Montserrat Nogueira

On the origin of serum CD26 and its altered concentration in cancer patients

Dipeptidyl peptidase IV (DPP-IV), assigned to the CD26 cluster, is expressed on epithelial cells and lymphocytes and is a multifunctional or pleiotropic protein. Its peptidase activity causes degradation of many biologically active peptides, e.g. some incretins secreted by the enteroendocrine system. DPP-IV has, therefore, become a novel therapeutic target for inhibitors that extend endogenously produced insulin half-life in diabetics, and several reviews have appeared in recent months concerning the clinical significance of CD26/DPP-IV. Biological fluids contain relatively high levels of soluble CD26 (sCD26). The physiological role of sCD26 and its relation, if any, to CD26 functions, remain poorly understood because whether the process for CD26 secretion and/or shedding from cell membranes is regulated or not is not known. Liver epithelium and lymphocytes are often cited as the most likely source of sCD26. It is important to establish which tissue or organ is the protein source as well as the circumstances that can provoke an abnormal presence/absence or altered levels in many diseases including cancer, so that sCD26 can be validated as a clinical marker or a therapeutic target. For example, we have previously reported low levels of sCD26 in the blood of colorectal cancer patients, which indicated the potential usefulness of the protein as a biomarker for this cancer in early diagnosis, monitoring and prognosis. Through this review, we envisage a role for sCD26 and the alteration of normal peptidase capacity (in clipping enteroendocrine or other peptides) in the complex crosstalk between the lymphoid lineage and, at least, some malignant tumours.

Fifteen years of prothymosin alpha: contradictory past and new horizons

Prothymosin alpha (ProTalpha) is a highly acidic and small protein of only 111 amino acids with an unusual primary structure. One would expected it to play an essential role in the organism, as it has a wide distribution and is high conserved among mammals, yet its exact function remains elusive. Despite the number of effects described for ProTalpha, intracellular and extracellular, none are accepted as its physiological role. Furthermore, many other aspects of its biology still remain obscure. In this review, we discuss the structural properties, location, gene family, functions and immunomodulatory activities of and cellular receptors for ProTalpha. These topics are addressed in an attempt to reconcile opposing outlooks while emphasizing those points where scant investigations do exist. We have also re-evaluated some previous results in light of the structural properties of ProTalpha and have found that molecular mimetism could be the underlying basis. This molecular mimicry hypothesis provides a clue that must not be overlooked for a realistic appraisal of future results.

Preoperative serum CD26 levels: diagnostic efficiency and predictive value for colorectal cancer.

CD26 is an ectoenzyme with dipeptidyl peptidase IV activity expressed on a variety of cell types. Although the function of the high concentration of serum-soluble CD26 (sCD26) is unknown, it may be related to the cleavage of biologically active polypeptides. As CD26 or enzymatic activity levels were previously associated with cancer, we examined the potential diagnostic and prognostic value of preoperative sCD26 measurements by ELISA in colorectal carcinoma patients. We found a highly significant difference between sCD26 levels in healthy donors (mean 559.7 ± 125.5 μg l−1) and cancer patients (mean 261.7 ± 138.1 μg l−1) (P < 0.001). A cut-off at 410 μg l−1 gave 90% sensitivity with 90% specificity which means that the diagnostic efficiency of sCD26 is higher than that shown by other markers, particularly in patients at early stages. Moreover, sCD26 as a variable is not related with Dukes’ stage classification, age, gender, tumour location or degree of differentiation. With a follow-up of 2 years until recurrence, preliminary data show that sCD26 can be managed as a prognostic variable of early carcinoma patients. In addition, the origin of sCD26 is discussed.

Cytokines regulate membrane adenosine deaminase on human activated lymphocytes

CD26 is a lymphocyte marker that can anchor adenosine deaminase (ADA) on the T cell surface. We found that ADA is regulated by cytokines on the cell surface during T cell activation. By means of flow cytometry, immunofluorescence, and immunoblotting techniques, we found that interleukin (IL)‐2 and IL‐12 up‐regulate ecto‐ADA and CD26 expression. In clear contrast, IL‐4 led to down‐regulation of lymphocyte surface ADA without modifying the level of CD26. Moreover, neither circulating ADA transcription nor mRNA translation was regulated by cytokines. These results, along with absence of total‐ADA modulation, the variable amount of ADA found in purified plasma membranes, and the different effect of Brefeldin A on the surface presence of ADA and CD26 indicated that cytokines regulate the translocation of ADA towards the cell surface through a mechanism not involving CD26. Ecto‐ADA protected activated lymphocytes from the toxic effects of extracellular adenosine. Therefore, this cell surface ADA control might constitute part of the fine immunoregulatory mechanism of adenosine‐mediated signaling through purinergic receptors in leukocytes.

lnterleukin-12 enhances CD26 expression and dipeptidyl peptidase IV function on human activated lymphocytes

Research of a cellular pathway activated by IL-12 which may result in new therapeutical approaches for IL-12, led us to find an intriguing relationship between IL-12 and CD26/DPPIV ectopeptidase on activated T cells. Both the percentage and median fluorescence intensity (MFI) of CD26+ cells in the PHA-stimulated PBMC or lymphoblasts increased when IL-12 (optimum dose, 2 ng/ml) was present. Maximum CD26 expression was observed on day-2 cultures of lymphoblasts, the presence of IL-12 receptor probably being necessary for this upregulation. In addition, CD26 upregulation correlated with enhanced DPPIV function. Enzyme affinity and secretion of the soluble form of DPPIV were not affected by IL-12. Kinetic behaviours of Ag expression and enzymatic activity support a different CD26 regulation pathway by IL-12. These data suggest that the correlation found in vivo between the CD26 expression and Th1-like immune responses is due to this IL-12-dependent upregulation.

