Franco Silvestris

Antibody Production and In Vitro Behavior of CD27-Defined B-Cell Subsets: Persistent Hepatitis C Virus Infection Changes the Rules

ABSTRACT There is growing interest in the tendency of B cells to change their functional program in response to overwhelming antigen loading, perhaps by regulating specific parameters, such as efficiency of activation, proliferation rate, differentiation to antibody-secreting cells (ASC), and rate of cell death in culture. We show that individuals persistently infected with hepatitis C virus (HCV) carry high levels of circulating immunoglobulin G (IgG) and IgG-secreting cells (IgG-ASC). Thus, generalized polyclonal activation of B-cell functions may be supposed. While IgGs include virus-related and unrelated antibodies, IgG-ASC do not include HCV-specific plasma cells. Despite signs of widespread activation, B cells do not accumulate and memory B cells seem to be reduced in the blood of HCV-infected individuals. This apparent discrepancy may reflect the unconventional activation kinetics and functional responsiveness of the CD27+ B-cell subset in vitro. Following stimulation with T-cell-derived signals in the absence of B-cell receptor (BCR) engagement, CD27+ B cells do not expand but rapidly differentiate to secrete Ig and then undergo apoptosis. We propose that their enhanced sensitivity to BCR-independent noncognate T-cell help maintains a constant level of nonspecific serum antibodies and ASC and serves as a backup mechanism of feedback inhibition to prevent exaggerated B-cell responses that could be the cause of significant immunopathology.

Deregulated expression of monocyte chemoattractant protein-1 (MCP-1) in arterial hypertension: role in endothelial inflammation and atheromasia.

Objective Arterial hypertension is recurrently associated with inflammation of the endothelium as an effect of the upregulation of functional molecules, including cytokines, adhesion molecules and chemokines. However, the role of monocyte chemoattractant protein-1 (MCP-1) in maintaining the inflammatory state of endothelial cells (EC) that leads to the progressive cardiovascular damage is unclear. Design Here, we investigated the expression of MCP-1, its major cell source as well as recurrence of a defined polymorphism (−2518 MCP-1) apparently linked to endothelial damage in several diseases. Methods Serum MCP-1 was measured by enzyme-linked immunosorbent assay (ELISA) in 740 hypertensive patients, subdivided according to their individual organ damage. Expression of both MCP-1 and its receptor CCR2 was evaluated in circulating ECs and macrophages by flow cytometry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), while gene variants of MCP-1 were revealed by PCR. Results Soluble MCP-1 was significantly elevated in patients with diffuse atheromasia. Furthermore, it was overexpressed by ECs activated to attract macrophages via the MCP-1/CCR2 pathway, whereas the −2518 MCP-1 polymorphism was correlated with atherosclerosis in most patients. Conclusions. Overexpression of MCP-1 is predominant in hypertensive patients with atheromasia in the form of a defined polymorphism. Measurement of MCP-1 may thus reflect the degree of endothelial damage, while early detection of such a polymorphism may acquire a prognostic value in the development of atherosclerosis.

Negative Regulation of the Osteoblast Function in Multiple Myeloma through the Repressor Gene E4BP4 Activated by Malignant Plasma Cells

Purpose: To explore the pathogenetic mechanisms that suppress the osteoblast function in multiple myeloma because osteogenesis results in defective new bone formation and repair. Experimental Design: Microarray gene analysis revealed the overexpression of E4BP4, a transcriptional repressor gene, in normal osteoblasts cocultured with myeloma cells that were releasing the parathyroid hormone-related protein (PTHrP). Thus, the effect of E4BP4 was assessed in PTHrP-stimulated osteoblasts by measuring the RNA levels of both Runx2 and Osterix as major osteoblast transcriptional activators. Because E4BP4 is a negative regulator of the cyclooxygenase-2 (COX-2) pathway that drives the expression of both Runx2 and Osterix, these factors were investigated after prostaglandin E2 treatment to overcome the COX-2 defect as well as in E4BP4-silenced osteoblasts. Finally, E4BP4, PTHrP, Osterix, and osteocalcin levels were measured in vivo in patients with bone disease together with the E4BP4 protein in bone biopsies. Results: E4BP4 was specifically induced by PTHrP and inhibited both Runx2 and Osterix, whereas E4BP4-silenced osteoblasts expressed functional levels of both factors. The prostaglandin E2 treatment of E4BP4-up-regulated osteoblasts promptly restored Runx2 and Osterix activities, suggesting that integrity of COX-2 pathway is essential for their transcription. Down-regulation of Osterix by E4BP4 was confirmed in vivo by its inverse levels in osteoblasts from myeloma patients with increased serum PTHrP, whose bone biopsies expressed the E4BP4 protein. Conclusions: Our data support the role of E4BP4 as osteoblast transcriptional repressor in inhibiting both Runx2 and Osterix in myeloma bone disease and correlate its effect with the increased PTHrP activity.

