Claudia Casnici

Alternative BCR/ABL Splice Variants in Philadelphia Chromosome–Positive Leukemias Result in Novel Tumor-Specific Fusion Proteins that May Represent Potential Targets for Immunotherapy Approaches

Imatinib currently represents the standard treatment in the early chronic phase of chronic myelogenous leukemia (CML), thanks to the high percentage of cytogenetic complete remission achieved, but it is yet unclear to what extent it can eradicate leukemia. Therefore, different vaccination strategies have been suggested, mainly based on the exploitment of the junctional peptides spanning the fusion region of the Bcr/Abl proteins. To identify new potential immunologic targets, 63 Philadelphia chromosome-positive patients and 6 BCR/ABL-positive cell lines were tested in nested reverse transcriptase PCR to detect the presence of BCR/ABL transcripts arising from the alternative splicing of the main BCR/ABL transcripts. We could detect BCR/ABL transcripts with junctions between BCR exon 1, 13, or 14 and ABL exon 4 in approximately 80% of patients and 84% of cell lines, beside the main fusion transcripts. Translation products of these transcripts were characterized at their COOH terminus by a large amino acid portion derived from the out of frame (OOF) reading of ABL gene. These proteins were detected in BCR/ABL-positive cell lines by immunoprecipitation and immunohistochemistry. Finally, we determined whether OOF-specific CD8+ T cells could be found in the peripheral blood of CML patients and whether they could acquire effector function following in vitro sensitization with OOF-derived peptides predicted to bind to human leucocyte antigen (HLA)-A2 and HLA-A3 molecules. We detected the presence of OOF-specific CD8+ T cells in four of four patients studied, and in one case, these T cells exhibited specific cytotoxic activity against both peptide-pulsed targets and autologous primary CML cells.

The apoptotic effect of somatostatin analogue SMS 201-995 on human lymphocytes

The antiproliferative effect of a synthetic octapeptide, somatostatin analogue SMS 201-995 (SMS), and its capacity to bind were evaluated on human peripheral blood lymphocytes (PBL) activated by phytohemoagglutinin (PHA). We then addressed our work to investigate if SMS inhibits PHA activation of PBL by a cytostatic rather than a cytotoxic mechanism. Consequently, we studied the cell cycle distribution and the activation of caspase-3, measuring the presence of the cleavage product of poly(ADP-ribose) polymerases (PARP), and we evaluated the presence of apoptotic DNA by using a monoclonal antibody specific for the single-stranded regions of DNA. All our results indicate that SMS induces apoptosis in activated lymphocytes.

Inhibitory effect of somatostatin on human T lymphocytes proliferation

Somatostatin (SOM) was originally described as a growth hormone release inhibiting factor, but SOM and its specific receptors (SOM-r) have been shown to be expressed on both normal and activated T and B lymphocytes and other immunocompetent cells. In the present study we have demonstrated that SOM strongly inhibits the proliferation of human T lymphocytes when stimulated by PHA, Con A or alloantigens. However, SOM was most effective when the T cells were stimulated by an alloantigen rather than a polyclonal activator such as PHA and ConA. Moreover, SOM strongly inhibited the expression of activation markers such as CD69 and CD25 that are expressed on T lymphocytes during alloantigen stimulation. SOM also inhibited both CD28 and CD2 mediated T cell proliferation. Whereas proliferation of T cells induced by the engagement of CD3 antigen using specific mAbs was only marginally affected. Our results would support the concept that in humans SOM plays a key role in the modulation of T cell activation by interfering with the antigen-independent pathways CD2 and CD28.

Inhibitory effect of pasireotide and octreotide on lymphocyte activation

Somatostatin (SST) regulates the function of the central and peripheral nervous system, the endocrine and exocrine organs, as well as the vascular and immune system. These actions are mediated by five specific membrane somatostatin receptors. This study compares the effects on human lymphocytes of two long-acting somatostatin analogues that have different receptor affinity: octreotide and pasireotide. Both analogues have an antiproliferative effect on human lymphocyte proliferation, but they act at different concentration and, while octreotide enhances IL10 and inhibits gamma IFN pasireotide inhibits IL2 and gamma IFN. In both sets of experiment the different behaviour of the two analogues could be due to their different affinity to the SSTR subtypes. Finally this study suggest that the growth inhibitory action of somatostatin analogues is an apoptotic phenomenon and it can be mediated by SSTR2a, in the case of octreotide, and by SSTR3 when pasireotide is used or it can be mediated by the heterodimerization of the two receptor.

