Characterization of the autoimmune response against the nerve tissue S100ß in patients with type 1 diabetes.

Type 1 diabetes results from destruction of insulin-producing beta cells in pancreatic islets and is characterized by islet cell autoimmunity. Autoreactivity against non-beta cell-specific antigens has also been reported, including targeting of the calcium-binding protein S100ß. In preclinical models, reactivity of this type is a key component of the early development of insulitis. To examine the nature of this response in type 1 diabetes, we identified naturally processed and presented peptide epitopes derived from S100ß, determined their affinity for the human leucocyte antigen (HLA)-DRB1*04:01 molecule and studied T cell responses in patients, together with healthy donors. We found that S100ß reactivity, characterized by interferon (IFN)-γ secretion, is a characteristic of type 1 diabetes of varying duration. Our results confirm S100ß as a target of the cellular autoimmune response in type 1 diabetes with the identification of new peptide epitopes targeted during the development of the disease, and support the preclinical findings that autoreactivity against non-beta cell-specific autoantigens may have a role in type 1 diabetes pathogenesis.

Alteration of the serum levels of the epidermal growth factor receptor and its ligands in patients with non-small cell lung cancer and head and neck carcinoma.

Serum levels of the soluble epidermal growth factor receptor (sEGFR) and its ligands epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha) and amphiregulin (AR) were measured in healthy donors and patients with non-small cell lung cancer (NSCLC) and head and neck carcinoma (HNC). In NSCLC, we found sEGFR and EGF levels significantly lowered in patients with respect to healthy donors. In HNC patients, significantly diminished levels were found in the case of sEGFR, EGF and also AR. In both malignancies, no significant association was found between the serum levels of the molecules and the patients' gender, age or smoking habit. Only a significant association was found between the decrease of sEGFR and the absence of distant metastasis in NSCLC and the tumour stage in HNC. The most interesting result was that combining sEGFR and EGF, sensitivities of 88% in NSCLC and 100% in HNC were reached without losing specificity (97.8% in both cases). The use of discriminant analysis and logistic regression improved the sensitivity for NSCLC and the specificity for HNC. These data demonstrate a potentially interesting value of the serum levels of sEGFR and EGF, especially when combined, as markers for NSCLC and HNC.

Identification of receptors for prothymosin alpha on human lymphocytes.

Prothymosin alpha (ProTalpha) is a highly conserved and widely distributed protein whose physiological functions remain elusive. In previous work we identified high and low affinity-binding sites for ProTalpha in lymphoid cells. Here we demonstrate, by affinity cross-linking and affinity chromatography, the existence of three binding partners (31, 29, and 19 kDa) for ProTalpha in the membrane of PHA-activated lymphoblasts. These surface molecules possess the expected affinity and specificity for a ProTalpha receptor. Examination of the expression of this complex of molecules by flow cytometry reveals that they bind ProTalpha in a specific and saturable way. In addition, the distribution of the receptor on the cell surface was studied by fluorescence microscopy; a cap-like structure at one of the poles of the cells was identified. These results represent a new and promising approach in the research on ProTalpha, opening the way toward the understanding of the molecular mechanism of action of this protein.

Serum interleukin-12, interleukin-15, soluble CD26, and adenosine deaminase in patients with rheumatoid arthritis.

CD26, a transmembrane ectoenzyme, is overexpressed on rheumatoid arthritis (RA) peripheral blood T cells. As it has been recently described that IL-12 and IL-15 upregulate CD26 in vitro, we hypothesized that this CD26 overexpression might be interleukin dependent. The concentrations of IL-12 and IL-15, and soluble CD26 and adenosine deaminase (enzymes related to CD26) were analyzed in the serum of 35 patients with active and inactive RA and of healthy control subjects. IL-12 and IL-15 levels were significantly higher in patients' serum, independently of disease activity, even in patients on steroid therapy, i.e., the present therapies cannot eradicate their origin. Soluble CD26 was significantly reduced and related to the disease activity. In particular, it correlated inversely with the number of swollen joints. Although these data did not support our hypothesis, they support that interleukins not only initiate RA pathology but they can also participate in the maintenance of this immune response.

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.

Cell surface human alpha-L-fucosidase.

