Caterina Barbera

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.

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.

Tetracycline inhibits the nitric oxide synthase activity induced by endotoxin in cultured murine macrophages

Here we investigate the effects of tetracycline base and of a semi-synthetic tetracycline derivative, doxycycline, on the induction of inducible nitric oxide synthase and, hence, on the production of nitric oxide (NO) by lipopolysaccharide in J774 macrophage cultured in vitro. The treatment of J774 line with tetracycline base (6.25-250 microM) or doxycycline (5-50 microM) dose-dependently decreased the lipopolysaccharide-stimulated (1 microg/ml) inducible NO synthase activity and, consequently, nitrite formation. For instance, the inhibition was 70% for tetracycline base at 250 microM and 68% for doxycycline at 50 microM. The inhibitory effect of tetracyclines was due neither to a reduction in the viability of the cells, studied as colorimetric 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay, nor to an indiscriminate inhibition of total protein synthesis, but to a specific decrease in inducible NO synthase protein content in the cells, as attested by the significant reduction of the expression of inducible NO synthase, assayed by sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. However, no effect of tetracyclines on inducible NO synthase mRNA accumulation could be demonstrated in lipopolysaccharide-stimulated macrophage line, suggesting that the inhibitory effect of tetracyclines on NO synthesis involves post-transcriptional events. The reduction in lipopolysaccharide-stimulated nitrite accumulation produced by tetracyclines was significantly less when they were applied 6 h after lipopolysaccharide and absent 12 h after lipopolysaccharide, indicating that tetracyclines modify an early event in inducible NO synthase activation operating after mRNA transcription. The findings presented in this study indicate that the modulation of NO synthesis is another possible pathway by which tetracyclines may function as anti-inflammatory compounds.

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.

Doxycycline Reduces Mortality to Lethal Endotoxemia by Reducing Nitric Oxide Synthesis via an Interleukin-10-Independent Mechanism

It was demonstrated that doxycycline protected BALB/c mice injected intraperitoneally with bacterial lipopolysaccharide (LPS) against lethal septic shock. Doxycycline (at 1.5 mg/kg) exerted its protective effect by inhibiting nitrate production by an interleukin-10-independent mechanism. Experiments carried out in vitro also indicated that doxycycline inhibited NO synthesis by LPS-activated macrophages without inducing any significant modification in interleukin-10 release. These data suggest that the direct inhibition of nitrate release is the main mechanism of the antiinflammatory activity of doxycycline in septic shock.

Chemically modified tetracyclines induce cytotoxic effects against J774 tumour cell line by activating the apoptotic pathway

Here, we have studied the effects of chemically modified tetracyclines (CMTs) on apoptosis both at the level of the cytoplasmic proteolytic caspase cascade, and on Bcl-2 and c-myc mRNA expression in the J774 macrophage cell line. The results indicate that CMTs induce morphological changes consistent with apoptotic events, as clearly demonstrated both by the acridine orange and ethidium bromide staining, and by TUNEL and fragmentation ELISA assays. Furthermore, the analysis of the cell cycle by flow cytometry shows an evident apoptotic sub-G0G1 peak, without important modifications in the cell cycle distribution. CMTs induce programmed cell death (PCD) in a dose-dependent manner and CMT-8 is the strongest among them. CMT-1 and CMT-8 activate mainly caspase-8 as attested by the inhibitory effects of Z-VAD-fmk and Z-IEDT-fmk on CMT-induced apoptosis. Part of CMT-induced PCD is due to the activation of caspase-9, since it is reduced by the specific caspase-9 inhibitor, Z-LEHD-fmk. Besides, CMTs increase Bcl-2 and c-myc mRNA expression. Collectively, these data indicate that CMTs are potentially anti-tumour agents, since they strongly trigger apoptosis both activating the proteolytic system of the caspase family and modulating genes involved in PCD regulation.

IL-15 in human visceral leishmaniasis caused by Leishmania infantum

Interleukin (IL)‐15 is a recently discovered cytokine with the ability to stimulate the proliferation activity of Th1 and/or Th2 lymphocytes. Here, we investigated the involvement of IL‐15 in the immune response to Leishmania infantum infection by studying patients with visceral leishmaniasis (VL). We found that IL‐15 is produced by leishmanial antigen (LAg)‐stimulated peripheral blood mononuclear cells (PBMC) from active VL patients at a significantly higher level than those produced by cells from healed VL subjects or healthy controls. A significant increase in IL‐15 serum blood levels was also observed in acute VL patients compared with healed ones. Furthermore, recombinant IL‐15 had an appreciable effect in vitro in reducing IL‐4 and increasing the production of IL‐12 in response to LAg, but it was ineffective in altering the production of interferon‐γ (IFN‐γ). The production of endogenous IL‐15 in acute VL patients appeared to be insufficient to activate both IFN‐γ and IL‐12, as attested by the absence of modification of these two cytokines by neutralization experiments in the presence of anti‐IL‐15 monoclonal antibodies (MoAB). On the contrary, the neutralization of IL‐15 increased IL‐4 production. Together, these results indicate that endogenous IL‐15 plays a role in the suppression of Th2‐type cytokines, even though it does not enhance the production of Th1 cytokines in acute VL patients. Since IL‐15, in the presence of anti‐IL‐4 MoAb, caused a further increase in IL‐12 production and led to a significant production of IFN‐γ, one of its indirect effects on Th1 cell activation could be due to the latter’s effect on Th2 cytokines such as IL‐4. Therefore, our observations indicate that there is a potential for IL‐15 to augment the T‐cell response to human intracellular pathogens.

Modulation of Nitric Oxide Production by Tetracyclines and Chemically Modified Tetracyclines

Chemically modified tetracyclines (CMTs) dose-dependently decreased inducible nitric oxide synthase (iNOS) and, consequently, nitric oxide (NO) formation by the lipopolysaccharide (LPS)-stimulated J774 line. The inhibitory effect was due to a specific reduction in the iNOS protein content in the cells, as attested by Western blot analysis and by the inhibition of iNOS mRNA accumulation. Furthermore, CMTs cause a dose-dependent increase in cell death in the J774 line mediated by the NO-independent apoptotic mechanism.