The Expression of GHS-R in Primary Neurons Is Dependent upon Maturation Stage and Regional Localization

Ghrelin is a hormone with a crucial role in the regulation of appetite, regulation of inflammation, glucose metabolism and cell proliferation. In the brain ghrelin neurons are located in the cortex (sensorimotor area, cingular gyrus), and the fibres of ghrelin neurons in hypothalamus project directly to the dorsal vagal complex (DVC). Ghrelin binds the growth hormone secretagogue receptor (GHS-R) a G-protein-coupled receptor with a widespread tissue distribution, indeed these receptors are localized both in nonnervous, organs/tissues (i.e. adipose tissue, myocardium, adrenals, gonads, lung, liver, arteries, stomach, pancreas, thyroid, and kidney) as well as in central nervous system (CNS) and higher levels of expression in the pituitary gland and the hypothalamus and lower levels of expression in other organs, including brain. A GHS-R specific monoclonal antibody has been developed and characterized and through it we demonstrate that GHS-R is expressed in primary neurons and that its expression is dependent upon their developmental stage and shows differences according to the brain region involved, with a more pronounced expression in hippocampal rather than cortical neurons. A characterization of GHS-R within the central nervous system is of extreme importance in order to gain insights on its role in the modulation of neurodegenerative events such as Alzheimer’s disease.

Evidence that SMS 201-995 enhances the immunosuppressive effect of FK506

Somatostatin (SS) was originally described as a growth hormone release inhibiting factor synthesised in the hypothalamus. Recently, SS and its receptor (SSTR) have been demonstrated in lymphoid tissues and seem to play a regulatory, largely inhibitory, role in immune responses. The aim of the present study was to check the immunosuppressive effect of a SS derived peptide, the octreotide (SMS 201-995) and to verify whether this molecule acted synergistically with FK506. An immunosuppressive effect of SMS was observed on the proliferation of rat spleen cells induced in vitro, either by polyclonal mitogens such as PHA or by alloantigens. With PHA stimulation, 10(-14) M SMS significantly enhanced the immunosuppressive action of 0.00001 microg/ml FK506. The addition of SMS in MLR (10(-11)-10(-9)M) increased the antiproliferative effect of both 0.0001 microg/ml and 0.00001 microg/ml FK506. In consideration of the extremely low concentration of both drugs that was required to obtain a good immunosuppression in vitro, we verified the association of FK506 and SMS in vivo in an allogeneic skin graft model that used Lewis (Lew) rats as donors and Brown Norway (BN) rats as recipients. BN treated with 0.1 mg/kg FK506 and 0.5-10 microg/kg SMS showed a significant increase in mean skin allograft survival time when compared to either a monotherapy or control group. None of the animals died or showed signs of drug-related toxicity. In conclusion, a combined therapy of SMS and FK506, administered at lower dosages than those that are considered therapeutic, led to an effective immunosuppression without any undesirable side effects.

Optimized “In Vitro” Culture Conditions for Human Rheumatoid Arthritis Synovial Fibroblasts

The composition of synovial fluid in rheumatoid arthritis (RA) is complex and strongly influences the microenvironment of joints and it is an inseparable element of the disease. Currently, “in vitro” studies are performed on RA cells cultured in the presence of either recombinant proinflammatory cytokines-conditioned medium or medium alone. In this study, we evaluated the use of synovial fluid, derived from RA patients, as optimal culture condition to perform “in vitro” studies on RA synovial fibroblasts. We observed that synovial fluid is more effective in inducing cell proliferation with respect to TNF-alpha or culture medium alone. Spontaneous apoptosis in fibroblasts was also decreased in response to synovial fluid. The expression of proinflammatory cytokines in the presence of synovial fluid was significantly elevated with respect to cells cultured with TNF-alpha or medium, and the overall morphology of cells was also modified. In addition, modulation of intracellular calcium dynamics elicited in response to synovial fluid or TNF-alpha exposure is different and suggests a role for the purinergic signalling in the modulation of the effects. These results emphasize the importance of using RA synovial fluid in “in vitro” studies involving RA cells, in order to reproduce faithfully the physiopathological environmental characteristic of RA joints.

