Allergy and the skin.

Allergic skin disorders include urticaria, angioedema, contact dermatitis and atopic dermatitis, but the model fitting most closely the systemic concept of allergy is atopic dermatitis (AD), the pathogenesis of which is linked to a complex interaction between skin barrier dysfunction and environmental factors such as allergens and microbes. In particular, an important advance was the demonstration that the mutation of the skin barrier protein filaggrin is related strictly to allergen sensitization and to the development of asthma in subjects with AD. The altered skin barrier function, caused by several factors, results in the passage of allergens through the skin and to systemic responses. A pivotal role in such a response is exerted by Langerhans cells which, via their immunoglobulin E (IgE) receptor, capture the allergens and present them to T cells. When T helper type 2 (Th2) cells are activated, the production of a proinflammatory cytokines and chemokines pattern sustains the persistence of inflammation. Known AD-related cytokines are interleukin (IL)-5, IL-13 and tumour necrosis factor (TNF)-alpha, with emerging importance for IL-17, which seems to drive airway inflammation following cutaneous exposure to antigens, and IL-31, which is expressed primarily in skin-homing Th2 cells. Skin-homing is another crucial event in AD, mediated by the cutaneous lymphocyte-associated antigens (CLA) receptor, which characterizes T cell subpopulations with different roles in AD and asthma.

Mechanisms of villous atrophy in autoimmune enteropathy and coeliac disease.

Since in coeliac disease mucosal flattening has been suggested to result from an increased enterocyte apoptosis triggered by Fas/Fas ligand system and perforin cytolytic granules, we looked for a similar mechanism in autoimmune enteropathy. Moreover, we tried to assess whether enterocyte autoantibodies, which are the hallmark of autoimmune enteropathy, may be involved in triggering enterocyte apoptosis in this condition. Immunohistochemical staining with anti-Fas, -FasL and -perforin MoAb, and TUNEL technique were applied on endoscopic duodenal biopsies of two autoimmune enteropathy patients, two untreated coeliac patients and two biopsied controls. Cytotoxicity assays were carried out by incubating peripheral blood mononuclear cells from a healthy subject (effectors) with enterocytes primed with patient or control sera (targets). In autoimmune enteropathy a large number of enterocytes were apoptotic, as in coeliac disease, whereas neither Fas/Fas ligand or perforin expressions were up-regulated. On the other hand, antibody-dependent cellular cytotoxicity assay revealed the ability of sera from patients with autoimmune enteropathy to mediate enterocyte death through apoptosis. These results point to enterocyte autoantibody-dependent cellular cytotoxicity as the prevalent mechanism of increased enterocyte apoptosis in autoimmune enteropathy but not in coeliac disease.

Pyrimidine-2,4,6-Triones: a new effective and selective class of matrix metalloproteinase inhibitors.

Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases that have been implicated in various disease processes. Different classes of MMP inhibitors, including hydroxamic acids, phosphinic acids and thiols, have been previously described. Most of these mimic peptides and most likely bind in a similar way to the corresponding peptide substrates. Here we describe pyrimidine-triones as a completely new class of metalloprotease inhibitors. While the pyrimidine-trione template is used as the zinc-chelating moiety, the substituents have been optimized to yield inhibitors comparable in their inhibition efficiency of matrix metalloproteinases to hydroxamic acid derivatives such as batimastat. However, they are much more specific for a small subgroup of MMPs, namely the gelatinases (MMP-2 and MMP-9).

Expression of cytokines, inducible nitric oxide synthase, and matrix metalloproteinases in pouchitis: effects of probiotic treatment.

OBJECTIVE: The efficacy of probiotic organisms in the treatment of pouchitis has been reported. In the present study, we evaluated the tissue levels of pro- and anti-inflammatory cytokines, nitric oxide synthase, and matrix metalloproteinases in control and inflamed pouches before and after antibiotic and probiotic treatment of patients with acute pouchitis. METHODS: Pouch biopsy samples were obtained from seven patients with pouchitis before and after antibiotic and probiotic treatment. Tissue samples from five patients with normal pouches were used as controls. Cytokines were determined by ELISA, matrix metalloproteinase activity was evaluated by zymograms, and nitric oxide synthase activity was determined by measuring arginine to citrulline conversion. RESULTS: Tissue levels of tumor necrosis factor a increased (p < 0.01) in pouchitis relative to uninflamed pouches and reduced after antibiotic and probiotic treatment. Also, interferon y and interleukin 1alpha (IL-1alpha) augmented in pouchitis, but their increase did not reach statistical significance. The latter, however, were lower (p < 0.05) after treatment with the antibiotics and probiotics. Tissue levels of IL-4 and IL-10 were unchanged in inflamed pouches and unaffected by antibiotic treatment. However, IL-10 increased (p < 0.05) after probiotic treatment. Moreover, inflamed pouches had higher levels of inducible nitric oxide synthase and gelatinase activities, which decreased after treatment. CONCLUSIONS: The ability of antibiotic and probiotic treatments to increase tissue levels of IL-10, at a higher level than those observed in control pouches, and to decrease, to levels present in control pouches, proinflammatory cytokine, inducible nitric oxide synthase, and matrix metalloproteinase activity may suggest a mechanism of action to explain the efficacy of this therapeutic regime in pouchitis.

