Fabrizio Spinozzi

The natural tyrosine kinase inhibitor genistein produces cell cycle arrest and apoptosis in Jurkat T-leukemia cells

Genistein, a natural isoflavonoid phytoestrogen, is a strong inhibitor of protein tyrosine kinases. We analyzed the effects of genistein on in vitro growth, cell-cycle progression and chromatin structure of Jurkat cells, a T-cell leukemia line with a constitutively increased tyrosine phosphorylation pattern. Exposure of in vitro cultured Jurkat cells to genistein resulted in a dose-dependent, growth inhibition. Cell-cycle analysis of genistein-treated cells revealed a G2/M arrest at low genistein concentrations (5-10 micrograms/ml), while at higher doses (20-30 micrograms/ml) there was also a perturbation in S-phase progression. The derangements in cell-cycle control were followed by apoptotic death of genistein-treated cells. Immunocytochemical analysis of cells stained with a FITC-conjugated anti-phosphotyrosine monoclonal antibody showed that 30 micrograms/ml genistein effectively inhibit tyrosine kinase activity in cultured Jurkat cells. Our results indicate that the natural isoflavone genistein antagonizes tumor cell growth through both cell-cycle arrest and induction of apoptosis and suggest that it could be a promising new agent in cancer therapy.

Human CD1-restricted T cell recognition of lipids from pollens

Plant pollens are an important source of environmental antigens that stimulate allergic responses. In addition to acting as vehicles for foreign protein antigens, they contain lipids that incorporate saturated and unsaturated fatty acids, which are necessary in the reproduction of higher plants. The CD1 family of nonpolymorphic major histocompatibility complex–related molecules is highly conserved in mammals, and has been shown to present microbial and self lipids to T cells. Here, we provide evidence that pollen lipids may be recognized as antigens by human T cells through a CD1-dependent pathway. Among phospholipids extracted from cypress grains, phosphatidyl-choline and phosphatidyl-ethanolamine were able to stimulate the proliferation of T cells from cypress-sensitive subjects. Recognition of phospholipids involved multiple cell types, mostly CD4+ T cell receptor for antigen (TCR)αβ+, some CD4−CD8− TCRγδ+, but rarely Vα24i + natural killer–T cells, and required CD1a+ and CD1d+ antigen presenting cell. The responding T cells secreted both interleukin (IL)-4 and interferon-γ, in some cases IL-10 and transforming growth factor-β, and could provide help for immunoglobulin E (IgE) production. Responses to pollen phospholipids were maximally evident in blood samples obtained from allergic subjects during pollinating season, uniformly absent in Mycobacterium tuberculosis–exposed health care workers, but occasionally seen in nonallergic subjects. Finally, allergic, but not normal subjects, displayed circulating specific IgE and cutaneous weal and flare reactions to phospholipids.

Manometric investigation of anorectal function in early and late stage Parkinson's disease

Abnormal gastrointestinal function is relatively frequent in Parkinsons disease, and constipation is a disturbing symptom in many patients. However, it remains to be established whether anorectal abnormalities are characteristic of the late stages of the disease. Clinical and anorectal manometric function were investigated in groups of early and late stage parkinsonian patients. Thirty one patients (19 men, 12 women, age range 22 to 89 years) entered the study. The disease severity was assessed by Hoehn and Yahr staging: there were four (12.9%) stage I, seven (22.6%) stage II, 10 (32.2%) stage III, and 10 (32.2%) stage IV patients. Anorectal variables were measured by standard manometric equipment and techniques. Values obtained in early stage patients (Hoehn and Yahr stage I and II) were compared with those obtained in late stage patients (Hoehn and Yahr stage III and IV). Overall, more than 70% of patients complained of chronic constipation, with chronic laxative use reported in more than 30%. Late stage patients were slightly older than their early stage counterparts. Pelvic floor dyssynergia was documented in more than 60% of patients. Manometric variables were not different in the two groups. In conclusion, defecatory dysfunction is frequent in Parkinsons disease, it is not confined to late stage patients, and it is found early in the course of the disease. This has potential implications for a targeted therapeutic approach.

