Giordano Bianchi

Ex vivo enrichment of mesenchymal cell progenitors by fibroblast growth factor 2.

Bone marrow stromal cells, obtained from postnatal bone marrow, contain progenitors able to differentiate into several mesenchymal lineages. Their use in gene and cell therapy requires their in vitro expansion and calls for the investigation of the culture conditions required to preserve these cells as a stem compartment with high differentiative potential during their life span. Here we report that fibroblast growth factor 2 (FGF-2)-supplemented bone marrow stromal cell primary cultures display an early increase in telomere size followed by a gradual decrease, whereas in control cultures telomere length steadily decreases with increasing population doublings. Together with clonogenic culture conditions, FGF-2 supplementation prolongs the life span of bone marrow stromal cells to more than 70 doublings and maintains their differentiation potential until 50 doublings. These results suggest that FGF-2 in vitro selects for the survival of a particular subset of cells enriched in pluripotent mesenchymal precursors and is useful in obtaining a large number of cells with preserved differentiation potential for mesenchymal tissue repair.

HUMAN MESENCHYMAL STEM CELLS INHIBIT NEUTROPHIL APOPTOSIS: A MODEL FOR NEUTROPHIL PRESERVATION IN THE BONE MARROW NICHE

Mesenchymal stem cells (MSC) establish close interactions with bone marrow sinusoids in a putative perivascular niche. These vessels contain a large storage pool of mature nonproliferating neutrophils. Here, we have investigated the effects of human bone marrow MSC on neutrophil survival and effector functions. MSC from healthy donors, at very low MSC:neutrophil ratios (up to 1:500), significantly inhibited apoptosis of resting and interleukin (IL)‐8‐activated neutrophils and dampened N‐formyl‐l‐methionin‐l‐leucyl‐l‐phenylalanine (f‐MLP)‐induced respiratory burst. The antiapoptotic activity of MSC did not require cell‐to‐cell contact, as shown by transwell experiments. Antibody neutralization experiments demonstrated that the key MSC‐derived soluble factor responsible for neutrophil protection from apoptosis was IL‐6, which signaled by activating STAT‐3 transcription factor. Furthermore, IL‐6 expression was detected in MSC by real‐time reverse transcription‐polymerase chain reaction and enzyme‐linked immunosorbent assay. Finally, recombinant IL‐6 was found to protect neutrophils from apoptosis in a dose‐dependent manner. MSC had no effect on neutrophil phagocytosis, expression of adhesion molecules, and chemotaxis in response to IL‐8, f‐MLP, or C5a. These results support the following conclusions: (a) in the bone marrow niche, MSC likely protect neutrophils of the storage pool from apoptosis, preserving their effector functions and preventing the excessive or inappropriate activation of the oxidative metabolism, and (b) a novel mechanism whereby the inflammatory potential of activated neutrophils is harnessed by inhibition of apoptosis and reactive oxygen species production without impairing phagocytosis and chemotaxis has been identified.

Cell Therapy for Bone Disease: A Review of Current Status

Bone marrow is a reservoir of pluripotent stem/progenitor cells for mesenchymal tissues. Upon in vitro expansion, in vivo bone‐forming efficiency of bone marrow stromal cells (BMSCs) is dramatically lower in comparison with fresh bone marrow, and their in vitro multidifferentiation potentials are gradually lost. Nevertheless, when BMSCs are isolated and expanded in the presence of fibroblast growth factor 2, the percentage of cells able to differentiate into the osteogenic, chondrogenic, and adipogenic lineages is greater. Osteogenic progenitors are not exclusive to skeletal tissues. We could also think of cells in different adult tissues as potentially capable of following an osteochondrogenic differentiation pathway, but, under normal physiological conditions, they are inhibited in this process by the environment and/or the adjacent cell populations. When, for some reason such as pathology, the environment changes dramatically and the inhibiting condition is removed, these cells could become osteoblasts. Bone is repaired via local delivery of cells within a scaffold. Bone formation was first assessed in small animal models. Large animal models were successively developed to prove the feasibility of the tissue engineering approach in a model closer to a real clinical situation. Eventually, pilot clinical studies were performed. Extremely appealing is the possibility of using mesenchymal progenitors in the therapy of genetic bone diseases via systemic infusion. There is experimental evidence to suggest that mesenchymal progenitors delivered by this route engraft with a very low efficiency and do not produce relevant and durable clinical effects. Under some conditions, where the local microenvironment is either altered (i.e., injury) or under important remodeling processes (i.e., fetal growth), engraftment of stem and progenitor cells seems to be enhanced. A better understanding of their engraftment mechanisms will, hopefully, extend the field of therapeutic applications of mesenchymal progenitors.

