A novel knock-in mouse model of cryopyrin-associated periodic syndromes with development of amyloidosis: Therapeutic efficacy of proton pump inhibitors.

BACKGROUND: Cryopyrin-associated periodic syndromes (CAPS) are a group of autoinflammatory diseases linked to gain-of-function mutations in the NOD-like receptor family, pyrin domain containing 3 (NLRP3) gene, which cause uncontrolled IL-1ß secretion. Proton pump inhibitors (PPIs), which are commonly used as inhibitors of gastric acid production, also have anti-inflammatory properties, protect mice from sepsis, and prevent IL-1ß secretion by monocytes from patients with CAPS. OBJECTIVE: We sought to develop a novel Nlrp3 knock-in (KI) mouse model of CAPS to study amyloidosis, a severe CAPS complication, and test novel therapeutic approaches. METHODS: We generated KI mice by engineering the N475K mutation, which is associated with the CAPS phenotype, into the mouse Nlrp3 gene. KI and wild-type mice received PPIs or PBS intraperitoneally and were analyzed for survival, inflammation, cytokine secretion, and amyloidosis development. RESULTS: Mutant Nlrp3 KI mice displayed features that recapitulate the immunologic and clinical phenotype of CAPS. They showed systemic inflammation with high levels of serum proinflammatory cytokines, inflammatory infiltrates in various organs, and amyloid deposits in the spleen, liver, and kidneys. Toll-like receptor stimulated macrophages from KI mice secreted high levels of IL-1ß, IL-18, and IL-1α but low amounts of IL-1 receptor antagonist. Treatment of KI mice with PPIs had a clear clinical effect, showing a reduction in inflammatory manifestations, regression of amyloid deposits, and normalization of proinflammatory and anti-inflammatory cytokine production by macrophages. CONCLUSION: Nlrp3 KI mice displayed a CAPS phenotype with many characteristics of autoinflammation, including amyloidosis. The therapeutic effectiveness of PPIs associated with a lack of toxicity indicates that these drugs could represent relevant adjuvants to the anti-IL-1 drugs in patients with CAPS and other IL-1-driven diseases.

IL-25 dampens the growth of human germinal center-derived B-cell non Hodgkin Lymphoma by curtailing neoangiogenesis.

Interleukin (IL)-25, a member of the IL-17 cytokine superfamily, is produced by immune and non-immune cells and exerts type 2 pro-inflammatory effects in vitro and in vivo. The IL-25 receptor(R) is composed of the IL-17RA/IL-17RB subunits. Previous work showed that germinal centre (GC)-derived B-cell non Hodgkin lymphomas (B-NHL) expressed IL-17AR, formed by IL-17RA and IL-17RC subunits, and IL-17A/IL-17AR axis promoted B-NHL growth by stimulating neoangiogenesis. Here, we have investigated expression and function of IL-25/IL-25R axis in lymph nodes from human GC-derived B-NHL, i.e. Follicular Lymphoma (FL,10 cases), Diffuse Large B Cell Lymphoma (6 cases) and Burkitt Lymphoma (3 cases). Tumor cells expressed IL-25R and IL-25 that was detected also in non-malignant cells by flow cytometry. Immunohistochemical studies confirmed expression of IL-25R and IL-25 in FL cells, and highlighted IL-25 expression in bystander elements of the FL microenvironment. IL-25 i) up-regulated phosphorylation of NFkBp65, STAT-1 and JNK in B-NHL cells; ii) inhibited in vitro proliferation of the latter cells; iii) exerted anti-tumor activity in two in vivo B-NHL models by dampening expression of pro-angiogenic molecules as VEGF-C, CXCL6 and ANGPT3. In conclusion, IL-25, that is intrinsically pro-angiogenic, inhibits B-NHL growth by reprogramming the angiogenic phenotype of B-NHL cells.

Interleukin-17A promotes the growth of human germinal center derived non-Hodgkin B cell lymphoma.