Increased Allergen-Specific, Steroid-Sensitive γδ T Cells in Bronchoalveolar Lavage Fluid from Patients with Asthma

Macrophages are the main cellular component in bronchoalveolar lavage fluid specimens obtained from normal patients. Other cell types, including T lymphocytes (CD3+) of both helper (CD4+) and suppressor-cytotoxic (CD8+) T-cell subsets, are also present, but the percentage of these cell types usually does not exceed 10%. Eosinophils, basophils and mast cells, and T lymphocytes have been found to be increased in bronchoalveolar lavage fluid samples in studies that have attempted to quantify the magnitude of the airway inflammatory response in patients with asthma [1, 2]. Recent findings suggest that T lymphocytes play a fundamental role in the induction of allergic inflammation. In fact, these cells not only recruit other specialized cells, such as eosinophils, by secreting interleukin-5 [1], they also promote local and systemic synthesis of IgE through the production of interleukin-4 [3, 4]. Lymphocytes with such functional activities are currently termed T-helper 2 (Th2), whereas the non-overlapping cell counterpart that secretes interferon- or interleukin-2 or both is termed T-helper 1 (Th1) and is involved in the delayed hypersensitivity immune reactions [3]. Although Th2-like activated T lymphocytes have been detected in the lungs of atopic asthmatic patients [5], it is still unclear whether these cells bear the or the less common T-cell receptor for antigen on their surface. This doubt is legitimated by the fact that many CD3+ intraepithelial lymphocytes found in the nasal mucosa of patients with allergic rhinitis are T cells that display the T-cell receptor heterodimer [6]. These findings prompted us to determine whether allergen-specific T lymphocytes are present in the bronchoalveolar lavage fluid of asthmatic patients and that, in these patients, they will be the major T-cell subset to disappear after systemic steroid treatment. Interestingly, our in vivo and in vitro studies provide evidence that apoptosis is the basic mechanism through which corticosteroid treatment acts on this T-lymphocyte type, thereby confirming the results of our previous investigations [7]. Methods Patients We studied 12 mildly symptomatic patients (6 children and 6 adults) with chronic asthma (FEV1 70% of that predicted for persons of their age and height) who were not receiving therapy with inhaled or oral corticosteroids, sodium cromoglycate, theophylline, or 2-agonists. Results of skin prick tests with purified Dermatophagoides pteronyssinus, D. farinae, and Parietaria judaica allergen extracts (Neo Abello, Madrid, Spain) were positive; results of an enzyme-linked immunosorbent assay (DPC Corporation, Los Angeles, California) for circulating allergen-specific IgE were also positive. None of the patients was a smoker or had had antecedent upper respiratory tract infection. Bronchoalveolar lavage fluid collection and fiberoptic bronchoscopy were done as previously described [8]. Ten healthy non-atopic, nonsmoking volunteers and patients diagnosed as having pulmonary sarcoidosis (n = 5) or extrinsic allergic alveolitis (n = 4), two pathologic conditions known to be associated with over-expanded lung CD4+ or CD8+ T-cell populations, were used as adult control groups. Age-matched uninfected children with cystic fibrosis (n = 5) or anatomic malformation (n = 4) of the airways served as controls for the cohort of asthmatic children. Bronchoalveolar lavage was repeated in 3 asthmatic patients after 1 week of therapy with deflazacort (Flantadin, Lepetit, Milano, Italy), 60 mg twice daily (a dose equivalent to 50 mg of prednisone), and then 3 weeks after therapy. We administered corticosteroids to these patients for exacerbation of their disease. We obtained informed consent from all study participants, and the clinical research was conducted in accordance with the local ethical committee guidelines. Lymphocyte Typing Pulmonary T cells were phenotyped with the following monoclonal antibodies: phycoerythrin-conjugated anti-CD3 (Ortho Pharmaceutical Corporation, Raritan, New Jersey), which recognizes up to 90% of T cells bearing the or T-cell receptor heterodimer; anti-CD4 and anti-CD8 (Ortho), which stain helper/inducer and cytotoxic T-cell subsets; and fluorescein-conjugated anti-TCR 1, anti-V1(a), and anti-V2(a) (T Cell Sciences, Cambridge, Massachusetts), which identify all T lymphocytes and the reciprocal subtypes expressing V1+ and V2+ gene products. In the analysis of two-color cytofluorimetric data (FACScan, Becton-Dickinson, Mountain View, California), we used the Lysis II program (Becton-Dickinson) to optimize gating of lymphocytes and to provide an objective means of excluding both debris and other cell types. T-Cell Enrichment In accordance with the manufacturers instructions (Dynal, A. S., Oslo, Norway), pulmonary mononuclear cells from 3 D. pteronyssinus-sensitive patients were enriched in TCR1-reactive T-cell subsets by negative magnetic immunoselection, using