Successful bone marrow transplantation reveals the lack of endothelial progenitor cells mobilization in a patient with critical limb ischemia: a case report.

Restoring blood flow to ischemic tissue is a prerequisite for treatment of ischemic diseases. Cell-based therapy based on bone marrow transplantation is a promising option for patients with critical limb ischemia (CLI). The efficacy of cell therapies to augment neovascularization seems to involve endothelial progenitor cells (EPCs); however, the mechanisms underlying the efficacy have not been fully elucidated. Herein we have described the case of a young patient with severe CLI, who experienced a 24-month beneficial clinical response to autologous bone marrow transplantation. The exceptional amelioration enabled him to perform standardized maximal treadmill exercise test that demonstrated lack of exercise-induced EPC mobilization, despite adequate stromal-derived factor 1 and vascular endothelial growth factor responses. Therefore, tissue ischemia is not sufficient to promote the recruitment of EPCs that have been demonstrated to be involved in the recovery from ischemia. The local implantation of marrow-derived elements may provide cells and/or trophic factors, which have the capacity to augment angiogenesis, opening new approaches to the etiopathogenesis of the disease.

Altered maturation of peripheral blood dendritic cells in patients with breast cancer.

Tumours have at least two mechanisms that can alter dendritic cell (DC) maturation and function. The first affects the ability of haematopoietic progenitors to differentiate into functional DCs; the second affects their differentiation from CD14+ monocytes, promoting an early but dysfunctional maturation. The aim of this study was to evaluate the in vivo relevance of these pathways in breast cancer patients. For this purpose, 53 patients with invasive breast cancer were compared to 68 healthy controls. To avoid isolation or culture procedures for enrichment of DCs, analyses were directly performed by flow cytometry on whole-blood samples. The expression of surface antigens and intracellular accumulation of regulatory cytokines upon LPS stimulation were evaluated. The number of DCs, and in particular of the myeloid subpopulation, was markedly reduced in cancer patients (P<0.001). Patient DCs were characterized by a more mature phenotype compared with controls (P=0.016), and had impaired production of IL-12 (P<0.001). These alterations were reverted by surgical resection of the tumour. To investigate the possible role of some tumour-related immunoactive soluble factors, we measured the plasmatic levels of vascular endothelial growth factor, IL-10 and spermine. A significant inverse correlation between spermine concentration and the percentage of DCs expressing IL-12 was found. Evidence was also obtained that in vitro exposure of monocyte-derived DCs to spermine promoted their activation and maturation, and impaired their function. Taken together, our results suggest that both the above-described mechanisms could concomitantly act in breast cancer to affect DC differentiation, and that spermine could be a mediator of dysfunctional maturation of DCs.

[Cytofluorimetric evaluation of peripheral blood dendritic cells in patients with Mediterranean Kaposi's sarcoma]. / Valutazione citofluorimetrica di cellule dendritiche circolanti in pazienti affetti da sarcoma di Kaposi mediterraneo.

AIM: Kaposi's sarcoma (KS) is a lympho-angioproliferative disorder characterized by angiomatous nodules and plaques that mainly affect the skin. The disease is consistently associated with human herpesvirus-8 (HHV8) and with a state of preexistent immunosuppression. Dendritic cells (DCs) have an instrumental role in the activation and function of both innate and adaptative immune responses. At least 2 distinct subsets have been characterized in peripheral blood based on phenotypic markers: myeloid DCs (CD11c+), associated with Ag uptake, T cell activation and ability to secrete IL-12, and plasmacytoid DCs, high virus-induced IFN-alpha producing cells. Because of the role of both DC subtypes in antiviral and antitumor induced responses, we hypothesized that DCs could be involved in the onset and evolution of KS. METHODS: Thirty-five patients with mediterranean KS assigned to different clinical stages were compared with 51 healthy control subjects. Peripheral blood DCs were quantified and functionally characterised by flow cytometry directly on whole blood samples. The production of the regulatory cytokines, IL-12 and IL-10, was assessed as intracellular accumulation after incubation with or without lipopolysaccharide (LPS). RESULTS: Myeloid DCs identified as lineage-/HLA-DR+/CD11c+ cells were significantly lower in KS patients than in controls (0.54+/-0.25 vs 0.69 +/-0.26% of the peripheral blood mononuclear cells; p<0.017). Furthermore, CD11c+ DCs were lower in patients with more diffuse disease. Plasmacytoid DCs, identified as lineage-/HLA-DR+/CD123+ cells, were lower in KS patients (0.23+/-0.19 vs 0.36+/-0.17; p<0.001). DCs from KS patients were more mature, as assessed by expression of the maturation marker CD83, and showed an impaired ability to produce IL-12 upon LPS stimulation, as compared with controls. CONCLUSION: The numerical and functional alterations of peripheral blood DCs observed in KS patients suggest an involvement of these cells in the onset and evolution of the disease.

Cytokine production in scleroderma patients: effects of therapy with either iloprost or nifedipine.