Tumor necrosis factor-alpha (TNF-α) induces integrin CD11b/CD18 (Mac-1) up-regulation and migration to the CC chemokine CCL3 (MIP-1α) on human neutrophils through defined signalling pathways

Strong evidence suggests that neutrophils may play an active role in acute and chronic inflammatory disorders, such as rheumatoid arthritis and atherosclerosis. Given the role of pro-inflammatory cytokine TNF-alpha in these inflammatory processes, we planned the present study to investigate the effect of short term incubation with TNF-alpha on neutrophil migration to CCL3, a chemokine produced in inflammatory sites and normally devoid of neutrophil chemotactic properties. We found that TNF-alpha primed neutrophils for migration to CCL3 via CCR5. TNF-alpha-induced migration was a consequence of the TNF-alpha-induced up-regulation of integrin CD11b/CD18 (Mac-1) on neutrophil surface. Furthermore, TNF-alpha activity was found to be strictly dependent on the activation of ERK 1/2 p44, cooperating with the intracellular pathways involving Src kinases, PI3K/Akt, p38 MAPK, well known as activated in response to classical chemoattractants (CXCL8) or priming agents (GM-CSF). On the contrary, the effect of TNF-alpha on neutrophil migration to CCL3 was not dependent on JNK 1/2. In conclusion, the present report shows that TNF-alpha unveils a previously unknown capacity of neutrophils to migrate to CCL3 through the intervention of Mac-1. TNF-alpha regulates Mac-1 up-regulation through signalling pathways, involving various kinases, but not JNK 1/2. Although highly speculative, ERK 1/2 p44 may represent a selective target for the pharmacologic manipulation of neutrophil-mediated adverse activities in TNF-alpha-mediated inflammatory states.

Induction of neutrophil chemotaxis by leptin: crucial role for p38 and Src kinases.

Abstract:  Leptin is involved in energy homeostasis, hematopoiesis, inflammation, and immunity. Although hypoleptinemia characterizing malnutrition has been strictly related to increased susceptibility to infection, other hyperleptinemic conditions, such as end‐stage renal disease (ESRD), are highly susceptible to bacterial infections. On the other hand, ESRD is characterized by neutrophil functional defects crucial for infectious morbidity, and several uremic toxins capable of depressing neutrophil functions have been identified. In the present study, we investigated leptins effects on neutrophil function. Our results show that leptin inhibits neutrophil migration in response to classical chemoattractants. Otherwise, leptin is endowed with chemotactic activity toward neutrophils. The two activities, inhibition of the cell response to chemokines and stimulation of neutrophil migration, could be detected at similar concentrations. On the contrary, neutrophils exposed to leptin did not display detectable [Ca2+]i mobilization, oxidant production, orβ2‐integrin upregulation. The results demonstrate that leptin is a pure chemoattractant devoid of secretagogue properties but capable of inhibiting neutrophil chemotaxis to classical neutrophilic chemoattractants. This effect is dependent on the activation of intracellular kinases involved in F‐actin polymerization and neutrophil locomotion. Indeed, p38 mitogen‐activated protein kinase (MAPK) and Src kinase, but not extracellular‐regulated kinase (ERK), were activated by short‐term incubation with leptin. Moreover, p38 MAPK inhibitor SB203580 and Src kinase inhibitor PP1, but not MEK inhibitor PD98059, blocked neutrophil chemotaxis toward leptin. Serum from patients with ESRD inhibits migration of normal neutrophils in response to N‐formyl‐methionine‐leucyl‐phenylalanine (FMLP) with a strict correlation between serum leptin levels and serum ability to suppress neutrophil locomotion. The serum inhibitory activity can be effectively prevented by immune‐depletion of leptin. Taking into account the crucial role of neutrophils in host defense, we show that leptin‐mediated ability of ERSD serum to inhibit neutrophil chemotaxis appears to be a mechanism contributing to neutrophil dysfunction in ESRD.

Nonleukoreduced red blood cell transfusion induces a sustained inhibition of neutrophil chemotaxis by stimulating in vivo production of transforming growth factor-β1 by neutrophils : role of the immunoglobulinlike transcript 1, sFasL, and sHLA-I

BACKGROUND: Red blood cell (RBC) transfusion has been linked to increased susceptibility to infections in critically ill patients and to augmented incidence of postoperative infections. The mechanisms by which transfusions can induce immunosuppression are only partially defined. Recently, it has been demonstrated that RBC supernatants inhibit neutrophil migration. Such inhibitory activity is due to transforming growth factor (TGF)‐β1 contained in the supernatants that desensitize neutrophils to subsequent chemotaxic stimulation.