a mixture of anti- T-cell receptor monoclonal antibodies. This procedure yielded a + T-cell population of greater than 85%, which was used for culture and apoptotic cell death experiments. Proliferation Assay To assess the allergen-specificity of bronchoalveolar lavage T cells, 1.2 105 mononuclear cells and 4 104 -enriched lung T cells (the latter co-cultured with 8 104 autologous macrophages) from three D. pteronyssinus-sensitive patients and, because of the very small percentage of T cells in normal bronchoalveolar lavage fluid, 1.2 105 mononuclear cells from three controls were seeded in microplates and cultured in medium alone (RPMI-1640 [Gibco, Grand Island, New York] supplemented with fetal calf serum, L-glutamine, Hepes buffer, and antibiotics) or in the presence of 1 mug/mL purified D. pteronyssinus (Neo Abello, Madrid, Spain) or unrelated allergen Lolium perenne (purified L. perenne, Neo Abello) from the Graminaceae family. After 60 hours of culture, the cells were pulsed for 16 hours with 0.5 muCi [3H]-thymidine and were harvested, and the radioactivity was measured by liquid scintillation. Results are expressed as net cpm (counts per minute) [3H]-thymidine incorporation and reflect absolute cpm [3H]-thymidine uptake minus background cpm (cpm incorporated in the absence of allergen). Evaluation of Apoptotic Cell Death Apoptotic cells were qualitatively evaluated by agarose gel DNA electrophoresis [7] and quantitatively assessed as described previously [9]. Briefly, 1 106 TCR1+ pulmonary T cells were incubated for 24 hours with medium alone or dexamethasone (107 M), resuspended in 1.5 mL hypotonic propidium iodide solution and propidium iodide fluorescence of individual nuclei measured in a FACScan flow cytometer (Becton-Dickinson). We carried out control experiments using macrophage-depleted pulmonary T cells from healthy persons. Statistical Analysis Study populations were stratified according to age class. We used the Kruskall-Wallis one-way analysis of variance (with a significance level of 0.05) for statistical evaluation of the differences in bronchoalveolar lavage T-lymphocyte subset distributions. Pairwise comparisons for measuring the significance of the differences among mean values ( SE) calculated in the various groups (asthmatic patients compared with controls) were done with the Mann-Whitney U-test. In asthmatic patients (n = 12), the overall relation between serum IgE levels (IU/mL) and the percentages of pulmonary T cells was analyzed using the Spearman correlation test. The Statistical Package for the Social Sciences (SPS, Chicago, Illinois), version 4.0, was used for all statistical computations. Results As shown in Table 1, a significant proportion of CD3+ T lymphocytes in patient samples double-stained for the TCR1 monoclonal antibody, which defines + T lymphocytes. More than half of these T cells were CD4+, primarily of the V1+ subset, whereas the remainder of T cells found in the lung of our asthmatic patients were CD4 CD8, as shown by the fact that they never reacted with anti-CD8 monoclonal antibody. In addition, the percentage of T cells expressing the V2 isoform of the T-cell receptor was negligible in all samples tested (data not shown). Statistically significant differences were also obtained when the absolute numbers of pulmonary T cells from patients with asthma (mean count in adults, 29.6 2.5 104; in children, 23.2 3.1 104) were compared with those of the corresponding controls (mean count in adults, 0.2 0.1 104; P < 0.001; in children, 0.3 0.1 104; P < 0.001). Table 1. Bronchoalveolar Lavage T-Lymphocyte Subset Distribution in Patients with Allergic Asthma* The bronchoalveolar lavage T-cell concentration was not correlated with either specific serum IgE levels or any other clinical or functional (spirometric) variables (data not shown). In contrast, a relation was found between total serum IgE concentrations and pulmonary T-lymphocyte numbers, but this did not reach statistical significance (r = 0.21; P = 0.06). In the three in vitro experiments that we carried out, pulmonary T cells from D. pteronyssinus-sensitive patients proliferated in response to the specific allergen, with a net [3H]-thymidine incorporation ranging from 2400 to 4850 cpm, but did not proliferate in the presence of unrelated (L. perenne) allergen extract, with a [3H]-thymidine incorporation ranging from 120 to 450 cpm. Furthermore, lung T cells abruptly decreased and + CD4+ and CD8+ T lymphocytes increased in these patients after 1 week of treatment with glucocorticoids. Interestingly, the absolute numbers of T cells did not increase again when therapy was discontinued (Figure 1). The T-cell subpopulation was sensitive to steroid-induced programmed cell death in vitro. In fact, 24-hour dexamethasone incubation followed by flow-cytometric analysis with propidium iodide staining showed that the propidium iodide fluorescence profiles of apoptotic nuclei increased from 7% to 35% in dexamethasone-treated bronchoalveolar -enriched T cells, but not in control

