopy evidences CaFak as a cytosolic protein that colocalizes on the membrane with the integrin-like VN receptors upon yeast adhesion to VN. The protein tyrosine kinase (PTK) inhibitors genistein and herbimycin A strongly inhibited C. albicans yeast adhesion to VN and EA.hy 926 endothelial cells. Moreover, engagement of alpha v beta 3 and alpha v beta 5 integrin-like on C. albicans either by specific monoclonal antibodies or upon adhesion to VN or EA.hy 926 endothelial cells stimulates CaFak tyrosine phosphorylation that is blocked by PTK inhibitor. A role for CaFak in C. albicans yeast adhesion was also supported by the failure of VN to stimulate its tyrosine phosphorylation in a C. albicans mutant showing normal levels of CaFak and VNR-like integrins but displaying reduced adhesiveness to VN and EA.hy 926 endothelial cells. Our results suggest that C. albicans Fak-like protein is involved in the control of yeast cell adhesion to VN and endothelial cells.     

Optimized determination of T cell epitope responses

Pools of overlapping peptides corresponding to specific antigens are frequently used to identify T cell immune responses to vaccines or pathogens. While the response to the entire pool of peptides provides important information, it is often desirable to also know to which individual peptides within the pool the immune responses are directed. In this report, we analyzed various ways of deconvoluting an immune response to a pool of peptides to determine the number of different peptides to which the T cells are responding. We used a Monte Carlo simulation to optimize the construction of peptide pools that could identify responses to individual peptides using the fewest numbers of assays and patient material. We find that the number of assays required to deconvolute a pool increases by the logarithm of the number of peptides within the pool; however, the optimum configuration of pools changes dramatically according to the number of responses to individual peptides that are expected to be in the sample. Our simulation will help in the design of clinical trials in which the breadth of the response is being measured, by allowing a calculation for the minimum amount of blood that needs to be collected. In addition, our results guide the design and implementation of the experiments to deconvolute the responses to individual peptide epitopes.     

Synthesis and characterization of new siloxane-modified addition polyimides

A new series of polyimides was synthesized by addition polymerization of 4,4′-(bismaleimido)-diphenylmethane, 1,4-piperazine and an amino-terminated polydimethylsiloxane. The synthesis was carried out in m-cresol solution following a two-step procedure. Copolymers containing 15 and 20 wt.-% of the polydimethylsiloxane elastomer were prepared, together with the unmodified base copolymer. Infrared spectroscopy, ¹³C and ¹H NMR, thermal and thermogravimetric analyses were used to characterize the copolymers. The DSC curves of all the samples showed an exotherm in the temperature range 230–280°C, attributable to curing and addition reactions involving the chain-end groups. Different glass transition temperatures (T_g) were observed, depending on the elastomer content. Thermogravimetric analysis indicated that no significant changes occur in the thermal stability of rubber-modified copolymers.     

Modulation of interferon-γ receptor during human T lymphocyte alloactivation

Previous work has shown that neutralization of physiologically secreted interferon(IFN)-γ or blockade of its receptor during T lymphocyte activation inhibits both proliferation and cytotoxic T lymphocyte generation, suggesting that IFN-γ plays a crucial role in T lymphocyte induction and differentiation. In this study, the kinetics of the surface expression of the 90-kDa IFN-γ receptor (IFN-γR) was followed during human mixed lymphocyte reaction (MLR) to alloantigens. IFN-γR mRNA is constitutively expressed on resting peripheral blood lymphocytes emerging from nylon wood column (NW-PBL) and its expression increases two- to threefold on alloactivated NW-PBL. IFN-γR protein is poorly expressed on the membrane of resting CD3⁺ cells, but up-modulates after 3-day MLR and sharply down-modulates at day 6. Both the p55 and the p75 chains of interleukin-2 receptor (IL-2R) were shown to up-modulate in parallel with IFN-γR, whereas they were still highly expressed at day 6. After alloactivation, IFN-γ and IL-2 secretion starts at 24 h, peaks at day 3 and decreases just when IFN-γR and IL-2R begin to up-modulate. Proliferation peaks at day 6. Lastly, stimulation with distinct cell populations showed that the intensity of lymphocyte proliferation, IFN-γR membrane up-modulation, and IFN-γ and IL-2 secretion are regulated in a parallel manner, thus suggesting that they are interrelated. Taken as whole these results demonstrate that increased expression of IFN-γR on T lymphocytes can be a critical event during their activation, and strongly support the hypothesis that IFN-γ/IFN-γR interaction provides a signal for its progression.     

