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Biomedical subjects

M Galdiero

Publications and source records attributed to M Galdiero.

At least 19 recordsLinked to original sources

New perspectives in the medical treatment of acromegaly.

Currently available medical treatment of acromegaly includes dopamine agonists, slow release formulation of somatostatin analogues and pegvisomant, a GH-receptor antagonist. Dopamine agonists are well tolerated, not expensive but poorly effective. Somatostatin analogues are highly effective in 60-70% of patients based on the receptor profile of individual tumors. Pegvisomant is reported to normalize IGF-I levels in nearly the totality of the patients, but is devoted of tumor shrinking effect. In a preliminary study in patients with acromegaly, a new somatostatin analogue with affinity to four of the five somatostatin receptors (SOM230) was shown to be similarly effective as octreotide in some patients and more effective than octreotide in other patients. Moreover, new molecules with selective activity on the somatostatin receptor type 2, or 5, or 1 have been reported in vitro to strongly suppress GH secretion. Other new promising alternatives are the chimeric compounds with both somatostatin receptor and dopamine receptor binding. These drugs have been also shown to possess strong GH-inhibitory activity in primary cultures from GH-secreting adenomas. These drugs are the future perspectives in the treatment of patients with GH-secreting or GH/PRL-secreting tumors.

Acromegaly↗

Interleukin-8 production by THP-1 cells stimulated by Salmonella enterica serovar Typhimurium porins is mediated by AP-1, NF-kappaB and MAPK pathways.

Interleukin-8 (IL-8) is released in response to inflammatory stimuli, such as bacterial products. Either porins or lipopolysaccharide (LPS) stimulated THP-1 cells to release IL-8 after 24 h. We have previously reported that stimulation of monocytic cells with Salmonella enterica serovar Typhimurium porins led to the activation of mitogen-activated protein kinase cascades and of protein tyrosine kinases (PTKs). In this report, we demonstrate, using two potent and selective inhibitors of MEK activation by Raf-1 (PD-098059) and p38 (SB-203580), that both ERK1/2 and p38 pathways play a key role in the production of IL-8 by porins and LPS. Porin-stimulated expression of activating protein 1 (AP-1) and correlated IL-8 release is also inhibited by PD-098059 or SB-203580 indicating that the Raf-1/MEK1-MEK2/MAPK cascade is required for their activation. Also PTKs modulate the pathway that control IL-8 gene expression, in fact its expression is abolished by tyrphostin. By using N-acetyl-leucinyl-leucinyl-norleucinal-H (ALLN) an inhibitor of nuclear factor-kappaB (NF-kappaB) activity, we also observed IL-8 release modulation. Our results elucidate some of the molecular mechanisms by which AP-1 and NF-kappaB regulate IL-8 release and open new strategies for the design of specific molecules that will modulate IL-8 effects in various infectious diseases.

Cell Line↗

Lipoteichoic acid and muramic acid modulate the expression of CD80/CD86 on THP-1 cells and CD28/CD152 on Jurkat cells.

The aim of this study is to evaluate the effect of lipoteichoic acid (LTA) and muramic acid (MA) on costimulatory molecules CD80/CD86 on THP-1 cells and CD28/CD152 on Jurkat cells. The interactions between these molecules strongly influence the immune response through the regulation of cytokine release which, on its own, is able to regulate the immunological response by a feedback mechanism. Our results show that LTA and MA regulate expression of CD86 on macrophages while the expression of CD80 remains unmodified. LTA and MA increase the expression of CD86 on THP-1 cells, a macrophage cell line. MA increased Jurkat T cells CD152 expression.

Antigens, CD↗

Porins and lipopolysaccharide from Salmonella typhimurium regulate the expression of CD80 and CD86 molecules on B cells and macrophages but not CD28 and CD152 on T cells.