Interleukin (IL)-17A belongs to IL-17 superfamily and binds the heterodimeric IL-17 receptor (R)(IL-17RA/IL-17RC). IL-17A promotes germinal center (GC) formation in mouse models of autoimmune or infectious diseases, but the role of IL-17A/IL-17AR complex in human neoplastic GC is unknown. In this study, we investigated expression and function of IL-17A/IL-17AR in the microenvironments of 44 B cell non-Hodgkin lymphomas (B-NHL) of GC origin (15 follicular lymphomas, 17 diffuse large B cells lymphomas and 12 Burkitt lymphomas) and 12 human tonsil GC. Furthermore, we investigated the role of IL-17A in two in vivo models of GC B cell lymphoma, generated by s.c. injection of SU-DHL-4 and OCI-Ly8 cell lines in Severe combined immunodeficiency (SCID)/Non Obese Diabetic (NOD) mice. We found that: (i) B-NHL cell fractions and tonsil GC B cells expressed IL-17RA/IL-17RC, (ii) IL-17A signaled in both cell types through NF-kBp65, but not p38, ERK-1/2, Akt or NF-kBp50/105, phosphorylation, (iii) IL-17A was expressed in T cells and mast cells from neoplastic and normal GC microenvironments, (iv) IL-17A rendered tonsil GC B cells competent to migrate to CXCL12 and CXCL13 by downregulating RGS16 expression; (v) IL-17A stimulated in vitro proliferation of primary B-NHL cells; (vi) IL-17A (1 µg/mouse-per dose) stimulated B-NHL growth in two in vivo models by enhancing tumor cell proliferation and neo-angiogenesis. This latter effect depended on IL-17A-mediated induction of pro-angiogenic gene expression in tumor cells and direct stimulation of endothelial cells. These data define a previously unrecognized role of human IL-17A in promoting growth of GC-derived B-NHL and modulating normal GC B cell trafficking.

Transplanted human adipose tissue-derived stem cells engraft and induce regeneration in mice olfactory neuroepithelium in response to dichlobenil subministration.

We used immunodeficient mice, whose dorsomedial olfactory region was permanently damaged by dichlobenil inoculation, to test the neuroregenerative properties of transplanted human adipose tissue-derived stem cells after 30 and 60 days. Analysis of polymerase chain reaction bands revealed that stem cells preferentially engrafted in the lesioned olfactory epithelium compared with undamaged mucosa of untreated transplanted mice. Although basal cell proliferation in untransplanted lesioned mice did not give rise to neuronal cells in the olfactory mucosa, we observed clusters of differentiating olfactory cells in transplanted mice. After 30 days, and even more at 60 days, epithelial thickness was partially recovered to normal values, as also the immunohistochemical properties. Functional reactivity to odorant stimulation was also confirmed through electro-olfactogram recording in the dorsomedial epithelium. Furthermore, we demonstrated that engrafted stem cells fused with mouse cells in the olfactory organ, even if heterokaryons detected were too rare to hypothesize they directly repopulated the lesioned epithelium. The data reported prove that the migrating transplanted stem cells were able to induce a neuroregenerative process in a specific lesioned sensory area, enforcing the perspective that they could become an available tool for stem cell therapy.

The antitumor potential of Interleukin-27 in prostate cancer.

Prostate cancer (PCa) is of increasing significance worldwide as a consequence of the population ageing. Fragile elderly patients may particularly benefit from noninvasive and well tolerable immunotherapeutic approaches. Preclinical studies have revealed that the immune-regulatory cytokine IL-27 may exert anti-tumor activities in a variety of tumor types without discernable toxicity. We, thus, investigated whether IL-27 may function as anti-tumor agent in human (h) PCa and analyzed the rationale for its clinical application. In vitro, IL-27 treatment significantly inhibited proliferation and reduced the angiogenic potential of hPCa cells by down-regulating the pro-angiogenesis-related genes fms-related tyrosine kinase (FLT)1, prostaglandin G/H synthase 1/cyclooxygenase-1 (PTGS1/COX-1) and fibroblast growth factor receptor (FGFR)3. In addition, IL-27 up-regulated the anti-angiogenesis-related genes such as CXCL10 and TIMP metallopeptidase inhibitor 3 (TIMP3). In vivo, IL-27 reduced proliferation and vascularization in association with ischemic necrosis of tumors developed after PC3 or DU145 cell injection in athymic nude mice. In patients' prostate tissues, IL-27R was expressed by normal epithelia and low grade PCa and lost by high tumor grade and stages. Nevertheless, IL-27R was expressed by CD11c(+), CD4(+) and CD8(+) leukocytes infiltrating the tumor and draining lymph nodes. These data lead to the conclusion that i) IL-27's anti-PCa potential may be fully exploited in patients with well-differentiated, localized IL-27R positive PCa, since in this case it may act on both cancerous epithelia and the tumor microenvironment; ii) PCa patients bearing high grade and stage tumor that lack IL-27R may benefit, however, from IL-27's immune-stimulatory properties.

