Viviana Ferlazzo

Differential requirements for antigen or homeostatic cytokines for proliferation and differentiation of human Vγ9Vδ2 naive, memory and effector T cell subsets

We have compared four human subsets of Vγ9Vδ2 T cells, naive (Tnaive, CD45RA+CD27+), central memory (TCM, CD45RA–CD27+), effector memory (TEM, CD45RA–CD27–) and terminally differentiated (TEMRA, CD45RA+CD27–), for their capacity to proliferate and differentiate in response to antigen or homeostatic cytokines. Cytokine responsiveness and IL‐15R expression were low in Tnaive cells and progressively increased from TCM to TEM and TEMRA cells. In contrast, the capacity to expand in response to antigen or cytokine stimulation showed a reciprocal pattern and was associated with resistance to cell death and Bcl‐2 expression. Whereas antigen‐stimulated cells acquired a TCM or TEM phenotype, IL‐15‐stimulated cells maintained their phenotype, with the exception of TCM cells, which expressed CD27 and CD45RA in various combinations. These results, together with ex vivo bromodeoxyuridine incorporation experiments, show that human Vγ9Vδ2 memory T cells have different proliferation and differentiation potentials in vitro and in vivo and that TEMRA cells are generated from the TCM subset upon homeostatic proliferation in the absence of antigen.

Tension-free hernia repair is associated with an increase in inflammatory response markers against the mesh ☆

BACKGROUND The purpose of this study was to evaluate the involvement of inflammatory mediators in patients undergoing Lichtenstein tension-free hernioplasty (LH) using polypropylene prosthetic materials or conventional Bassini hernia repair (BH). METHODS Thirty patients male with unilateral inguinal hernia without complications or recurrence were included in this study. Randomly, patients underwent LH or BH. Peripheral venous bloods samples were collected 24 hours prior to surgery and then 6, 24, 48 and 168 hours postoperatively. RESULTS We present evidences that LH patients showed a higher increased serum level of fibrinogen, C-reactive protein, alpha-1-antitrypsin, and interleukin-6 than BH patients. Postoperative visual analogue scales for pain were reduced on mobilization for patients undergoing LH compared with BH. Neutrophils were significantly increased only in LH compared with baseline. Ceruloplasmin, transferrin, and albumin levels were unmodified after BH or LH. CONCLUSIONS In conclusion our data show that although LH induces less pain and more rapid postoperative recovery, it is associated with an higher inflammatory response compared with BH, likely due to polypropylene mesh.

Reciprocal stimulation of γδ T cells and dendritic cells during the anti-mycobacterial immune response

γδ T cells and dendritic cells (DC) are two distinct cell types of innate immunity that participate in early phases of immune response against Mycobacterium tuberculosis infection. Here we show that a close functional relationship exists between these cell populations. Using an in vitro coculture system, Vγ1 T cells from Tcrb–/– mice were found to be activated by DC infected in vitro with BCG, as indicated by the elevated CD69 expression, IFN‐γ secretion and cytotoxic activity. This activation process was due to a non‐cognate mechanism since it required neither cell to cell contact nor interaction between the TCR and a specific antigen, but was mediated by DC‐derived IL‐12. Reciprocally, Vγ1 T cells provided a key cytokine, IFN‐γ, which increased IL‐12 production by BCG‐infected DC. Moreover, exposure of BCG‐infected DC to Vγ1 T cells conditioned the former to prime a significantly stronger anti‐mycobacterial CD8 T cell response. Consequently, stimulation of γδ T cells and their non‐cognate interaction with DC could be applied as an immune adjuvant strategy to optimize vaccine‐induced CD8 T cell immunity.

Estrogen regulates cytokine production and apoptosis in PMA-differentiated, macrophage-like U937 cells

We have investigated the effects of sex steroids, estradiol (E2), and testosterone (T) on the synthesis of tumor necrosis factor alpha (TNF‐α) and interleukin‐10 (IL‐10) in phorbol‐myristate‐acetate (PMA)‐differentiated human monoblastic U937 cells. The ability of both hormones to modulate the viability and programmed cell death of macrophage‐like PMA‐differentiated U937 cells was also inspected. E2 increased TNF‐α synthesis, whereas T had no effect on the production of this cytokine. The combination of E2 and its antagonist tamoxifen or ICI‐182,789 completely abolished the induction of TNF‐α, while combination of T and its antagonist Casodex (CSDX) did not significantly affect TNF‐α production by U937 cells. Exposure of cells to E2 resulted in a dose‐dependent decrease of IL‐10 synthesis, while again T did not show any detectable effect. In addition, E2 induced a significant increase of apoptosis in macrophage‐like U937 cells and this increase was inhibited by the simultaneous addition of either tamoxifen or ICI‐182. In contrast, T alone or in combination with CSDX did not modify apoptotic rates of U937 cells. This evidence, taken together, suggests that estrogens, but not androgens, exert a pro‐inflammatory action through the modulation of TNF‐α and IL‐10, and regulate the immune effector cells by the induction of programmed cell death. J. Cell. Biochem. 90: 187–196, 2003.