Circulating gamma/delta T lymphocytes from systemic sclerosis (SSc) patients display a T helper (Th) 1 polarization.

Systemic sclerosis (SSc) is a connective tissue disease in which immune system activation is evidenced by high levels of different cytokines in the sera and/or in the supernatants of cultured peripheral blood mononuclear cells (PBMC) and by the presence of specific autoantibodies. gamma/delta T cells accumulate in the lung and the skin of SSc patients suggesting their potential role in the development and maintenance of the disease. The aim of this study was to assess cytokine production and cytotoxic activity of circulating gamma/delta T lymphocytes obtained from SSc patients and to evaluate their potential role during this disorder. Our results showed that both the proportion and the absolute number of IFN-gamma gamma/delta-producing cells (i.e. displaying a Th1 polarization) in SSc was significantly higher than either the proportion and the absolute number of IL-4 gamma/delta-producing cells in SSc or the proportion and the absolute number of IFN-gamma gamma/delta-producing cells in healthy controls (P < 0.05 for both groups). Furthermore, the cytotoxic activity of enriched gamma/delta T cells was significantly increased in SSc patients compared with controls. The results concerning the Vdelta1+ T cell subset paralleled those of total gamma/delta T lymphocytes. In contrast, alpha/beta T cells from SSc and control subjects displayed Th2 cytokine production. All these findings were independent of both disease subset and clinical status. Our data demonstrate that, although SSc is generally considered a Th2 autoimmune disease, Th1 polarization of gamma/delta T cells and an increase in their cytotoxic activity is observed in SSc, suggesting that gamma/delta T cells could have a relatively autonomous role in the pathogenesis in this disease.

Apoptosis and peripheral blood lymphocyte depletion in coeliac disease.

In coeliac disease (CD) immunological abnormalities are not confined to the small bowel and it has been suggested that changes in peripheral blood lymphocytes (PBL), such as lymphopenia and increased T-cell activation, may predispose to malignant or autoimmune complications of this condition. In the light of the recent findings about the Fas-Fas ligand (FasL) system in regulating lymphocyte homeostasis, the aim of the present study was to investigate peripheral lymphocyte Fas-mediated apoptosis in CD to establish whether the homeostatic role of apoptosis in peripheral T-cell selection is maintained. Moreover, because a soluble form of Fas has been described to be functionally implicated in the Fas signalling system, suggesting a relationship between some disorders and soluble Fas function, we measured levels of soluble Fas in sera of coeliac patients and analysed the relationship between these levels and the proportions of apoptotic and Fas(+) PBL to further explore the function of the Fas-FasL pathway in this condition. Finally, we evaluated whether the increased prevalence of anticardiolipin antibodies, recently described in CD, could be related to PBL apoptosis in this condition. We demonstrated an increased apoptosis and higher levels of Fas and FasL expression in PBL isolated from untreated coeliac patients when compared to treated coeliac patients and controls. In addition, low levels of soluble Fas and a significant positive correlation between anticardiolipin antibodies and PBL apoptosis were found in untreated CD. Then, our results showed an increased susceptibility of PBL to undergo Fas-mediated apoptosis in active CD. This increased apoptosis could be responsible for both lymphopenia and immunogenic exposure of phospholipids with subsequent production of autoantibodies.

Intraepithelial and lamina propria lymphocytes show distinct patterns of apoptosis whereas both populations are active in Fas based cytotoxicity in coeliac disease.