OBJECTIVE: The aim of this study was to evaluate the effect of porins from Salmonella typhimurium on costimulatory molecules such as CD80/CD86 and CD28/CD152. The interactions between these molecules are able to influence the immune response through the regulation of cytokines release which, on their own, are able to regulate the immunological response by a feedback mechanism. METHODS: S. typhimurium strain SH5014 (a rough lipopolysaccharide (LPS) producing strain) was used as the source of porins and LPS. Peripheral blood mononuclear cells were obtained from healthy adult donors. THP1 cells were obtained from ATCC (Rockville, MD, USA). Immunofluorescence and flow cytometry were performed using a FACS IV (Becton-Dickinson, Mountain View, CA, USA). RESULTS: Our results show that porins of S. typhimurium increase the expression of CD86 and the expression of CD80 both on B lymphocytes and macrophages, while the expression of CD28 and CD152 on T lymphocytes was unaltered. The expression of CD80 and CD86 is dose-dependent and starts after 24 h post treatment, peaks at 48 h and goes back to the basal value after 72 h. CONCLUSIONS: S. typhimurium porins are able to induce a high expression of costimulatory molecules (CD80 and CD86) on lymphocytes and macrophages.

Antigens, CD↗

Monocytic activation of protein tyrosine kinase, protein kinase A and protein kinase C induced by porins isolated from Salmonella enterica serovar Typhimurium.

OBJECTIVES: In the present study a monocytic cell line, U937, was used to investigate the possible involvement of protein tyrosine kinases (NT-PTKs), protein kinase A (PKA) and protein kinase C (PKC) in cell signaling pathways following Salmonella enterica serovar Typhimurium porin stimulation. METHODS: Different concentrations of porins and lipopolysaccharide (LPS) were analysed to evaluate changes in PTK activity by a non radioactive tyrosine kinase assay and in PKA and PKC phosphorylation by Western blotting analysis. The inhibitors of PTK, PKA and PKC activation used, were: 3,4-dihydroxybenzylidene-malononitrile (tyrphostin 23), inhibitor of epidermal growth factor (EGF) receptor tyrosine kinase activity; dihychloride (H-89), a selective inhibitor of PKA which is useful to discriminate between the effects of PKC and PKA; diacylglycerol kinase inhibitor II (R59949), which is useful for elucidating roles of PKC; calphostin C, a specific inhibitor of PKC. RESULTS: Porins of the outer membrane of the ST were isolated to be used as a stimulus in the performed experiments. Following porin treatment, a dose-dependent increase in PTK, PKA and PKC activation was observed. U937 monocytes pretreated with inhibitors induced an evident decrease in PTK activity and PKA and PKC phosphorylation pattern in porin stimulated monocytes. CONCLUSIONS: Our data support the important role played by NT-PTK, PKA and PKC in transducing the activating signal in macrophages stimulated with porins through the activation of the mitogen-activated protein kinase (MAPK) pathway that participate in the regulation of gene expression.

Blotting, Western↗

Apoptosis modulation by mycolic acid, tuberculostearic acid and trehalose 6,6'-dimycolate.

The object of our study is to demonstrate that some components of M. tuberculosis, such as cord factor or mycolic acid or whole bacteria can prolong cell survival compared to controls. The cells treated with cord factor or mycolic acid at a concentration of 5 microg/ml were 65+/-8% viable reaching 70+/-8% at a concentration of 10 microg/ml. The cells treated with heat killed mycobacteria were 70+/-8% viable; while control cells exhibited a viability 50+/-7%. Conversely, tuberculostearic acid induced early cell death. The results also demonstrated a dose-dependent effect on the viability or induction of macrophage apoptosis. We also showed that prolonged viability of the treated cells with mycolic acid or cord factor (+20+/-4% and +25+/-5%, respectively) was correlated with a significant increase in Bcl-2 expression. The treated cells with whole bacteria presented a Bcl-2 expression of 40+/-6%, while Fas expression was not changed compared to controls. This study confirm that at the site of mycobacterial infection, necrosis, apoptosis or prolonged survival of the cells depend on the quantity and quality of the molecules expressed by the mycobacteria; whether necrosis or apoptosis or prolonged survival is more or less favorable to the host likely depends on several factors regarding the inflammatory and immune response, both markedly stimulated by mycobacteria.

Animals↗

p53 and c-myc activation by Pasteurella haemolytica leukotoxin is correlated with bovine mononuclear cells apoptosis.