Androgens affect muscle, motor neuron, and survival in a mouse model of SOD1-related amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective loss of upper and lower motor neurons and skeletal muscle atrophy. Epidemiologic and experimental evidence suggest the involvement of androgens in ALS pathogenesis, but the mechanism through which androgens modify the ALS phenotype is unknown. Here, we show that androgen ablation by surgical castration extends survival and disease duration of a transgenic mouse model of ALS expressing mutant human SOD1 (hSOD1-G93A). Furthermore, long-term treatment of orchiectomized hSOD1-G93A mice with nandrolone decanoate (ND), an anabolic androgenic steroid, worsened disease manifestations. ND treatment induced muscle fiber hypertrophy but caused motor neuron death. ND negatively affected survival, thereby dissociating skeletal muscle pathology from life span in this ALS mouse model. Interestingly, orchiectomy decreased androgen receptor levels in the spinal cord and muscle, whereas ND treatment had the opposite effect. Notably, stimulation with ND promoted the recruitment of endogenous androgen receptor into biochemical complexes that were insoluble in sodium dodecyl sulfate, a finding consistent with protein aggregation. Overall, our results shed light on the role of androgens as modifiers of ALS pathogenesis via dysregulation of androgen receptor homeostasis.

Interleukin-27 inhibits the growth of pediatric acute myeloid leukemia in NOD/SCID/Il2rg-/- mice.

PURPOSE: Acute myeloid leukemia (AML) accounts for more than half of fatal cases in all pediatric leukemia patients; this observation highlights the need of more effective therapies. Thus, we investigated whether interleukin (IL)-27, an immunomodulatory cytokine, functions as an antitumor agent against pediatric AML cells. EXPERIMENTAL DESIGN: Expression of WSX-1 and gp130 on AML cells from 16 pediatric patients was studied by flow cytometry. Modulation of leukemia cell proliferation or apoptosis upon IL-27 treatment in vitro was tested by bromodeoxyuridine/propidium iodide (PI) and Ki67, or Annexin V/PI staining and flow cytometric analysis. The angiogenic potential of AML cells treated or not with IL-27 was studied by chorioallantoic membrane assay and PCR array. In vivo studies were carried out using nonobese diabetic/severe combined immunodeficient (NOD/SCID)/Il2rg(-/-) mice injected intravenously with five pediatric AML cell samples. Leukemic cells engrafted in PBS and IL-27-treated animals were studied by immunohistochemical/morphologic analysis and by PCR array for expression angiogenic/dissemination-related genes. RESULTS: We provided the first demonstration that (i) AML cells injected into NOD/SCID/Il2rg(-/-) mice gave rise to leukemia dissemination that was severely hampered by IL-27, (ii) compared with controls, leukemia cells harvested from IL-27-treated mice showed significant reduction of their angiogenic and spreading related genes, and (iii) similarly to what was observed in vivo, IL-27 reduced in vitro AML cell proliferation and modulated the expression of different genes involved in the angiogenic/spreading process. CONCLUSION: These results provide an experimental rationale for the development of future clinical trials aimed at evaluating the toxicity and efficacy of IL-27.

Enhanced anti-neuroblastoma activity of a fenretinide complexed form after intravenous administration.