The acid alpha-L-fucosidase is usually found as a soluble component of lysosomes where fucoglycoconjugates are degraded. In the present investigation, we have demonstrated the existence of a cell surface protein with enzymatic alpha-L-fucosidase activity that crossreacts specifically with a rabbit anti-(alpha-L-fucosidase) Ig. By different approaches, this alpha-L-fucosidase, which represents 10-20% of the total cellular fucosidase activity, was detected in all the tested human cells (hemopoietic, epithelial, mesenchymal). Two bands of approximately 43-49 kDa were observed, although theoretical data support the possibility of having the same genetic origin that the known 50 to 55-kDa Mr alpha-L-fucosidase. We speculate about an alternative traffic pathway for the plasma membrane alpha-L-fucosidase to work on the rapid turnover of glycoproteins.

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 microg l(-1)) and cancer patients (mean 261.7 +/- 138.1 microg l(-1)) (P< 0.001). A cut-off at 410 microg 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.

Mechanisms of CD26/dipeptidyl peptidase IV cytokine-dependent regulation on human activated lymphocytes.

Among the cellular pathways activated by IL-12, we had previously found that both the percentage and intensity of CD26(+)cells in the PHA-stimulated T cells increased when IL-12 was present (independently of its CD4 or CD8 phenotype). Now, we examined the molecular mechanisms of this IL-12-mediated effect. The IL-12 regulation pathway is dependent of de novo protein synthesis and independent of cytokine secretion. Our results show two transcripts for CD26 in PBMC for the first time and no regulation by ILs at this level. Furthermore, secretion of the serum forms of CD26/DPPIV were not affected by IL-12. Interestingly, assays with neutralizing mAbs against TNF-alpha suggest that this cytokine negatively modulates CD26 expression. The fact that translation and probably translocation of CD26 toward the cell surface can be regulated by IL-12 and TNF-alpha reveals new aspects about the control of this T(H1)marker.

Thymic peptides and preparations: an update.

The possibilities of thymic peptides in human therapy are still being described. Here, we focus on their general characteristics and on recent advances in this area.

Expression of A2B adenosine receptors in human lymphocytes: their role in T cell activation.

Extracellular adenosine has a key role in the development and function of the cells of the immune system. Many of the adenosine actions seem to be mediated by specific surface receptors positively coupled to adenylate cyclase: A2A and A2B. Despite the fact that A2A receptors (A2ARs) can be easily studied due to the availability of the specific agonist CGS21680, a pharmacological and physiological characterization of adenosine A2B receptors (A2BRs) in lymphocytes has not been possible due to the lack of suitable reagents. Here we report the generation and characterization of a polyclonal antipeptide antibody raised against the third extracellular loop of the A2BR human clone which is useful for immunocytochemical studies. This antibody has permitted the detection of A2BR+ cells in lymphocyte samples isolated from human peripheral blood. The pharmacology of cAMP-producing compounds is consistent with the presence of functional A2BRs but not of A2A receptors in these human cells. The percentage of A2BR-expressing cells was similar in the CD4(+) or CD8(+) T cell subpopulations. Interestingly activation signals delivered by either phytohemagglutinin or anti-T cell receptor/CD3 complex antibodies led to a significant increase in both the percentage of cells expressing the receptor and the intensity of the labeling. These receptors are functional since interleukin-2 production in these cells is reduced by NECA but not by R-PIA or CGS21680. These results show that A2BR expression is regulated in T cell activation and suggest that the role of adenosine in lymphocyte deactivation is mediated by A2BRs.

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.

Interleukin-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.

Binding of 125I-prothymosin alpha to lymphoblasts through the non-thymosin alpha 1 sequence.

The important immunological activities of Thymosin alpha 1 (T alpha 1), a peptide derived from the thymus, led to its use in combination therapies in cancer patients. Prothymosin alpha (ProT alpha) is a highly acidic polypeptide, first isolated as the putative precursor of T alpha 1. However ProT alpha is now known to be more immunoreactive than T alpha 1 in certain in vivo and in vitro assays. Recent results indicate that ProT alpha may be useful to design future therapeutic interventions in cancer patients if the mechanisms underlying these effects are puzzled out. With this in mind, we radiolabeled ProT alpha to obtain a high specific activity and a high biological activity for 125I-ProT alpha. Moreover, we also obtained autoantibodies exhibiting high titers and an unique specificity for anti-ProT alpha and anti-T alpha 1. With both tools we studied the presence of binding sites for ProT alpha on the surface of lymphoblast cells. We conclude that ProT alpha binds through the non-T alpha 1 sequence.