CD1-Restricted Recognition of Exogenous and Self-Lipid Antigens by Duodenal γδ+ T Lymphocytes

γδ T cells are present in the mucosal intestinal epithelia and secrete factors necessary to maintain tissue integrity. Ags recognized by these cells are poorly defined, although in mice non-classical MHC class I molecules have been implicated. Since MHC class I-like CD1 receptors are widely expressed at the surface of epithelial and dendritic intestinal cells and have the capacity to present lipid Ags to T cells, we hypothesized that these molecules might present autologous and/or exogenous phospholipids to intestinal γδ T lymphocytes. Intraepithelial T lymphocytes from normal human duodenal mucosal biopsies were cloned and exposed to natural and synthetic phospholipids using CD1a-, CD1b-, CD1c- or CD1d-transfected C1R lymphoblastoid or HeLa cell lines as APCs. Their cytolytic properties and regulatory cytokine secretion were also examined. Most clones obtained from duodenal mucosa (up to 70%) were TCRαβ+, and either CD4+ or CD8+, whereas 20% were CD4−CD8− (6 clones) or TCRγδ+ (12 clones). A relevant percentage (up to 66%) of TCRγδ+ but few (<5%) TCRαβ+ T cell clones responded to synthetic and/or natural phospholipids presented by CD1 molecules, as measured by both [3H]thymidine incorporation and IL-4 release assays. A Th1-like cytolytic and functional activity along with the ability to secrete regulatory cytokines was observed in most phospholipid-specific γδ T cell clones. Thus, a substantial percentage of TCRγδ+ but few TCRαβ+ from human duodenal mucosa recognize exogenous phospholipids in a CD1-restricted fashion. This adaptive response could contribute to mucosal homeostasis, but could also favor the emergence of inflammatory or allergic intestinal diseases.

Activation of cord T lymphocytes: II. Cellular and molecular analysis of the defective response induced by anti-CD3 monoclonal antibody

Despite the fact that the percentage of circulating CD3-positive cells is similar in cord and adult blood, the proliferative response induced by anti-CD3 monoclonal antibody (mAb) was impaired in the majority of human cord peripheral blood mononuclear cell (PBMC) samples we tested. The cell proliferative defect was associated with low interleukin 2 (IL 2) gene expression and scant IL 2 production. However, interleukin 2 receptor was fully expressed at both the mRNA and protein levels. Such a finding is consistent with the observation that exogenous recombinant IL 2 is able to boost the anti-CD3-mediated response of cord PBMC. Furthermore, when anti-CD3 and phorbol myristate acetate (PMA) were added together, they exerted a very marked synergistic effect on both the proliferation of, and IL 2 production by, cord PBMC. The addition of allogeneic antigen presenting cells plus soluble anti-CD3 or Sepharose-coupled anti-CD3 mAb to the cord T cell cultures had no significant effect on proliferation, whereas both elicited good mitogenesis of adult T cells. Moreover, addition of exogenous recombinant interleukin 1 to anti-CD3-stimulated T cells failed to trigger any proliferation in either adult or cord samples. Since the combination of PMA and calcium ionophore A23187 is effective in triggering optimal proliferation of cord T cells, the defect would seem to be associated with a failure in transmembrane transduction of the activation signals provided by the anti-CD3 stimulus for the cord T cell.

