Antonella D'Ambrosio

Cholera toxin B subunit promotes the induction of regulatory T cells by preventing human dendritic cell maturation

Cholera toxin B subunit (CTB) is an efficient mucosal carrier molecule for the generation of immune responses to linked antigens. There is also good evidence that CTB acts as an immunosuppressant, as it is able to down‐modulate human monocyte/macrophage cell line activation and to suppress Th1‐type responses. In the present study, we examined the possibility that recombinant CTB (rCTB) may affect human dendritic cell (DC) functions in response to LPS stimulation and may induce the generation of DC with the capacity to generate CD4+ regulatory T cells (Tregs). Our findings show that rCTB partially prevents the LPS‐induced maturation process of monocyte‐derived DC (MDDC) and decreases their IL‐12 production with no relevant effect on IL‐10 production. LPS‐stimulated MDDC pretreated with rCTB are able to promote the induction of low proliferating T cells, which show an enhanced IL‐10 production associated with a reduced IFN‐γ production and the same high levels of TGF‐β as the control. These T cells suppress proliferation of activated autologous T cells. Transwell experiments and blockade of IL‐10R and TGF‐β showed that the immunomodulatory effect is mediated by soluble factors. Thus, T cells induced by rCTB‐conditioned MDDC acquire a regulatory phenotype and activity similar to those described for type 1 Tregs.

Peripheral blood biomarkers in multiple sclerosis.

Multiple sclerosis is the most common autoimmune disorder affecting the central nervous system. The heterogeneity of pathophysiological processes in MS contributes to the highly variable course of the disease and unpredictable response to therapies. The major focus of the research on MS is the identification of biomarkers in biological fluids, such as cerebrospinal fluid or blood, to guide patient management reliably. Because of the difficulties in obtaining spinal fluid samples and the necessity for lumbar puncture to make a diagnosis has reduced, the research of blood-based biomarkers may provide increasingly important tools for clinical practice. However, currently there are no clearly established MS blood-based biomarkers. The availability of reliable biomarkers could radically alter the management of MS at critical phases of the disease spectrum, allowing for intervention strategies that may prevent evolution to long-term neurological disability. This article provides an overview of this research field and focuses on recent advances in blood-based biomarker research.

N-ACETYL-CYSTEINE ENHANCES CELL ADHESION PROPERTIES OF EPITHELIAL AND LYMPHOID CELLS

In this paper, we show that the antioxidant N‐acetyl‐cysteine (NAC) is capable of enhancing the adhesion properties of the epithelial cell line A431 and of the lymphocytic cells with cytotoxic activitv from human peripheral blood: the natural killer (NK) cells. This effect leads to an increased efficiency of A431 cells to form a monolayer and of NK cells to kill their targets. In both cases a specific effect of NAC was found in the distribution of those molecules of the cytoskeleton which are generally involved in cell substrate and cell‐to‐cell contact region formation, e.g., the actin microfilaments. NAC could thus behave as a drug influencing certain cytoskeleton‐dependent cell processes in a non‐histotype dependent manner.

Thiol supplier N-acetylcysteine enhances conjugate formation between natural killer cells and K562 or U937 targets but increases the lytic function only against the latter

In this in vitro study, an evaluation of the importance of intracellular oxidative balance on cell-mediated cytotoxicity was performed by analyzing the effects of the antioxidant N-acetylcysteine (NAC), a specific thiol supplier, on natural killer (NK) cell-mediated cytotoxicity. The results obtained indicate that an enhancement of target cell (TC) killing can be detected when a pre-exposure of effector cells (EC) to NAC was performed. However, this effect seems to depend upon the TC type used. In fact, the increase of EC activity was detected against the differentiated U937 TC while no changes were detected by the same effectors against K562 cells. The mechanism of this enhancement seems to be ascribable to an increased ability of NAC-exposed NK cells to form conjugates (binding) which, in turn, appears to be due to a specific effect of NAC on actin microfilaments. A role for NAC as a cytoskeleton thiol-modifier contributing to the activation of effector cells can thus be hypothesized.

Diltiazem modulates monokine production in human mixed lymphocyte culture

BACKGROUND Calcium channel blockers are widely used in transplantation. Their immunosuppressive activity is well known and has been demonstrated both in vitro and in vivo. Nevertheless, their effect on cytokine production has never been reported. METHODS One-way mixed lymphocyte cultures (MLCs) have been obtained from healthy human subjects. Cytokine production has been assessed by three different methods: by enzyme-linked immunosorbent assay on supernatants of MLC, by enzyme-linked immunospot method on MLC cells for measuring cytokine-producing cells, and by the reverse transcription polymerase chain reaction technique on MLC cells for measuring cytokine mRNAs. RESULTS An interesting effect on proinflammatory monokines was observed: in this study, we demonstrate that the calcium antagonist diltiazem enhances interleukin-1beta and slightly reduces interleukin-6 production in MLC, but it has no effect on tumor necrosis factor-alpha levels. CONCLUSION For the first time, a modulation of monokine production by diltiazem can be demonstrated. This evidence suggests that calcium antagonist drugs may exert effects on monocytes and possibly on other antigen-presenting cells.