To analyse the role of Pasteurella haemolytica Leukotoxin (LKT) in the mechanism of apoptotic cell death of bovine lymphocytes, we evaluated DNA fragmentation and p53 and c-myc expression. P. haemolytica strain ATCC 14003 was cultivated for LKT production. DNA fragmentation was analysed by electrophoresis on Agarose gel. DNA strand breaks in individual apoptotic cells were also detected by an in situ Terminal deoxy nucleotidyl Transferase (TdT). The Polymerase Chain Reaction (PCR) procedure was used for verified p53 and c-myc activation by P. haemolytica LKT. LKT was able to induce DNA fragmentation in a dose and time-dependent fashion. The greatest apoptotic effect was obtained using LKT at a concentration of 0.25 U. The results show that p53 and c-myc activation by LKT is correlated with apoptosis of bovine lymphocytes and monocytes. Our data suggest that LKT may have an important role in the bacterial virulence of Pasteurella haemolytica.

Animals↗

Coinfection with BHV-1 modulates cell adhesion and invasion by P. multocida and Mannheimia (Pasteurella) haemolytica.

Viruses are thought to facilitate bacterial infections of the respiratory tract. The present study shows the effect of BHV-1 on Pasteurella multocida and Mannheimia haemolytica adherence and invasion of MDBK cells. The virus-infected MDBK cells become more susceptible to the adherence of both species of Pasteurella. The observed adherence increase depends on the length of virus pre-incubation time and on virus concentration. When MDBK cells are not infected with virus, they are only invaded by P. multocida, while M. haemolytica is not able to penetrate. The viral infection favours also the invasion by M. haemolytica.

Animals↗

Induction of tyrosine phosphorylated proteins in THP-1 cells by Salmonella typhimurium, Pasteurella haemolytica and Haemophilus influenzae porins.

The effect of porins purified from Salmonella typhimurium, Pasteurella haemolytica and Haemophilus influenzae on induction of tyrosine phosphorylation in THP-1 cells and C3H/HeJ macrophage was investigated. Incubation of porins at concentration of 1.0-5.0 microg ml(-1) with either THP-1 or macrophage from C3H/HeJ mice resulted in tyrosine phosphorylation of specific host cell proteins. After porin stimulation a pattern of tyrosine phosphorylated proteins appeared in the soluble cytoplasmic fraction, in the membrane fraction and in the insoluble protein fraction. The observed effects were dependent on the porin concentrations; they reached a maximal expression at 3 min and declined at 60 min. Porin and lipopolysaccharide treatments induce a similar phosphorylation pattern in all of the three cellular fractions studied. A difference can be observed in the cytoplasmic fraction bands of 50-60 kDa, which are more evident after treatment with lipopolysaccharide, and in the insoluble fraction band of 80 kDa and the cytoplasmic fraction band of 250 kDa, which are more evident after treatment with porins. The events of tyrosine protein phosphorylation were present in macrophage from lipopolysaccaride-hyporesponsive C3H/HeJ mice stimulated with porins, while they were markedly reduced when the cells were stimulated with lipopolysaccharide. Staurosporine, genistein and cytochalasin D induced in the three cellular fractions a different inhibition pattern of tyrosine protein phosphorylation in porin stimulated cells. Porins extracted from the three bacterial species investigated behave in a similar way as stimuli more or less potent; Hib porin seems to be the most powerful stimulator and, moreover, it specifically induces phosphorylation of a 55 kDa band.

Animals↗

Porins and lipopolysaccharide induce apoptosis in human spermatozoa.

Treatment of human spermatozoa with porins or lipopolysaccharide (LPS) increases spontaneous apoptosis in these cells. Porins and LPS were extracted from Salmonella enterica serovar Typhimurium and Pasteurella multocida and were mixed with human spermatozoa for detection of levels of apoptosis.

Apoptosis↗

Haemophilus influenzae porin contributes to signaling of the inflammatory cascade in rat brain.

In the present study we observed that the Haemophilus influenzae type b (Hib) porin, among the different surface bacterial components, is involved in the pathophysiology of bacterial meningitis. This study demonstrates that inoculation of Hib porin into the fourth cerebral ventricle causes the simultaneous expression of interleukin-1alpha (IL-1alpha), tumor necrosis factor alpha (TNF-alpha), and macrophage inflammatory protein 2 (MIP-2) at 6 h after inoculation. At 24 h, the expression of MIP-2 decreases while the expression of IL-1alpha and TNF-alpha increases. The mRNA expression of IL-1alpha, TNF-alpha, and MIP-2 is correlated with injury to the blood-brain barrier as demonstrated by the appearance of serum proteins and leukocytes in cerebrospinal fluid and by the increase in brain water content.