Abnormal gastrointestinal motility in patients with celiac sprue

No study to date has objectively investigated whether the motor behavior of the small bowel is abnormal in celiac sprue. The purpose of this study was to systematically address this topic by means of intraluminal pressure recordings in a series of such patients. Sixteen subjects (nine adults, seven children, age range 2–69 years) with celiac sprue were recruited and studied while untreated. Manometric examination was carried out for 6 hr during fasting and 3 hr after a meal. Adult celiac patients displayed a significantly (mean ±sem) greater frequency of migrating motor complexes in comparison to controls during fasting (4.44±1.6 vs 2.45±0.20,P<0.01), whereas no differences were found in the pediatric group with respect to this variable. Fasting motor abnormalities, chiefly represented by discrete clustered contractions, giant jejunal contractions, and bursts of nonpropagated contractions, were discovered in a high percentage in both groups of celiac subjects (89% in adults and 44% in children, respectively). Similar abnormalities were observed in the postprandial period, especially in adults. In conclusion, patients with celiac sprue frequently display discrete gastrointestinal motor abnormalities, which though perhaps nonspecific may account for several symptoms complained of by such patients.

Expression and functional role of 1F7 (CD26) antigen on peripheral blood and synovial fluid T cells in rheumatoid arthritis patients.

The expression and the functional role of the CD26 (1F7) T cell surface molecule, an ectoenzyme which seems to represent a functional collagen receptor of T lymphocytes and to have a role in T cell activation, were analysed in both peripheral blood (PB) and synovial fluid (SF) T cell samples from patients with active and inactive rheumatoid arthritis (RA). Although patients with active disease displayed higher percentages of PB CD26+ CD4+ T cells than inactive RA and control subjects, CD26 antigen expression on RA SF T lymphocytes was low. The anti‐1F7 binding to the T cell surface, that led to CD26 antigen modulation and enhancement of both IL‐2 synthesis by, and 3H‐TdR incorporation of, anti‐CD3‐ or anti‐CD2‐triggered PB T cells in RA and control subjects, was unable to affect significantly both expression and functional activity of RA SF T lymphocytes. Since the 1F7 antigen spontaneously reappeared on the surface of unstimulated SFT cells after 2‐5 days of culturing, the low IF7 antigen expression of anti‐lF7 in the SF T cell compartment may be the result of in vivo molecule modulation exerted by the natural ligand in the joint, with important implications for T cell activation and lymphokine synthesis.

γδ T cells, allergen recognition and airway inflammation

Abstract The ability of T-cell receptor (TCR) γδ cells to recognize foreign non-peptide antigens suggests a role in mucosal surveillance. However, their expansion, together with the presence of antigen-presenting cells (APCs) expressing CD1 antigens (a ligand for γδ TCRs), in the respiratory tract of allergic subjects also suggests a pathogenic role for these cells in allergic diseases. Here, the hypothesis is presented that γδ T cells and CD1 + APCs may operationally distinguish healthy from atopic status.

Phenotypic dissection of cord blood immunoregulatory T-cell subsets by using a two-color immunofluorescence study

Expression of TQ1(Leu8) and 2H4 antigens on human cord blood T-cell subsets was evaluated by a double immunofluorescence analysis. In normal adult blood all of the helper function for B-cell differentiation is confined to the smaller OKT4+TQ1-(Leu8-) cell subset, while the OKT4+TQ1+(Leu8+) cell subpopulation includes a subset of suppressor inducer 2H4+(JRA+) cells. Our results indicated that the OKT4+TQ1-(Leu8-) cell subpopulation was decreased and the reciprocal OKT4+TQ1+(Leu8+) cell subset was markedly increased in cord blood E-rosetting OKT3+ cell population. A rise in the number of cord OKT4+2H4+ cells was also found. In addition, TQ1 antigen was present on OKT3+E-, a less mature, cord T-cell subset, not present in adult blood. These findings may not only be of help in understanding lymphoid cell development during ontogeny, but also may agree with the reported strong-suppressor and weak-helper activities exerted by the T-cell subsets circulating in human cord blood.