OBJECTIVES: The major limitation to successful chemotherapy of neuroblastoma (NB) is the toxicity and the poor bioavailability of traditional drugs. METHODS: We synthesised an amphiphilic dextrin derivative (DX-OL) able to host fenretinide (4-HPR) by complexation. In this study, we have investigated the effects of 4-HPR-loaded amphipilic dextrin (DX-OL/4-HPR) in comparison with 4-HPR alone both in vitro on human NB cells and in vivo in pseudometastatic NB models. The haemolysis assay was used as a measure of the potential damage caused by the pharmaceutical formulation in vivo. Pharmacokinetic experiments were performed to assess drug plasma levels in mice treated with free or complexed 4-HPR. KEY FINDINGS: DX-OL/4-HPR exerted a more potent cytotoxic activity on NB cells. Complexed 4-HPR significantly increased the proportion of sub-G1 cells with respect to free 4-HPR. Dextrin derivatives showed no haemolytic activity, indicating their suitability for parenteral administration. DX-OL/4-HPR increased the lifespan and the long-term survival of treated mice over controls. The analysis of drug plasma levels indicates that the complexed drug has a higher AUC due to a reduced clearance from the blood. CONCLUSIONS: Our data suggest that DX-OL/4-HPR is an injectable formulation that is able to improve drug aqueous solubility and bioavailability.

Direct inhibition of human acute myeloid leukemia cell growth by IL-12.

Acute myeloid leukemia is a haematopoietic malignancy originating from the transformation of myeloid progenitors that proliferate and accumulate in the bone marrow. In AML patients the survival rate at 5 years is 40-50% highlighting the need for novel therapies. In this study we have asked whether IL-12, an immuno-modulatory cytokine with anti-tumor activity, may inhibit directly AML cell growth. We show that the human AML cell lines U937, K562 and THP-1 expressed both chains of the IL-12 receptor (R), i.e. IL-12Rß1 and IL-12Rß2. IL-12 inhibited the angiogenic potential of AML cells in vitro, but did not affect their survival or proliferation. In vivo experiments were performed using SCID-NOD mice injected intraperitoneally (i.p.) with the human U937 AML cell line and subsequently treated with human recombinant IL-12 or PBS i.p. Histological, immunohistochemical and flow cytometric analyses on explanted tumors revealed that IL-12 reduced new vessel formation, induced apoptosis and inhibited tumor cell proliferation. Studies on a panel of angiogenesis related genes in explanted tumors using PCR arrays showed significantly down-regulated expression of numerous pro-angiogenic genes including VEGF-C, IL-6, IL-8, CXCL1, CXCL6 and alanyl aminopeptidase in IL-12 vs PBS treated mice. This study shows for the first time that IL-12 targets directly AML cell growth and paves the way to further investigation of IL-12 as potential drug for AML treatment.

Interleukin-23 acts as antitumor agent on childhood B-acute lymphoblastic leukemia cells.

Interleukin (IL)-23 is a proinflammatory cytokine belonging to the IL-12 superfamily. The antitumor activity of IL-23 is controversial, and it is unknown whether or not the cytokine can act directly on tumor cells. The aim of this study was to investigate the potential direct antitumor activity of IL-23 in pediatric B-acute lymphoblastic leukemia (B-ALL) cells and to unravel the molecular mechanisms involved. Here, we show, for the first time, that IL-23R is up-regulated in primary B-ALL cells, compared with normal early B lymphocytes, and that IL-23 dampens directly tumor growth in vitro and in vivo through the inhibition of tumor cell proliferation and induction of apoptosis. The latter finding is related to IL-23-induced up-regulation of miR15a expression and the consequent down-regulation of BCL-2 protein expression in pediatric B-ALL cells. This study demonstrates that IL-23 possesses antileukemic activity and unravels the underlying mechanisms. Thus, IL-23 may be a candidate novel drug for the treatment of B-ALL patients unresponsive to current therapeutic standards.

Interleukin-27 acts as multifunctional antitumor agent in multiple myeloma.

PURPOSE: Multiple myeloma (MM) derives from plasmablast/plasma cells that accumulate in the bone marrow. Different microenvironmental factors may promote metastatic dissemination especially to the skeleton, causing bone destruction. The balance between osteoclast and osteoblast activity represents a critical issue in bone remodeling. Thus, we investigated whether interluekin-27 (IL-27) may function as an antitumor agent by acting directly on MM cells and/or on osteoclasts/osteoblasts. EXPERIMENTAL DESIGN: The IL-27 direct antitumor activity on MM cells was investigated in terms of angiogenesis, proliferation, apoptosis, and chemotaxis. The IL-27 activity on osteoclast/osteoblast differentiation and function was also tested. In vivo studies were done using severe combined immunodeficient/nonobese diabetic mice injected with MM cell lines. Tumors from IL-27- and PBS-treated mice were analyzed by immunohistochemistry and PCR array. RESULTS: We showed that IL-27 (a) strongly inhibited tumor growth of primary MM cells and MM cell lines through inhibition of angiogenesis, (b) inhibited osteoclast differentiation and activity and induced osteoblast proliferation, and (c) damped in vivo tumorigenicity of human MM cell lines through inhibition of angiogenesis. CONCLUSIONS: These findings show that IL-27 may represent a novel therapeutic agent capable of inhibiting directly MM cell growth as well as osteoclast differentiation and activity.