Anti-inflammatory effects of chemically modified tetracyclines by the inhibition of nitric oxide and interleukin-12 synthesis in J774 cell line

We investigated the effects of chemically modified tetracyclines (CMTs) on the production of nitric oxide (NO) and on the synthesis of some cytokines: tumour necrosis factor alpha (TNF-alpha), interleukin(IL)-10 and IL-12 in lipopolysaccharide (LPS)-treated J774 cell line. Furthermore, we studied the ability of these drugs to modify the viability in LPS-stimulated J774 macrophages. CMTs decreased, in a dose-dependent manner, inducible NO synthase (iNOS) activity and, consequently, nitrite formation in J774 cultures. The CMT-induced decrease in NO production is due to the inhibition of enzyme activity rather than to a direct effect on enzyme expression. The absence of the inhibition in mRNA accumulation indicates that the inhibiting activity is mainly post-transcriptional. CMTs were unable to modulate TNF-alpha and IL-10 synthesis and they were not effective in modifying the transcription of relative mRNA in J774 macrophages. On the contrary, IL-12 mRNA expression was significantly increased by CMT-1 and CMT-8 with LPS activation. Since IL-12 protein secretion was inhibited by CMTs, these compounds interfere in the blocking of post-transcriptional events. The studies on cell viability showed that various CMTs induced a dose-dependent decrease in J774 macrophage viability. The cytotoxic activity was present even though NO production was inhibited by CMTs. These compounds appear to be able to activate apoptosis in aNO-independent way. Altogether, these results indicate that CMTs can exert anti-inflammatory effects by inhibiting NO synthesis, and they are able to modify cell viability by exerting a strong apoptotic activity.

Anti-inflammatory effects of annexin-1: stimulation of IL-10 release and inhibition of nitric oxide synthesis.

Annexin-1 (ANX-1) is an anti-inflammatory protein induced by glucocorticoids. Like glucocorticoids, ANX-1 and derived peptides inhibit eicosanoid synthesis, block leukocyte migration and induce apoptosis of inflammatory cells. Cytokines may possess either pro-inflammatory, i.e. interleukin(IL)-1beta, tumor necrosis factor (TNF)-alpha, IL-12 or anti-inflammatory properties, i.e. IL-4, IL-10. The experiments described in the present study have been performed to answer the question whether the anti-inflammatory action of ANX-1 may be mediated, at least in part, by the release of IL-10. In macrophage (J774) cell line cultures primed with lipolysaccharide (LPS), recombinant ANX-1 stimulated IL-10 release in a dose- and time-dependent manner. In the same cells, the protein and its derived N-terminal peptide (amino acids 2-26) dose-dependently inhibited the release of nitric oxide (NO). Furthermore, both the whole protein and the peptide down-regulated the mRNA expression of the inducible nitric oxide sythase (iNOS). The peptide was also able to inhibit the expression of IL-12 mRNA. These results suggest that some of the anti-inflammatory effects of ANX-1 may be mediated by the release of IL-10, which, in turn, inhibits iNOS mRNA expression and, hence, NO release. In addition, ANX-1-stimulated IL-10 release may also be responsible for the inhibition of IL-12 mRNA expression and, consequently, IL-12 synthesis.

Association between the HLA-DR alleles and longevity: a study in Sardinian population.

Human longevity may be correlated with optimal functioning of the immune system, suggesting that genetic determinants of longevity also resides in those polymorphisms for the immune system genes that regulate immune responses as histocompatibility (HLA) antigens. However, conflicting results have been obtained. Some well planned and designed association studies performed in Caucasians suggest that longevity is associated with positive selection of alleles (i.e. HLA-DR11) or haplotypes (i.e. HLA-B8,DR3) that confer resistance to infectious diseases, respectively, via peptide presentation or via antigen non-specific control of immune response. Association studies are subjected to a number of possible confounding factors, the homogeneity of the population in term of geographical origin among others. Because of the lack of large-scale heterogeneity, the Sardinians represent a suitable population for association studies addressed to dissect the complex traits as longevity. Thus, we have evaluated, by the amplification refractory mutation system/polymerase chain reaction, HLA-DR frequencies in 120 centenarians (79 women and 41 men) and 86 controls (53 women and 33 men) from Sardinia, to validate, in this very homogeneous population, the associations between HLA alleles or haplotypes and longevity observed in other Caucasoid populations. No significant differences were obtained by analysing the differences between Centenarians and controls except for HLA-DRB1*15 that was increased in centenarians. However, the significance was not maintained by multiplying P values for the number of alleles under study. Thus, in Sardinian centenarians, we were not able to confirm the findings observed in the well planned and designed studies performed in other Caucasoid populations. Besides, HLA HFE gene polymorphisms have been recently demonstrated to be associated with longevity in the Sicilian population but not in Danish one. On the whole these findings clearly show that HLA/longevity associations are population-specific, being heavily affected by the population-specific genetic and environmental history. So, in our opinion, HLA genes might be considered survival genes not longevity genes.