Interactions between DMPC liposomes and the serum blood proteins HSA and IgG.

The interaction between two serum blood proteins, namely human serum albumin (HSA) and human immunoglobulin G (IgG), with 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) liposomes has been studied in detail using dynamic light scattering, flow cytometry, enzyme-linked immunosorbent assay (ELISA), electrophoretic mobility, differential scanning calorimetry (DSC), and surface tension measurements. HSA and IgG interact with liposomes forming molecular aggregates that remain stable at protein concentrations beyond those of total liposome coverage. Both HSA and IgG penetrate into the liposome bilayer. An ELISA assay indicates that the Fc region of IgG is the one that is immersed in the DMPC membrane. The liposome-protein interaction is mainly of electrostatic nature, but an important hydrophobic contribution is also present.

Interleukin-dependent modulation of HLA-DR expression on CD4 and CD8 activated T cells

Interleukins (IL) regulate differentT‐cell surface Ag known as activation markers that have distinct functional roles. In this paper, while studying the influence of some cytokines(IL‐12, IL‐2 and IL‐4) on the expression of several markers [CD69,CD25, CD26, CD3, human leukocyte antigen (HLA‐DR), CD45R0] in in vitro activated human T lymphocytes, we observed two groups of donors responding to phytohaemagglutinin (PHA) activation with high or low HLA‐DRAg expression. We also found that CD4 and CD8 populations had different HLA‐DR densities under PHA activation (particularly the high HLA‐DR‐expressing group). Interleukins, in a dose‐dependent manner (IL‐2 partially), upregulated these HLA‐DR levels. In 5 day cultures, IL‐12and IL‐2 enhanced the CD8/CD4 ratio of activated T cells, which was responsible, in part, for the IL‐dependent HLA‐DR upregulation. IL‐12 and IL‐2 also upregulated the HLA‐DR expression at the molecular level on CD8, and IL‐12 downregulated it on CD4 cells. It seems that IL‐4 upregulated HLA‐DR by shortening the mitogen‐dependent regulation kinetics. We hypothesize that the different effect of each IL on HLA‐DR expression might be related to the regulation of the dose of antigenic peptide presentation and, thus, also influence TH1/TH2 dominance.

CD26: a negative selection marker for human Treg cells.

A major obstacle hampering the therapeutic application of regulatory T (Treg) cells is the lack of suitable extracellular markers, which complicates their identification/isolation. Treg cells are normally isolated via CD25 (IL‐2Rα) targeting, but this protein is also expressed by activated CD4+ effector T (Teff) lymphocytes. Other extracellular (positive or negative) Treg selection markers (e.g., HLA‐DR, CD127) are also nonspecific. CD26 is an extracellular peptidase whose high expression has been traditionally used as an indicator of immune activation and effector functions in T cells. Now, we provide flow cytometry data showing high levels of CD26 within CD4+CD25− or CD4+FoxP3−/low effector T (Teff) lymphocytes, but negative or low levels (CD26−/low) in Treg cells selected according to the CD4+CD25high or the CD4+FoxP3high phenotype. Unlike the negative marker CD127 (IL‐7Rα), which is down modulated in CD4+ Teff lymphocytes after TCR triggering, most of these cells upregulate CD26 and take a CD4+CD25+/highCD26+ phenotype upon activation. In contrast, there is only a slight upregulation within Treg cells (CD4+CD25highCD26−/low). Thus, differences in CD26 levels between Treg and Teff subsets are stable, and assessment of this marker, in combination with others like CD25, FoxP3, or CD127, may be useful during the quantitative evaluation or the isolation of Treg cells in samples containing activated Teff lymphocytes (e.g., from patients with autoimmune/inflammatory diseases).

Prothymosin α receptors on peripheral blood mononuclear cells

125I‐Labeled prothymosin α (ProTα) was used to study the presence and characteristics of receptors for ProTα on human peripheral blood mononuclear cells (PBMC). The kinetics of 125I‐ProTα binding to PBMC was fast at 37°C, whilst it required 50 min to reach equilibrium at 4°C and room temperature. Analysis of steady state binding data by the method of Scatchard and by unlabeled ProTα competition experiments identified two binding sites with an apparent equilibrium dissociation constant of 216–321 pM for the high‐affinity receptor and of 11.4–21.1 nM for the low‐affinity one; the sites per cell ranged from 1,479 to 1,519 and from 47,547 to 56,169, respectively. The kinetically derived equilibrium dissociation constant agreed with these data and showed no interaction between receptors.

Interleukin-12-dependent activation of human lymphocyte subsets

Recently, we reported that IL-12 increased expression and function of CD26/DPPIV, this may be a new cellular pathway of the Th1-like immune responses. Here, we looked for a specific subset which would respond to CD26 upregulation by IL-12. Contrary to previously described results, under our culture conditions (1 microg/ml of PHA), IL-12 enhanced preferentially the CD8 cell proliferation. By using dual fluorescence analysis, IL-12-dependent CD26 expression was found in both CD4 and CD8 (previously CD26+ or CD26-) activated T cells and, moreover, the CD45RO percentage was unaffected. However, the density of CD45RO Ag (which was reported to coexpress with CD26) was impaired. These effects can be implicated in the biological functions of IL-12 and provide some clinical possibilities.