Optimedin: a novel olfactomedin-related protein that interacts with myocilin

Mutations in the MYOC gene may lead to juvenile open-angle glaucoma with high intraocular pressure, and are detected in about 4% of people with adult onset glaucoma. Most of these mutations are found in the third exon of the gene encoding the olfactomedin-like domain located at the C terminus of the protein. Another olfactomedin-related protein, known as noelin or pancortin, is involved in the generation of neural crest cells. Here we describe the identification of a novel olfactomedin-related gene, named optimedin, located on chromosome 1p21 in humans. Optimedin and noelin are both expressed in brain and retina. However, unlike noelin, rat optimedin is also highly expressed in the epithelial cells of the iris and the ciliary body in close proximity to the sites of Myoc expression. In the human eye, optimedin is expressed in the retina and the trabecular meshwork. Both optimedin and myocilin are localized in Golgi and are secreted proteins. The presence of mutant myocilin interferes with secretion of optimedin in transfected cells. Optimedin and myocilin interact with each other in vitro as judged by the GST pulldown, co-immunoprecipitation and far-western binding assays. The C-terminal olfactomedin domains are essential for interaction between optimedin and myocilin, while the N-terminal domains of both proteins are involved in the formation of protein homodimers. We suggest that optimedin may be a candidate gene for disorders involving the anterior segment of the eye and the retina.     

Soluble Antiapoptotic Molecules and Immune Activation in Chronic Heart Failure and Unstable Angina Pectoris

Programmed myocyte cell death and activation of the immune system have been shown to occur in patients with congestive heart failure. Besides, unstable angina episodes are likely to be associated with immune activation. Our aim was to evaluate the role of changes in circulating levels of soluble Fas (sFas), suggestive of an enhanced inhibitory response to ongoing apoptosis, and soluble IL2 receptor (sIL2-R), indicative of T-lymphocyte activation, in chronic heart failure and unstable angina pectoris. Thirty patients affected by chronic heart failure (20 idiopathic and 10 ischemic cardiomyopathy) and 13 patients with unstable angina were evaluated. Twenty healthy individuals matched for age and gender were used as controls. A complete biochemical determination of indexes of myocardial damage including cardiac troponin I (cTnI) and creatine kinase (MB/CK) was performed. The results demonstrated that mean levels of sFas and sIL2-R were significantly increased in patients affected by chronic heart failure and unstable angina and were not associated with changes in renal function or with serum levels of cTnI. Highest values of sFas were found in NYHA class IV patients (IV NYHA class = 7.39 ± 0.52 vs. controls = 1.34 ± 0.12 ng/ml; P < 0.01) and more elevated in idiopathic than in ischemic cardiomyopathy (3.64 ± 0.40 vs. 1.82 ± 0.37 ng/ml; P < 0.01). Moreover, in chronic heart failure patients sFas and ejection fraction were negatively correlated (P = 0.01), whereas sFas and sIL2-R were positively correlated (P < 0.01). In unstable angina patients too, sFas and sIL2-R appeared to be correlated (P = 0.03); whereas sFas (angina group = 3.18 ± 0.39 vs. controls = 1.34 ± 0.12 ng/ml; P < 0.01) and sIL2-R (angina group = 0.46 ± 0.11 vs. controls = 0.00 UI/ml; P < 0.01) were higher in angina group than in controls. In most of the cases, the increase of sFas was associated with comparable changes in sIL2-R serum levels, indicating that the activation of Fas system is strictly associated with autoimmune–inflammatory reactions. This phenomenon, both in chronic heart failure and in unstable angina, occurs in the absence of biochemical evidences of myocardial damage and seems to parallel the activation of T cell. Soluble Fas could have a role in sustaining inflammatory response and in prolonging the detrimental effects correlated with it in chronic heart failure and angina pectoris.