Cytokines and growth factors in wound drainage fluid from patients undergoing incisional hernia repair

Knowing the dynamics of growth factor and cytokine secretion within the site of a surgical operation is important, as they play a crucial role in the pathophysiology of wound healing and are a target for modifying the repair response. The aim of this study was to evaluate the production of several cytokines and growth factors in the drainage wound fluid from patients undergoing incisional hernia repair: namely, interleukin (IL)‐6, IL‐10, IL‐1α, IL‐1 ra, interferon‐γ, vascular endothelial growth factors and basic fibroblast growth factor. Ten female patients with abdominal midline incisional hernia undergoing surgical repair were included in this study. In all cases, a closed‐suction drain was inserted in the wound below the fascia and removed on postoperative day 4. Wound fluid was collected on postoperative days 1–4 and the amount was recorded each time. Growth factors and cytokines production was evaluated as the whole amount produced over a 24‐hour period. In all patients, the amount of drain fluid from surgical wounds was more copious the first day after surgery, it decreased significantly afterward. The presence of all cytokines was highest on postoperative day 1, decreasing over the following days. More specifically, the production of IL‐1 ra, IL‐6, IL‐1α, and IL‐10 on postoperative day 1 fell sharply on postoperative days 3 and 4, whereas, after an initial reduction, interferon‐γ showed an increase from day 2 onward. Vascular endothelial‐derived growth factor production increased progressively after the operation reaching statistical significance only on day 4. As for basic fibroblast growth factor, it showed an opposite pattern: it was higher on postoperative day 1 decreasing thereafter. This analysis of cytokine and growth factor production in the drain fluid will lead us to a better evaluation of the events that follow a surgical wound and to a better understanding of the healing process.

Effects of chemically modified tetracyclines (CMTs) in sensitive, multidrug resistant and apoptosis resistant leukaemia cell lines

Recently discovered chemically modified tetracyclines (CMTs) have shown in vitro and in vivo anti‐proliferative and anti‐tumour activities. Here, we evaluated in vitro the anti‐proliferative and apoptotic activity of six different dedimethylamino chemically modified tetracyclines (CMT‐1, CMT‐3, CMT‐5, CMT‐6, CMT‐7 and CMT‐8) in sensitive and multidrug resistant myeloid leukaemia cells (HL60 and HL60R) in vitro. Three of these compounds (CMT‐5, CMT‐6, CMT‐7) showed low cytotoxic activity both in sensitive and in resistant cells, CMT‐3 was endowed with a high anti‐proliferative activity only in sensitive cells and was moderately effective as apoptosis inducing agent, with an activity similar to that shown by doxycycline. On the contrary, CMT‐1 and CMT‐8 were very effective as programmed cell death inducing agents. The apoptotic pathway activated by these compounds involved the activation of caspases, especially caspase‐9 and, for CMT‐1, also the activation of Fas. Interestingly CMT‐8, but not CMT‐1, was able to induce apoptosis in multidrug resistant HL60R and in Fas‐ligand resistant HUT78B1 cell lines. These properties, together with others previously described (e.g. anti‐metastatic and anti‐osteolytic activities), suggest that CMT‐8 may have important applications in the clinical management of cancer. The comparative analysis of structure‐activity relationship of CMT‐8 and doxycycline suggests that the C‐5 hydroxy moiety may play an important role in conferring activity in multidrug resistant cells. These findings appear to support the hypothesis that CMT‐8 may represent an interesting lead for the development of a new class of potent apoptosis inducer agents active in multidrug resistant and Fas‐ligand resistant malignancies.

CD4+ CCR5+ and CD4+ CCR3+ lymphocyte subset and monocyte apoptosis in patients with acute visceral leishmaniasis

The potential involvement of apoptosis in the pathogenesis of visceral leishmaniasis (VL) was examined by studying spontaneous and Leishmania antigen (LAg)‐induced apoptosis using cryopreserved peripheral blood mononuclear cells (PBMC) of Sicilian patients with VL. Results indicate that monocytes and T lymphocytes from acute VL patients show a significantly higher level of apoptosis compared with that observed in healed subjects. The percentage of apoptotic cells was higher in monocytes than in T lymphocytes. T cells involved in programmed cell death (PCD) were mainly of the CD4+ phenotype. In particular, the T helper 1‐type (Th1) subset, as evaluated by chemokine receptor‐5 (CCR5) expression, is involved in this process. Cell death in Th1‐type uses a CD95‐mediated mechanism. Furthermore, Th1‐type CCR5+ cells are prone to cell suicide in an autocrine or paracrine way, as attested by enhanced expression of CD95L in acute VL patients. The reduction in Th1‐type cells by apoptosis was confirmed by the decrease in interferon‐γ secretion. In conclusion, apoptosis of monocytes, CD4+ and CD4+ CCR5+ T cells could be involved in the failure of cell mediated immunity that is responsible for severe immune‐depression in VL.