Lamina Propria CD4+LAP+ Regulatory T Cells Are Increased in Active Ulcerative Colitis but Show Increased IL-17 Expression and Reduced Suppressor Activity

BACKGROUND A CD4+CD25- regulatory T cell population expressing the surface TGF-β in its latent form LAP+ [latency associated peptide] cells was proved to be protective in experimental colitis and to be suppressive of human peripheral blood [PB] T proliferation. We investigated the frequency and function of lamina propria [LP] CD4+LAP+ T cells in inflammatory bowel disease [IBD] patients. METHODS Specimens from patients undergoing colonoscopy or bowel resection for IBD and colonic cancer were used as source of lamina propria mononuclear cells [LPMC]. The ulcerative colitis [UC] group was divided according to endoscopic activity evaluated with modified Baron Score. IL-17, IFN-γ, IL-10, LAP, and Foxp3 expression in CD3+CD8- [CD4] or CD3+/CD4+ gated cell population was assessed by immunofluorescence. The ability of FACS-sorted LP CD3+CD8-[CD4] LAP+CD25- to inhibit stimulated autologous PB CD3+CD8-[CD4] LAP- CD25- cells proliferation was assessed. RESULTS LP CD4LAP+ cells were significantly increased, when compared with controls, in active UC patients and not in Crohns disease patients. The majority of LP CD4+LAP+ cells were Foxp3-. The percentage of IL-17+ cells in LP CD3+CD8-[CD4] LAP+ cells was significantly higher in active UC patients when compared with controls. LP CD3+CD8-[CD4]LAP+CD25- isolated from UC patients showed reduced or no ability to inhibit autologous PB CD3+CD8-[CD4]LAP-CD25- cell proliferation when compared with controls. Removal of IL-17+ cells from LP CD3+CD8-[CD4] LAP+ cells increases their suppressive ability. CONCLUSIONS The percentage of LP CD4LAP+ cells is increased in active UC, showing reduced suppressor activity due to their increased proportion of intracellular IL-17 expression.

Modulation of Human Peripheral Blood Mononuclear Cell Proliferative Response by Diltiazem : In Vitro Comparison in Younger versus Older Subjects

BackgroundThe gradual aging of population has increased the number of elderly patients receiving kidney transplants. In elderly transplant recipients, careful immunosuppression has to be maintained to avoid both rejection and adverse effects. Clinical protocols after kidney transplantation include use of the calcium channel antagonist diltiazem to ameliorate the hypertensive effect and nephrotoxicity of the immunosuppressant agent ciclosporin (cyclosporine).ObjectiveSince immune response can be impaired by senescence, we evaluated the influence of diltiazem on lymphocyte proliferation both alone and in the presence of ciclosporin in younger versus older subjects.MethodsPeripheral blood mononuclear cells (PBMC) from younger healthy donors (aged 19–24 years) and older subjects (aged 59–65 years) were isolated and stimulated with mitogens, recombinant human interleukin-2 (IL-2), purified protein derivative (PPD) antigen from Mycobacterium tuberculosis, and anti-CD3 monoclonal antibody (αCD3 moAb) in the presence or absence of 10−4, 10−5, 10−6, 10−7 mol/L concentrations of diltiazem. In some experiments, lymphocytes from younger and older subjects were used as responder cells in an allogeneic mixed lymphocyte reaction (MLR) in the presence of different concentrations of diltiazem and 10 ng/mL of ciclosporin.ResultsWe found that PBMC from older subjects were more susceptible to immunosuppression induced by low concentrations of diltiazem when mitogens were used to stimulate cells. In particular, when pokeweed mitogen was used, diltiazem 10−7 mol/L was associated with statistically significant immunosuppression in older subjects compared with younger subjects. This effect was not observed when IL-2, PPD antigen and αCD3 moAb were used as stimulators. Moreover, in the allogeneic MLR, we found no differences between younger and older subjects when the 10−5, 10−6 and 10−7 mol/L concentrations of diltiazem were used alone or in the presence of ciclosporin. Only addition of the suprather-apeutic 10−4 mol/L concentration of diltiazem to ciclosporin was associated with statistically significant immunosuppression in older versus younger subjects.DiscussionOur results show that PBMC from older subjects are no more susceptible than PBMC from younger subjects to therapeutic doses of diltiazem when T-cell receptors are directly or indirectly involved. On the contrary, when PBMC activation was not mediated by T-cell receptor involvement, as in the case of pokeweed mitogen, susceptibility to a therapeutic concentration of diltiazem in older subjects was enhanced. Moreover, co-administration of therapeutic doses of diltiazem and ciclosporin in an MLR showed no significant differences between younger and older subjects in an in vitro model of lymphocyte response to allogeneic transplantation.ConclusionSince we found no variations in immunosuppression between older and younger subjects when therapeutic doses of diltiazem were added to ciclosporin, our data do not discourage the use of diltiazem in older kidney transplant recipients receiving ciclosporin therapy.