Animals↗

Neutrophil adhesion and transmigration through bovine endothelial cells in vitro by protein H and LPS of Pasteurella multocida.

This study describes an in vitro investigation on the role of Pasteurella multocida cells and its isolated protein H and LPS on neutrophil adhesion and migration through bovine endothelial cell monolayers. P. multicoda cells, protein H and LPS increased the adhesion and transmigration of neutrophils through BAEC. The bacteria/cell ratio of 100 for P. multocida, protein H concentration 0.05-0.2 microM and LPS concentration 0.5-1.0 microM respectively, induced the maximum adhesion and transmigration of neutrophils through BAEC. The optimal time of incubation with bacteria or bacterial products was 4-6 h. Our results confirm the role of Gram-negative bacteria and of components of the outer membrane such as protein H or LPS in activating the neutrophils and in promoting the adhesion and cells transmigration from the vessels to the site of inflammation.

Animals↗

Role of Pasteurella multocida, Pasteurella haemolytica and Salmonella typhimurium porins on inducible nitric oxide release by murine macrophages.

The aim of this study was to verify whether Pasteurella haemolytica, P. multocida and Salmonella typhimurium porins could affect the inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) release by murine resident peritoneal macrophages in vitro. We also compared their effect with that elicited by P. haemolytica, P. multocida and S. typhimurium lipopolysaccharide (LPS) whose biological activity is well known. Variations in NO release and iNOS mRNA expression due to variable concentrations of porins were recorded and compared. We also investigated the synergism between bacterial products and interferon gamma (IFN-gamma). With this aim cells were incubated with porins together with murine rIFN-gamma prior to assessing the presence of NO in the supernatant and mRNA analysis. Porins in themselves were not able to induce NO release by resident peritoneal macrophages. Incubation of macrophages with IFN-gamma in the presence of porins increased NO release, whereas incubation in the presence of the arginine analog N(G)-monomethyl-L-arginine (NMA) inhibited NO release. The greatest NO release was obtained using porins at a concentration of 5 microg/mL. Porins, together with IFN-gamma, were also able to upregulate the mRNA expression of iNOS. Our findings suggest that gram-negative porins are able to modulate inflammatory and immunological responses by affecting the release of NO and the expression of iNOS gene in activated macrophages.

Animals↗

Modulation of anthracycline activity in canine mammary tumour cells in vitro by medroxyprogesterone acetate.

Failure of chemotherapy with anthracyclines as a result of drug resistance and toxicity is a major problem in the clinical management of neoplasia. The aim of the present study was to evaluate the activity of medroxyprogesterone acetate (MPA) as a chemosensitiser on anthracycline cytotoxicity. The study investigated whether such an effect could be related to an increase in lipid peroxidation, nitric oxide production, membrane fluidity and intracellular anthracycline concentration. The results showed that anthracyclines decreased nitric oxide production but increased membrane viscosity (polarisation constant) and lipid hydroperoxide formation in canine mammary tumour cells. Moreover, it was found that both drug-induced cytotoxicity and membrane viscosity increased in the presence of MPA. Conversely, lipid hydroperoxides decreased in MPA-supplemented cells. Medroxyprogesterone acetate did not show any effect on nitric oxide production. The two anthracyclines used (doxorubicin and idarubicin) showed differential intranuclear accumulation in canine mammary tumour cells, and MPA significantly modified intracellular concentration of anthracyclines.

Animals↗

Porins and lipopolysaccharide (LPS) from Salmonella typhimurium induce leucocyte transmigration through human endothelial cells in vitro.

Bacteria or bacterial products may constitute important inducers of surface molecule expression on endothelial cells and leucocytes. This study was undertaken to determine the effects of the Salmonella typhimurium porins, LPS-S and LPS-R on the transendothelial migration of leucocytes through human umbilical vein endothelial cells (HUVEC). Treatment of the HUVEC with either porins or LPS-S or LPS-R increased the transmigration of different leucocyte populations, in particular that of neutrophils. The maximal increase occurred using LPS-S treatment, whereas porin stimulation fell between LPS-S and LPS-R. The transmigration increase was dose-dependent and reached its maximum at about 100-1000 ng/ml of stimulus. Optimal endothelial activation occurred after 2-4 h and 4-6 h using LPS and porin, respectively. Stimulation of leucocytes with either porins or LPS slightly increased their transmigration through non-activated endothelial cells. Transmigration increased remarkably during the simultaneous stimulation of endothelial cells by IL-1ss together with either porins or LPS. To assess participation of E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and leucocyte adhesion complex (CD11/18) in porin- or LPS-mediated leucocyte migration, blocking MoAbs were used. Each blocking MoAb partially and selectively decreased leucocyte transmigration. The obtained results contribute to clarify some aspects of the inflammatory process at sites of infection.