Natural killer cells kill human melanoma cells with characteristics of cancer stem cells.

Experimental and clinical data suggest that tumours harbour a cell population retaining stem cell characteristics that can drive tumorigenesis. CD133 is considered an important cancer stem cells (CSC)-associated marker. In a large variety of human malignancies, including melanoma, CD133(+) cells have been reported to comprise CSC. In this study, we show that melanoma cell lines are highly heterogeneous for the expression of several stem cell-associated markers including CD133, c-kit/CD117 and p75 neurotrophin receptor/CD271. Since no information is available on the ability of NK cells to recognize and lyse melanoma stem cells, we assessed whether melanoma cell lines, characterized by stem cell-like features, were susceptible to lysis by IL-2-activated NK cells. We show that activated NK cells efficiently kill malignant melanoma cell lines that were enriched in putative CSC by the use of different selection methods (i.e. CD133 expression, radioresistance or the ability to form melanospheres in stem cell-supportive medium). NK cell-mediated recognition and lysis of melanoma cells involved different combinations of activating NK receptors. Since CSC have been reported to be both drug resistant and radioresistant, our present data suggest that NK-based adoptive immunotherapy could represent a novel therapeutic approach to possibly eradicate metastatic melanoma.

Human cord blood CD133+ stem cells transplanted to nod-scid mice provide conditions for regeneration of olfactory neuroepithelium after permanent damage induced by dichlobenil.

The herbicide dichlobenil selectively causes necrosis of the dorsomedial part of olfactory neuroepithelium (NE) with permanent damage to the underlying mucosa, whereas the lateral part of the olfactory region and the nasal respiratory mucosa remain undamaged. We investigated here whether human umbilical cord blood CD133(+) stem cells (HSC) injected intravenously to nod-scid mice pretreated with dichlobenil may engraft the olfactory mucosa and contribute to the regeneration of the damaged NE. We tested HLA-DQalpha1 DNA and three human microsatellites (Combined DNA Index System) as indicators of engrafted cells, finding polymerase chain reaction evidence of chimaerism in various tissues of the host, including the olfactory mucosa and bulb, at 7 and 31 days following HSC transplantation. Histology, immunohistochemistry, and lectin staining revealed the morphological recovery of the dorsomedial region of the NE in dichlobenil-treated mice that received HSC, contrasting with the lack of regeneration in similarly injured areas as these remained damaged in control nontransplanted mice. FISH analysis, to detect human genomic sequences from different chromosomes, confirmed persistent engraftment of the regenerating olfactory area with chimeric cells. Electro-olfactograms in response to odorants, to test the functionality of the olfactory NE, confirmed the functional damage of the dorsomedial area in dichlobenil-treated mice and the functional recovery of the same area in transplanted mice. These findings support the concept that transplanted HSC migrating to the damaged olfactory area provide conditions facilitating the recovery from olfactory receptor cell loss.

Inhibition of non-neuronal alpha7-nicotinic receptor reduces tumorigenicity in A549 NSCLC xenografts.