Proapoptotic Activity of a Monomeric Smac Mimetic on Human Fibroblast-Like Synoviocytes from Patients with Rheumatoid Arthritis

ABSTRACTInhibitors of apoptosis proteins (IAPs) block cell death in response to diverse stimuli. The mitochondrial protein, second mitochondria-derived activator of caspase (Smac), negatively regulates IAP inhibition of caspase activity. We investigated the proapoptotic activity of a synthetic Smac (Smac 066) on fibroblast-like synoviocytes (FLS) derived from patients with active rheumatoid arthritis (RA). We found that Smac 066 induced significant apoptosis in all RA-FLS samples. Furthermore, IAPs, which are upregulated in RA-FLS, were downregulated by Smac 066. This suggested that IAPs upregulation was responsible for RA-FLS sensitivity to Smac 066. Next, we analysed caspase activation and found that Smac 066 was associated with caspase 8 and caspase 3 activities. We then investigated the mechanism underlying Smac 066 downregulation of IAPs in RA-FLS with an apoptotic pathway array. Interestingly, Smac 066 significantly upregulated IGFBP-5, a protein involved in differentiation, apoptosis, and osteoblastic activation. Smac 066 may represent a new therapeutic approach to RA treatment.

Regulation of human peripheral blood lymphocytes IL-10 BY SMS 201-995

The somatostatin analog SMS 201-995 inhibits human peripheral blood lymphocytes (PBL) proliferation and here we demonstrate that it induces a significant increase in T cells IL-10 release as is evidenced in double fluorescence experiments. Seizing IL-10 by monoclonal antibody, SMS does not affect lymphocyte proliferation, suggesting that this cytokine is involved in the antiproliferative effect of these analog. We previously demonstrated that SMS inhibits T cells acting on the CD28 rather than the CD3-mediated signal in exactly the same way as does IL-10. Thus SMS inhibits human PBL activation by inducing IL-10 release and the consequent inhibition of the CD28 co-stimulatory pathway providing new perspectives on developing immunosuppressive strategies.

Smac127 Has Proapoptotic and Anti-Inflammatory Effects on Rheumatoid Arthritis Fibroblast-Like Synoviocytes

Rheumatoid arthritis (RA) is characterized by synovial inflammation and hyperplasia. Fibroblast-like synoviocytes (FLSs) are apoptosis-resistant and contribute to the pathogenesis of RA by producing cytokines and proteolytic enzymes, which degrade the extracellular matrix. We evaluated the proapoptotic and anti-inflammatory activity of the small molecule Smac127 on RA-FLSs cultured in synovial fluid (SF), in order to reproduce the physiopathological environmental characteristic of RA joints. In this context, Smac127 induces apoptosis by inhibiting apoptosis proteins (IAPs). This inhibition activates caspase 3 and restores the apoptotic pathway. In addition, Smac127 induces a significant inhibition of the secretion of IL-15 and IL-6, stimulation of pannus formation, and damage of bone and cartilage in RA. Also the secretion of the anti-inflammatory cytokine IL-10 is dramatically increased in the presence of Smac127. The cartilage destruction in RA patients is partly mediated by metalloproteinases; here we show that the MMP-1 production by fibroblasts cultured in SF is significantly antagonized by Smac127. Conversely, this molecule has no significant effects on RANKL and OPG production. Our observations demonstrate that Smac127 has beneficial regulatory effects on inflammatory state of RA-FLSs and suggest a potential use of Smac127 for the control of inflammation and disease progression in RA.