Prothymosin alpha receptors on lymphocytes.

Recent results indicate that prothymosin alpha (ProT alpha) may be useful in designing future therapeutic interventions in cancer patients and in potentiating the immune system. We described recently the presence and characteristics of two binding sites for ProT alpha on human peripheral blood mononuclear cells (PBMC). In search of a receptor upregulation, we decided to corroborate this finding on two lymphocytic populations, (PHA-activated) lymphoblasts and YT cells. The kinetics of [125I]ProT alpha binding to lymphoblasts were fast at room temperature but with YT cells were slower. Analysis of steady-state binding data identified two binding sites in lymphoblasts with an apparent equilibrium Kd of 44-75 pM and 4228-9143 sites per cell for the high-affinity receptor and 1.7-2.9 nM and 20,534-35,044 sites per cell for the low-affinity receptor. However, it identified only one site with a Kd of 265-435 pM and 8318-27,237 sites per cell in YT cells. We conclude that exists a ProT alpha receptor in the CD3+ T cell population, and this presence is regulated. After binding to cell surface, [125I]ProT alpha is internalized in a short period of time and then degraded; therefore, we conclude that the dynamics of ProT alpha receptor turnover in part determines the concentration of ProT alpha available to induce its enhancing effects.

The presence and cytotoxicity of CD16+ CD2- subset from PBL and NK cells in long-term IL-2 cultures enhanced by Prothymosin-alpha.

The aim of this study was to characterize the LAK cell subpopulation on which Prothymosin-alpha (ProT alpha) exerts its enhancing effect on cytotoxicity. We investigated the role of ProT alpha on LAK induction from peripheral blood lymphocytes (PBL) and NK-enriched cells, both cultured for 5 weeks with IL-2 and ProT alpha. The different cultures were separated into several subsets throughout the culture time using two color fluorescence activated cell sorting (FACS) and CD56, CD16, CD2 and CD8 monoclonal antibodies. Each cell subset was then tested for cytotoxicity against K562 and Daudi cells. The CD16+ CD2- subset from both, PBL and NK-enriched cells, was the only subset on which ProT alpha had an effect, significantly enhancing this subpopulation. Within the CD16 population, the cells CD16+ CD2+ were the most cytotoxic, although CD16+ CD2- cells were also cytotoxic. ProT alpha only potentiated the cytotoxicity of CD16+ CD2- subset significantly, with 29% and 41% for K562 and Daudi cells, respectively.

Interleukin-2 killer cells: in vitro evaluation of combination with prothymosin alpha.

Interleukin-2 (IL-2) is a potent inducer of lymphokine-activated killer (LAK) activity against both NK-sensitive and NK-resistant tumor cell lines. IL-2 therapy has been associated with clinical responses, but these responses have occurred only with high, toxic doses of the cytokine. Since prothymosin alpha (ProT alpha) is able to enhance the spontaneous NK activity of cells from normal donors, we studied the effect of ProT alpha on LAK activity by IL-2. The lysis of K562 and Daudi cells by effector cells cultured with IL-2 was increased time dependently when cultures also contained ProT alpha. PBLC was separated by discontinuous density centrifugation in the LGL-enriched fractions. Within the CD16+ population, all effector cell precursors were CD2+, but the effector population after IL-2 or IL-2 + ProT alpha activation also contained CD16+CD2- cells; the CD2 molecule is thus indispensable for induction of LAK activity by IL-2 or IL-2 + ProT alpha but not for the action of activated LAK cells (or for the enhancing effect of ProT alpha). Within the CD3- CD16+ LGL population, 5 micrograms/ml ProT alpha plus 50 U/ml IL-2 was able to induce p70 IL-2R expression to a similar extent to 100 U/ml of IL-2. The use of ProT alpha to enhance the induction of LAK activity by IL-2 may be able to improve immunotherapy of cancer.

Prothymosin alpha receptors on peripheral blood mononuclear cells.

125I-Labeled prothymosin alpha (ProT alpha) was used to study the presence and characteristics of receptors for ProT alpha on human peripheral blood mononuclear cells (PBMC). The kinetics of 125I-ProT alpha binding to PBMC was fast at 37 degrees C, whilst it required 50 min to reach equilibrium at 4 degrees C and room temperature. Analysis of steady state binding data by the method of Scatchard and by unlabeled ProT alpha 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.