BACKGROUND: Lamina propria (LPLs) and intraepithelial (IELs) lymphocytes are markedly increased in coeliac mucosa, and are thought to play a crucial role in the generation of villous atrophy in coeliac disease (CD). However, the mechanisms by which they mediate the killing of enterocytes in this condition are still poorly characterised. AIM: We investigated Fas mediated cytotoxicity and apoptosis of both LPLs and IELs, isolated from 10 untreated coeliac patients, 10 coeliac patients on a gluten free diet, and 10 biopsied controls. METHODS: Fas and Fas ligand expression were assessed by flow cytometry and immunocytochemistry. Lymphocyte cytotoxicity against Fas expressing Jurkat cells was determined by the Jam test. The effect of the antagonist ZB4 anti-Fas antibody on apoptotic activity exerted by coeliac lymphocytes against enterocytes was analysed. Lymphocyte apoptosis was assessed by oligonucleosome ELISA. RESULTS: LPLs and IELs showed increased apoptotic activity and higher levels of Fas ligand expression in untreated CD compared with treated CD patients and controls. Enterocyte apoptosis observed after coculturing coeliac lymphocytes and enterocytes in the presence of ZB4 antibody was reduced. In active CD, LPLs manifested increased apoptosis whereas IELs showed decreased apoptosis. CONCLUSIONS: Our results support the involvement of the Fas/Fas ligand system in CD associated enterocyte apoptosis. Increased LPL apoptosis is likely to downregulate mucosal inflammation whereas decreased IEL apoptosis could be responsible for autoimmune and malignant complications of CD.

Increased enterocyte apoptosis and Fas-Fas ligand system in celiac disease.

Our aim was to evaluate whether increased enterocyte apoptosis was responsible for mucosal flattening in celiac disease (CD), and, since the mechanisms responsible for tissue injury in this condition are unknown, we studied the possibility that the Fas-Fas ligand (FasL) system may be involved. Endoscopic duodenal biopsy specimens from 12 patients with untreated and 12 with treated CD and 12 control subjects were evaluated for enterocyte apoptosis by the terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine triphosphate nick-end labeling assay and for Fas and FasL expression by immunohistochemistry. A coculture of isolated enterocytes (targets) and purified lamina propria mononuclear cells (LPMCs) (effectors) was performed in the absence or presence of an antagonistic ZB4 anti-Fas antibody. We found a significant correlation between the degree of villous atrophy, morphometrically evaluated, and the level of enterocyte apoptosis, suggesting that mucosal flattening is a consequence of exaggerated epithelial cell death. Most celiac enterocytes express Fas, and LPMCs express FasL. The abolishment of enterocyte apoptosis observed in the presence of ZB4 antibody suggests that enterocytes are potential targets of lymphocyte infiltrate. These results directly demonstrate that FasL-mediated apoptosis is a major mechanism responsible for enterocyte death in CD.

Apoptotic effects of selected strains of lactic acid bacteria on a human T leukemia cell line are associated with bacterial arginine deiminase and/or sphingomyelinase activities.

The aim of the present work was, first, to analyze the apoptotic effect in vitro of sonicated preparations of selected strains of lactic acid bacteria on normal and tumor human lymphocytes. Incubation with bacterial samples led to a relevant time-dependent apoptotic cell death of Jurkat cells but not normal human peripheral blood lymphocytes. Lactobacillus brevis (CD2) samples were more efficient in inducing apoptosis of Jurkat cells than were samples of Streptococcus thermophilus (S244). In an attempt to characterize the mechanisms underlying these effects, we found that the apoptotic death-inducing ability of S244 preparations could be attributed to the ability of high levels of neutral sphingomyelinase activity to generate relevant amounts of ceramide, a known apoptotic death messenger, in Jurkat cells. On the other hand, our results indicate that apoptosis induced by CD2 samples could also be associated with high levels of arginine deiminase activity, which in turn was able to downregulate polyamine synthesis in Jurkat cells.

Cytolytic mechanisms of intraepithelial lymphocytes in coeliac disease (CoD).