Antibodies, Monoclonal↗

Effect of transforming growth factor beta on experimental Salmonella typhimurium infection in mice.

We have investigated the effect of the in vivo administration of recombinant transforming growth factor beta (rTGF-beta) on the pathogenic mechanisms involved in Salmonella typhimurium experimental infection in mice. The protective response elicited by macrophages was induced by rTGF-beta1 by 2 days after experimental infection, as demonstrated by an increased NO production, while the humoral protective effect began with cytokine mRNA expression 2 days after the challenge and continued after 5 days with cytokine release and lymphocyte activation. We demonstrated that all mice who received rTGF-beta1 survived 7 days after infection. The number of bacteria recovered in the spleens and in the livers of rTGF-beta1-treated mice 2 and 5 days after infection was significantly smaller than that found in the same organs after phosphate-buffered saline (PBS) inoculation. Furthermore, 2 and 5 days after infection, splenic macrophages from rTGF-beta1-treated mice showed a greater NO production than did those from PBS-treated mice. The effect of rTGF-beta1 on S. typhimurium infection in mice was correlated with the expression of cell costimulatory CD28 molecules. Five days after S. typhimurium infection, the percentage of CD28(+)-expressing T cells in splenic lymphocytes from rTGF-beta1-treated mice increased with respect to that from control mice. Gamma interferon (IFN-gamma) mRNA was present in a greater amount in spleen cells from rTGF-beta1-treated mice after 2 days, although the intensity of the band decreased 5 days after the challenge. A similar pattern was obtained with the mRNAs for interleukin-1alpha (IL-1alpha), IL-6, TGF-beta, and inducible nitric oxide synthase, which showed greater expression in cells obtained from rTGF-beta1-treated and S. typhimurium-infected mice 2 days after challenge. The treatment with rTGF-beta1 induced an increase in IL-1alpha and IFN-gamma release in the supernatant of splenocyte cultures 5 days after the experimental infection with S. typhimurium. Moreover, we demonstrated that 5 days after infection, the IFN-gamma titer was significantly greater in the sera of rTGF-beta-treated mice than in those of PBS-treated mice. Also, hsp60 showed greater expression 2 days after the challenge in splenocytes from rTGF-beta1-treated mice. The role played by proinflammatory and immunoregulatory cytokines and by CD28 is discussed.

Animals↗

Role of Pasteurella multocida porin on cytokine expression and release by murine splenocytes.

The aim of this study was to verify whether Pasteurella multocida porin can affect the expression and release of IL-1alpha, IL-6, TNF-alpha, IL-4, IFN-gamma, IL-10 and IL-12 by murine splenocytes in vitro. P. multocida porin and lipopolysaccharide (LPS) were able to induce the release of IL-1alpha, IL-6, TNF-alpha, IFN-gamma and IL-12 in a dose-dependent fashion. The greatest release of these cytokines was obtained using P. multocida porin at a concentration of 5 microg ml(-1) and LPS at a concentration of 1 microg ml(-1). The time-courses of release showed that P. multocida LPS was able to stimulate the production of IL-1alpha, IL-6, TNF-alpha, IFN-gamma and IL-12 earlier than porin and at a greater rate. No effect was observed on IL-4 and IL-10 release under the same experimental conditions. P. multocida porin and LPS were also able to up-regulate the mRNA expression of IL-1alpha, IL-6, TNF-alpha, IFN-gamma and IL-12 p40. Our findings suggest that P. multocida porin is able to modulate inflammatory and immunological responses by affecting the release of several cytokines and the expression of their genes.

Animals↗

Salmonella typhimurium porin internalization by leukocytes.

The location of Salmonella typhimurium porins has been found in human monocytes and lymphocytes by means of high resolution autoradiography. The results indicate that traces of porins are frequently visible on ultrathin sections of treated human lymphomonocytes and that they are located especially in the nuclear areas.

Adult↗