OBJECTIVE: To compare the long-term effects of intermittent infusion of iloprost with those of oral nifedipine on the in vitro production of cytokines in patients with systemic sclerosis (SSc), and to evaluate their relationship with the effects of the two treatments on clinical parameters. METHODS: The production of cytokines by alloactivated circulating mononucleated cells was assessed before and after one year of treatment in a subset of 31 patients enrolled in a 12-month randomized clinical trial. Nineteen patients were treated with a 5-day (8 hr per day), 2.0 ng/kg per minute infusion followed by a 1-day infusion every 6 weeks; 12 patients were treated with an oral slow-release formulation of nifedipine, 20 mg twice daily. Quantitative determinations of interleukin-1 beta (IL1-beta) and interleukin-6 (IL6) in the culture supernatants were performed with a commercial ELISA; the levels of tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) were measured by specific radioimmunometric assays. RESULTS: The production of IL1-beta was significantly lower in the iloprost group than in the nifedipine group. Both the cutaneous fibrosis and the capillaroscopic patterns were better in patients treated with iloprost than in patients treated with nifedipine. There was a significant positive covariance between IL1-beta changes and the changes in both the skin score and the capillaroscopic score. CONCLUSION: There are several mechanisms by which iloprost could exert its clinical efficacy. Vasodilatation and inhibition of platelet aggregation are certainly important, but they are transient. We suggest that the long-lasting modulation of the cytokine network observed in the present study could be another potential mechanism responsible for the persistent efficacy of iloprost despite its intermittent administration.

Differential effects of cyclo-oxygenase pathway metabolites on cytokine production by T lymphocytes.

Cyclo-oxygenase pathway metabolites released in the microenvironment by activated platelets and endothelial cells are potential local modulators of the immune response. In the present study, we have investigated the modulatory role of PGE2, iloprost (prostacyclin analogue), U-46619 (thromboxane analogue) on the release of IL-2, IFN-gamma, TNF-alpha and IL-6 by T lymphocytes. Our results show that PGE2 and prostacyclin differ in the regulation of cytokine production. PGE2 inhibited the release of IL-2 and IFN-gamma, while iloprost did not affect their production. The addition of PGE2 or iloprost greatly decreased the amount of TNF-alpha measured in the supernatants, although the rates of inhibition differed according to the kind of stimulation. Unlike that of PGE2, inhibition by iloprost was stronger in alloactivated cultures than in PHA-stimulated ones. In vitro IL-6 production was stimulated by PGE2 in alloactivated cultures and by iloprost, whatever the stimulus. These results are probably due to other cellular subsets contaminating the T-lymphocyte preparations. After complete removal of monocytes from cell cultures, there were inhibitory effects of lloprost and PGE2 on IL-6 released in the supernatants. We did not observe any significant effect of thromboxane analogue on the production of either cytokine.

Common variable immunodeficiency following Epstein-Barr virus infection.

The authors present a case of a patient who developed recurrent bacterial upper respiratory and pulmonary infections and marked hypogammaglobulinemia with a gradual decrease of serum IgG, IgA and IgM some months after acute Epstein-Barr virus infection. Test for identification of lymphocyte subpopulation showed increased CD8+ T-cells with a surface phenotype (CD8+, CD57+, HLA-DR+) characteristic of virus-induced, activated cytotoxic cells. Viral investigations showed a positive anti-EBNA titer, an IgG titer anti-VCA of 1:40, a negative IgG titer anti-EA and human immunodeficiency virus negativity. The authors conclude that these clinical features are indicative of possible common variable immunodeficiency following Epstein-Barr virus infection.

Association between polymorphisms in the TNF region and systemic lupus erythematosus in the Italian population.

The purpose of this work was to the hypothesis that MHC encoded susceptibility factors for SLE lie in the TNF region. An association study was performed by analyzing 123 northern Italian SLE patients and 199 matched controls for three TNF markers: two polymorphisms in the TNFA promoter and the TNFa microsatellite. Haplotypic combinations of TNF markers were also compared in patients and controls. No significant association was observed considering either the whole SLE panel or SLE clinical and immunological subtypes. Three TNF-238/A homozygous patients were detected, while no homozygote was present in controls. The clinical and immunological phenotype of the three -238/A homozygotes suggests that the -238/AA genotype is a marker of a particular clinical subtype.

Secretion of cytokines upon allogeneic stimulation: effect of aging.

The aim of the present study was to investigate the in vitro mitotic response and cytokine production after allogeneic stimulation of peripheral blood mononuclear cells (PBMC) from healthy elderly subjects. Interleukin-1 (IL-1), interleukin-2 (IL-2), gamma-Interferon (gamma-IFN), and Tumor Necrosis Factor alpha (TNF-alpha) were detected in the supernatants of mixed lymphocyte cultures (MLC). The in vitro proliferative response was significantly reduced in the elderly subjects. The amount of IL-2 detected in the supernatant from cultures of cells from elderly donors was higher than for young controls, as were the production of cytokines predominantly secreted by the macrophage population (IL-1 and TNF-alpha). There was no difference in the production of gamma-IFN by cells of elderly and young subjects.

Markers of procoagulant imbalance in patients with localized melanomas and autoimmune disorders.

Hypercoagulability can be defined as a condition of procoagulant imbalance due to heightened enzymatic activation of coagulation zymogens, but with no laboratory evidence of fibrin deposition nor clinical signs of thrombosis. The imbalance can be detected by measuring the plasma levels of prothrombin fragment 1 + 2 (F1 + 2), fibrinopeptide A (FPA) and thrombin-antithrombin III (TAT) complexes. The aims of this study were to establish the frequency of existence and biochemical pattern of hypercoagulability in patients with cancer and autoimmune disorders, clinical conditions associated with an increased risk of thrombosis, and to ascertain the most sensitive method for its diagnosis. In approximately one-fourth of the patients hypercoagulability was identified by finding high levels of FPA F1 + 2 or TAT unaccompanied by signs of fibrin deposition (expressed by normal levels of D-dimer). In a smaller proportion of patients (approximately 10%), the concomitant presence of high levels of D-dimer indicated that the activation of the coagulation cascade had gone beyond the stage of heightened enzymatic activity to the point of cross-linked fibrin deposition. Of the markers used to detect hypercoagulability. FPA seems to be the most sensitive, being significantly increased in all clinical conditions studied.