The effector arm of the mucosal immune system comprises lymphocytes scattered at intraepithelial and lamina propria levels. Intraepithelial lymphocytes (IEL) are a large population of oligoclonal resting cells which exhibit phenotypic and functional characteristics of cytolytic T cells when activated. Several mechanisms have been demonstrated to account for their cytotoxicity. Among them, one is mediated by perforin and granzyme molecules, another is mediated by Fas ligand (FasL) which delivers apoptotic signals through Fas receptor on target cells. There is good evidence that a flat intestinal mucosa may be produced by activated T cells. The aim of our study was to evaluate FasL and perforin expression by IEL, and its possible correlation with the increased enterocyte apoptosis in coeliac mucosa. Endoscopic duodenal biopsy specimens from 10 untreated coeliac patients, 10 treated coeliac patients, and 10 biopsied controls were evaluated for enterocyte apoptosis by terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine triphosphate nick end label method, for perforin expression by immunohistochemistry, and for FasL expression by immunocytochemistry. In untreated CoD there was a significant increase of percentage of both FasL+ and perforin+ IEL which positively correlated with enterocyte apoptosis in comparison with controls. All these parameters were significantly lower in treated CoD, even though they did not normalize. Our study demonstrates that in untreated CoD FasL and perforin expression by IEL is increased, and significantly correlates with the level of enterocyte apoptosis.

Acetyl-L-carnitine administration increases insulin-like growth factor 1 levels in asymptomatic HIV-1-infected subjects: correlation with its suppressive effect on lymphocyte apoptosis and ceramide generation.

The aim of this study was to investigate the impact of long-term acetyl-L-carnitine administration on CD4 and CD8 absolute counts, apoptosis, and insulin-like growth factor-1 (IGF-1) serum levels in HIV-1-infected subjects. The generation of cell-associated ceramide and HIV-1 viremia were also investigated. Eleven asymptomatic, HIV-1-infected subjects were treated daily with acetyl-L-carnitine (3 g) for 5 months. Immunologic and virologic measures and safety were monitored at the start of the treatment and then on days 90 and 150. Altogether our findings suggest that acetyl-L-carnitine administration has a substantial impact on the main immunologic abnormality associated with HIV infection, the loss of CD4 cells, by reducing the rate of apoptotic lymphocyte death. The reduction of ceramide generation and the increase of the serum levels of IGF-1, a major survival factor able to protect cells from apoptosis by different stimuli and conditions, could represent two important mechanisms underlying the observed anti-apoptotic effects of acetyl-L-carnitine.

Interleukin-2-activated rat natural killer cells express inducible nitric oxide synthase that contributes to cytotoxic function and interferon-gamma production.

Natural killer (NK) cells are large granular lymphocytes capable of destroying cells infected by virus or bacteria and susceptible tumor cells without prior sensitization and restriction by major histocompatability complex (MHC) antigens. Their cytotoxic activity could be strongly enhanced by interleukin-2 (IL-2). Previous findings, even if obtained with indirect experimental approaches, have suggested a possible involvement of the inducible nitric oxide (iNOS) pathway in the NK-mediated target cell killing. The aim of the present study was first to directly examine the induction of iNOS in IL-2-activated rat NK cells isolated from peripheral blood (PB-NK) or spleen (S-NK), and second to investigate the involvement of the iNOS-derived NO in the cytotoxic function of these cells. Our findings clearly indicate the induction of iNOS expression in IL-2-activated PB-NK and S-NK cells, as evaluated either at mRNA and protein levels. Accordingly, significantly high levels of iNOS activity were shown, as detected by the L-arginine to L-citrulline conversion in appropriate assay conditions. The consequent NO generation appears to partially account for NK cell-mediated DNA fragmentation and lysis of sensitive tumor target cells. In fact, functional inhibition of iNOS through specific inhibitors, as well as the almost complete abrogation of its expression through a specific iNOS mRNA oligodeoxynucleotide antisense, significantly reduced the lytic activity of IL-2-activated NK cells. Moreover, IL-2-induced interferon-gamma production appears also to be dependent, at least in part, on iNOS induction.

Circulating soluble factor-inhibiting natural killer (NK) activity of fresh peripheral blood mononuclear cells (PBMC) from inflammatory bowel disease (IBD) patients.

This study was performed in order to assess the cytotoxic activity, both natural (NK) and antibody-dependent (ADCC), of PBMC from 38 IBD patients and correlate it with their clinical features. Cytotoxicity assays were performed using sensitive target cells for NK and ADCC activities. In some experiments, highly purified NK cells, obtained both by Percoll density gradient and by co-culturing non-adherent PBMC with RPMI 8866 feeder cells, were used as effector cells. Furthermore, we evaluated NK cell parameters such as number, surface expression of adhesion molecules (CD11a/CD18, CD49d and CD54) and response to different stimuli. We observed a decreased NK cytotoxicity of PBMC from IBD patients, both in ulcerative colitis (UC) and Crohn's disease (CD), independently of the clinical activity of disease. In contrast, the ADCC lytic activity was within normal range. The lower NK cytotoxic activity observed in our IBD patients cannot be related to a decreased number of NK cells, surface expression of adhesion molecules, defective response to IL-2 and maturative defect. Decreased NK activity was induced in PBMC of controls when serum of patients was added and this was unrelated to monocyte-derived modulating factor(s). Our data show a decreased natural killing by fresh PBMC from IBD patients. This lower activity seems to be unrelated to a primary NK cell defect, since purified NK cells exhibited normal levels of killing. It might be hypothesized that serum factors, possibly derived from lymphocytes, with inhibitory properties on NK activity, might be functionally active in the blood of IBD patients, thus modulating NK activity.