Delayed apoptosis of human monocytes exposed to immune complexes is reversed by oxaprozin: role of the Akt/IκB kinase/nuclear factor κB pathway

Background and purpose:  Monocytes‐macrophages play a key role in the initiation and persistence of inflammatory reactions. Consequently, these cells represent an attractive therapeutic target for switching off overwhelming inflammatory responses. Non‐steroidal anti‐inflammatory drugs (NSAIDs) are among the most common drugs for the symptomatic treatment of rheumatic diseases. Their effects have been explained on the basis of cyclooxygenase (COX) inhibition. However, some of the actions of these drugs are not related to inhibition of prostaglandin synthesis.

Insulin primes human neutrophils for CCL3-induced migration: crucial role for JNK 1/2.

Abstract:  The present article shows that a short‐term exposure of purified human neutrophils to recombinant insulin conferred on these cells both the ability to migrate and the capacity to mobilize [Ca2+]i in response to CCL3, a chemokine per se ineffective with native neutrophils. Furthermore, the effects of recombinant insulin were reproduced by short‐term incubation with sera from adult patients with metabolic syndrome, known to be characterized by a hyperinsulinemic state. A strict linear correlation (P < 0.01) between sera insulin levels and seras ability to induce neutrophil locomotion was indeed found. Our data also suggest that (i) insulin primed neutrophils for migration to CCL3 via the selective activation of JNK 1/2, as shown by the use of inhibitors and kinase activation assay; (ii) the activation of Src kinases was necessary but not sufficient for CCL3‐induced locomotory activity; (iii) PI3K‐Akt, ERK 1/2, and p38 MAPK were not involved in insulin‐induced migratory competence. In summary, we provided evidence that the exposition of neutrophils to insulin, as it occurs in hyperinsulinemic conditions, confers the competence of the cells to migrate in response to CCL3, known to be generated near atherosclerotic plaques. As neutrophils have been recently suggested to be involved in breaking unstable atherosclerotic plaques, the present findings contribute to the understanding of the pathophysiology of plaque instability. Finally, biochemical analysis herein carried out raises the hypothesis of JNK 1/2 as an attractive therapeutic target.

Immune Complexes Induce Monocyte Survival through Defined Intracellular Pathways

Abstract:  Monocytes recruitment and survival at sites of inflammation are determinant for the persistence of inflammatory reactions. Immune‐complexes (ICs), whose tissue deposition is involved in a variety of autoimmune diseases, activate monocytes through the interaction with Fcγ‐receptor triggering the secretion of several inflammatory modulators and favoring their tissue accumulation by inhibiting the apoptosis. To elucidate the intracellular pathways governing this process, on the basis of our previous findings regarding the dose‐dependent inhibition of apoptosis in IC‐activated monocytes, we have investigated the role of PI3K/Akt pathway, MAP kinases, nuclear factor‐κB (NF‐κB), and caspase 3, 8, and 9. Here we show that IC‐activated monocytes underwent apoptosis at a rate comparable to that of resting monocytes in the presence of LY294002, a selective inhibitor of PI3K, as well in the presence of Akt inhibitor, PD98059 inhibitor of ERK1/2, and SB203580 inhibitor of p38. Moreover, IC‐triggered phosphorylation of Akt, ERK1/2, and p38 MAP kinase was demonstrated on Western blot analysis. SN50, an inhibitor of NF‐κB translocation and BMS345541, a specific inhibitor of IKK, also abolished the apoptosis protection conferred by ICs. In parallel, ICs induced an increase in NF‐κB activation, as shown by EMSA, together with the expression of XIAP, as shown by Western blot, though indicating that in monocytes IC protection from apoptosis is NF‐κB dependent. Finally, the activity of caspase 3, 8, and 9 resulted inhibited in IC‐activated monocytes. These results disclose a signaling route triggered by ICs which can be involved in the pathophysiology of inflammatory diseases and can represent a target for therapy of IC‐mediated diseases.

Bone marrow stromal cells and their use in regenerating bone tissue

We have used autologous BMSC/bioceramic composites to treat full-thickness gaps of long bones, first in a large animal model (tibia diaphysis in adult sheep) and than in few selected patients. A complete integration of ceramic with bone was observed by X-ray, histology, microradiography and SEM studies. In addition a good functional recovery was obtained. The healing process involved four main steps: 1) bone formation on the outer surface of the implant; 2) bone formation in the inner cylinder canal; 3) formation of fissures and cracks in the implant body (integration and disintegration mechanisms); 4) bone formation in the bioceramics pores. The results obtained are very promising and could represent a real and significant improvement in the repair of critical size long bone defects.