β3 Integrin Subunit Mediates the Bone-Resorbing Function Exerted by Cultured Myeloma Plasma Cells

alpha(v)beta(3) integrin was investigated in multiple myeloma in relation to the in vitro osteoclast-like activity of malignant plasma cells. Myeloma cells from patients with skeleton involvement overexpressed alpha(v)beta(3) and produced erosion pits on bone substrates, whereas this effect was not observed by cells from patients with no evidence of bone disease. We therefore explored the alpha(v)beta(3) transcriptional pathway in the bone-resorbing cells. Silencing of beta(3) chain abrogated the ability to produce erosion pits and extracellular signal-regulated kinase 1/2 phosphorylation resulting in the defective function of cFos and nuclear factor activator T cell 1, the terminal effectors of osteoclast activation. A similar defect occurred in constitutively beta(3)-deficient cells from patients with no skeleton disease. Microarray gene analysis of beta(3)(+) myeloma cells showed that several osteoclast-related genes were up-regulated. Their functions include the activation of receptor pathways beta(3) and c-fms that regulate several osteoclast functions. These data emphasize the postulated role of myeloma cells in multiple myeloma bone disease and suggest that their osteoclast-like activity is regulated, at least in vitro, by the beta(3) subunit of the integrin.

Role of Active Drug Transporters in Refractory Multiple Myeloma

Drug resistance is a major drawback for cancer chemotherapy protocols and previous studies have demonstrated the overexpression of the P-glycoprotein (P-gp) as mechanism by which myeloma cells develop multidrug resistance (MDR). However, other molecules may apparently promote MDR in multiple myeloma (MM). They include both lung resistance-related protein (LRP) and p53 activation. The inhibition of P-gp in MM patients treated with melphalan (PAM) has been associated to increased toxicity, whereas defective apoptosis due to down-modulation of the NF-kB is a feature of MDR+ myeloma cells. On the contrary, clinical trials with proteasome inhibitors have been successfully carried out to overcome MDR despite their toxicity profile. Recently, sigma receptors (sigmaR)(S), namely sigmaR(1) and sigmaR(2), have been found to be overexpressed in breast cancer cells. In addition, their levels correlate with both P-gp upregulation and MDR development. By contrast, selective inhibitors of sigmaR(S) as PB28, disrupt the P-gp signals and restore the apoptosis machinery in malignant cells. We have reviewed the major pathogenetic events promoting MDR in MM and focused on the sigmaR(S) as potential mechanism driving this function. We demonstrate that MDR+ myeloma cells overexpress the sigmaR(2) and that the treatment with PB28 induces P-gp down-modulation through the activation of the caspases enrolled in both extrinsic and intrinsic apoptotic pathways. Thus, sigmaR(2) inhibitors may be tentatively proposed for the treatment of PAM-resistant MM patients.

Distribution of anti-F(ab')2 antibodies and the 16/6 idiotype in systemic lupus erythematosus (SLE) probands and kindreds.

Levels of serum anti-F(ab′)2 antibodies and expression of the 16/6 anti-DNA idiotype were studied in 103 sera from first-degree relatives of 17 systemic lupus erythematosus (SLE) kindreds. Among healthy SLE relatives, 35.9% showed anti-F(ab′)2 elevations and 24%, Id 16/6 expression. Forty-three and two-tenths percent of healthy SLE relatives with elevated anti-F(ab′)2 also showed expression of 16/6; when Id 16/6 was positive, 16 of 25 relatives (64%) showed parallel elevations of anti-F(ab′)2. However, within individual families, distribution patterns of elevated anti-F(ab′)2 and Id 16/6 often did not coincide. Affinity-isolated anti-F(ab′)2 from four members of a single SLE kindred showed relative enrichment for Id 16/6 in only two of the four individuals studied. Moreover, none of the isolated anti-F(ab′)2 antibodies within this kindred or another kindred showing 16/6 Id expression reacted directly with 16/6 Id. Our studies suggest that whereas both anti-F(ab′)2 and Id 16/6 are increased within SLE kindreds, expression of the two does not always coincide. Furthermore, anti-F(ab′)2 antibodies do not show direct reactivity with Id 16/6. A number of anti-DNA idiotypic markers may play a role in idiotypic networks among such SLE kindreds.

Cytotherapies in multiple myeloma: a complementary approach to current treatments?