Modulation of nitric oxide synthase by nitric oxide donor compounds in migraine.

A controlled study was performed to assess the involvement of the nitric oxide pathway in migraine pathophysiology. Thirteen patients with migraine without aura and seven clinically healthy subjects (C) were selected. All of the migraine patients were studied both before, during an asymptomatic phase (t0), and 1 h after the administration of 5 mg isosorbide dinitrate, a nitric oxide donor able to induce an experimental migraine attack (t1). The nitric oxide levels were analyzed as nitrite accumulation in serum samples, in peripheral blood mononuclear cell extracts, and culture supernatants. Basal nitrite levels in serum samples and peripheral blood mononuclear cell culture supernatants or migraine patients and healthy subjects indicated that migraine patients possess an activated nitric oxide synthesis pathway (t0 vs. CF = 8.16, P < 0.01 and F = 16.2, P < 0.01, respectively). As expected, in the migraine patients treated with the nitric oxide donor, a marked increase of nitrite levels was observed in sera (t1 vs. t0 P < 0.05, t = 3.05). In contrast, during the nitric oxide donor-induced migraine attacks a statistically significant decrease of nitrite levels in peripheral blood mononuclear cell culture supernatants was observed (t1 vs. t0 P < 0.01, t = -4.03), whereas a significant increase of nitrite in total cell extracts was detected (t1 vs. t0 P < 0.001, t = -6.89). These preliminary data suggest that nitric oxide could be involved in the neurovascular modifications leading to a migraine attack.

NKR-P1A stimulation of arachidonate-generating enzymes in rat NK cells is associated with granule release and cytotoxic activity.

NKR-P1A protein has been implicated in the triggering of NK-mediated natural killing contributing to target cell recognition by NK cells. The aim of the present work was to assess whether NKR-P1A receptor triggering also induced arachidonic acid (AA) generation and to determine the possible role of this event on granule release and cytotoxicity. We demonstrated that activation of fresh peripheral blood rat NK cells by cross-linking with the anti-NKR-P1A 3.2.3 mAb induced calcium-dependent AA release, which is due to the activation of cytosolic phospholipase A2 (cPLA2), secretory phospholipase A2 (sPLA2), and diacylglycerol/monoacylglycerol lipase. We also documented the presence of a type II sPLA2 activity in the supernatant fluids from NKR-P1A-activated rat NK cells, suggesting that AA and lysophospholipids could be mobilized from the outside of the cell. The involvement of AA-generating enzymes in NKR-P1A-induced cytotoxic functions was also investigated. Treatment of effector cells with arachidonyl trifluoromethylketone, a cPLA2 inhibitor; p-bromophenacylbromide, a sPLA2 inhibitor; or RHC80267, a diacylglycerol lipase inhibitor, led to a partial inhibition of the redirected lysis against P815 target cells as well the granule content release induced by NKR-P1A cross-linking. A complete abolishment of these events was observed when the cells were simultaneously incubated with all three inhibitors. Taken together, our results support a crucial role for the arachidonate-generating enzymes in the induction of lytic activity of NK cells directly or by leading to generation of additional mediators that can play a role in the context of NK cell activation and cytotoxic functions.

Role of nitric oxide in cell-mediated tumor cytotoxicity.

Strong and increasing evidence shows that nitric oxide (NO) contributes to immune function, and in particular to 'non-specific host defense'. The aim of the present review was to focus the current understanding of the role of NO as a biochemical effector of L-arginine-dependent cell-mediated immune responses to neoplastic cells in vitro and in vivo. The cytokine-inducible nitric oxide synthase (NOS) seems to mainly be implicated in the cytotoxic activity of almost all the effector cells involved in tumor cell killing. The cytotoxic actions of NO against tumor cells appear to be related mainly to inhibition of several heme-containing enzymes of the mitochondrial electron transport complex and the citric acid cycle.