Introduction: Based on their tumor tropism, mesenchymal stem cells (MSCs) have been proposed as carriers of cytotoxic molecules in pioneering strategies of anti-cancer gene therapy. Similar to solid tumors, MSCs, genetically modified to stably express the TNF-related apoptosis-inducing ligand (TRAIL), have been applied to counter-attack multiple myeloma (MM) in vitro and envisioned as a promising strategy for future anti-MM treatments. Areas covered: Accumulating evidence based on the detection of genetic and functional abnormalities in MSCs from MM patients points to the supportive function of MSCs in both the development and progression of MM, driven by chronic interplays with malignant cells within the marrow milieu. In this review, we revisit the function of MSCs in the pathophysiology of MM and explore the pivotal mechanisms of their interaction with myeloma cells. We also discuss the therapeutic significance of novel strategies using TRAIL-engineered MSCs in this cancer model, dissecting their role as new tools for future treatments against MM. Expert opinion: A cytotherapy based on TRAIL-engineered MSCs against MM may be successfully combined with either conventional approaches of autologous stem cell transplantation or with novel anti-MM drugs. Intensive preclinical investigations are required to identify the best sources as well as modalities of MSC administration, thus defining the translational suitability of this strategy in the clinical setting.

Molecular localization of human IgG anti-F(ab′)2 reactivity with variable- and constant-region λ light-chain epitopes

Human IgG antibodies reacting with antigenic determinants on F(ab′)2 fragments represent generic antiidiotypic antibodies present in the serum of normal individuals. Additionally, the titers of these antibodies in the sera of patients with systemic lupus erythematosus (SLE) are inversely related to disease activity. Because these autoantibodies recognize predominantly light chain-related epitopes, especially λ type, we synthesized constant (C)λ- and variable (V)λ-related overlapping 7-mer peptides on polypropylene pins to determine anti-F(ab′)2-reactive epitopes on humanλ light chains. ELISA reactivity of affinity-purified anti-F(ab′)2 antibodies obtained from normal individuals and from patients with SLE, as well as murine anti-human light-chain monoclonal antibodies specific for Cλ and Vλ subgroup-related determinants, was tested using the overlapping 7-mers of humanλ light-chain sequence. The patterns of reactivity against Cλ-related peptides were similar in both normal and SLE-derived anti-F(ab′)2 antibodies. However, reactivity profiles against Vλ-related peptides were distinctively different between the normal and the SLE-associated anti-F(ab′)2 autoantibodies. A decrease in reactivity among the SLE IgG anti-F(ab′)2 antibodies was noted for particular amino acid Vλ complementarity-determining region (CDR) residues, including glycine at positions 27 and 54, alanine at 16 and 37, and tyrosine at 28 and 91. This different pattern of reactivity from normal may indicate that in SLE there is a failure of antiidiotypic control mechanisms, as reflected by a defect in production of antibody to immunodominant Vλ CDR residues.

Exosomes in melanoma: a role in tumor progression, metastasis and impaired immune system activity

Exosomes (Exo) are small vesicles produced by melanoma cells and the accessory cells of the tumor microenvironment. They emerge via both classical and direct pathways and actively participate in tumor colonisation of distant tissues. The proteins, nucleic acids, cytokines and growth factors engulfed by Exo are transferred to recipient cells, where they drive numerous functions required for the tumor escape from immune system control and tumor progression. By positively or negatively modulating immune cell properties, Exo provoke immune suppression and, in turn, defective dendritic cell (DC) functions. Together, these effects limit the cytotoxicity of T-cells and expand both T-regulatory and myeloid-derived suppressor populations. They also hinder perforin and granzyme production by natural killer cells. Finally, Exo also control the organotropism of melanoma cells. The distinct phenotypic properties of Exo can be exploited both for diagnostic purposes and in the early identification of melanoma patients likely to respond to immunotherapy. The potential therapeutic application of Exo derived from DCs has been demonstrated in vaccination trials, which showed an increase in anti-melanoma activity with respect to circulating tumor cells. However, additional studies are required before Exo can be effectively used in diagnostic and therapeutic applications in melanoma.

Human anti-F(ab′)2 antibodies show preferential reactivity for F(ab′)2 molecules bearing λ light chains

In order to evaluate binding specificities of anti-F(ab′)2 antibodies from patients with systemic lupus erythematosus (SLE) and from normal healthy controls, F(ab′)2 fragments were prepared from 24 IgG myelomas with defined isoelectric points, DNA-associated idiotypes, and κ/λ light chain types. Using ELISA and hemagglutination assays, anti-F(ab′)2 antibodies from 12 healthy controls and 29 SLE patients were observed to exhibit preferential binding (λ > κ) to myeloma F(ab′)2 fragments composed of λ light chains (P < 0.0001). No correlation of anti-F(ab′)2 binding and presence of cationic, neutral, or anionic isoelectric points or for DNA-associated idiotypes on monoclonal F(ab′)2 was detected. Anti-F(ab′)2 antibodies, often elevated in SLE during remission, show preferential specificity for F(ab′)2 fragments bearing λ light chains.