Nicotinic acetylcholine receptors (nAChR) are expressed on bronchial epithelial and non-small cell lung cancer cells and are involved in cell growth regulation. Nicotine (classical nAChR agonist) induced cell proliferation, whereas nAChR antagonists, d- tubocurarine or alpha-cobratoxin (alpha-CbT), induced cell death. In the current study, we further explored the antitumor potential mechanisms and activities of alpha-CbT. NOD/SCID mice were grafted intraperitoneally or orthotopically and treated with alpha-CbT. alpha-CbT treatment [0.04 ng/kg or 0.12 ng/kg] induced a strong reduction in tumor size ( approximately 90%) in comparison with mice treated with the vehicle alone. Tumor inhibition was related to severe induction of apoptosis. Moreover, neoangiogenesis was strongly inhibited (reduction of cells positive to vascular endothelial growth factor and CD31). Biochemical analyses of the cells, isolated by the primary lung tumor in alpha-CbT-treated mice, showed apoptosis features characterized by: (i) inhibition of BAD phosphorylation at Ser(112) and Ser(136); (ii) BAD dissociation from 14-3-3; (iii) BAD association with BCL-XL; and (iv) cleavage of caspase-9. Moreover, these cells were unable to grow in soft agar and develop tumor, when reinjected into mice. The small interfering RNA-mediated silencing of the alpha7-nAChR gene confirmed that alpha-CbT specifically inhibited the alpha7-nAChR-mediated survival pathway in A549 cells. Furthermore, the specificity of alpha-CbT is reinforced by the lack of effect of short chain toxin (Erabutoxin-a). Once more, the no effect of the low-affinity R33E-modified alpha-CbT strengthened the specificity of this inhibition. Although alpha7-nAChR antagonists, such as alpha-CbT, are unlikely to be a primary therapy, it may provide lead compounds for the design of clinically useful drugs.

Cochlear repair by transplantation of human cord blood CD133+ cells to nod-scid mice made deaf with kanamycin and noise.

We investigated the fate of human cord blood CD133+ hematopoietic stem cells (HSC) transplanted intravenously (IV) into irradiated nod-scid mice previously made deaf by ototoxic treatment with kanamycin and/ or intense noise, to verify whether HSC engraft the cochlea and contribute to inner ear restoration, in vivo. We tested the presence of HLA.DQalpha1 by PCR, used for traceability of engrafted cells, finding evidence that HSC migrated to various host tissues, including the organ of Corti (OC). By histology, antibody and lectin-staining analysis, we confirmed that HSC IV transplantation in mice previously damaged by ototoxic agents correlated with the repair process and stimulation ex novo of morphological recovery in the inner ear, while the cochlea of control oto-injured, nontransplanted mice remained seriously damaged. Dual color FISH analysis also provided evidence of positive engraftment in the inner ear and in various mouse tissues, also revealing small numbers of heterokaryons, probably derived from fusion of donor with endogenous cells, for up to 2 months following transplantation. These observations offer the first evidence that transplanted human HSC migrating to the inner ear of oto-injured mice may provide conditions for the resumption of deafened cochlea, emerging as a potential strategy for inner ear rehabilitation.

Targeting alpha7-nicotinic receptor for the treatment of pleural mesothelioma.

Human malignant pleural mesothelioma (MPM) is a dreadful disease and there is still no standard therapy available for a consistent therapeutic approach. This research is aimed at the evaluation of the potential therapeutic effect of a specific nicotinic receptor (nAChR) antagonist, namely alpha-Cobratoxin (alpha-CbT). Its effectiveness was tested in mesothelioma cell lines and in primary mesothelioma cells in vitro, as well as in vivo, in orthotopically xenotransplanted NOD/SCID mice. Cells showed alpha7-nAChR expression and their growth was significantly inhibited by alpha-CbT. Severe induction of apoptosis was observed after exposure to alpha-CbT [IC(80-90)]. Apoptosis was characterised by: change in mitochondrial potential, caspase-3 cleavage, down-regulation of mRNA and protein for survivin, XIAP, IAP1, IAP2 and Bcl-XL, inhibition by caspase-3 inhibitor. In vivo, the alpha-CbT acute LD(50) was 0.15 mg/kg. The LD(100) [0.24 mg/kg] induced fatal respiratory failure and massive kidney necrosis. Phase II experiments with 0.12 ng/kg alpha-CbT (1/1000 of LD(10)) were done in 53 xenotransplanted mice, inhibiting tumour development as confirmed by chest X-ray examinations, autopsy and microscopical findings. The growth of human proliferating T lymphocytes and of mesothelial cells in primary culture was not affected by alpha-CbT. Non-immunogenic derivatives of the alpha-CbT molecule need to be developed for possible human use.

Constitutive expression of IL-12R beta 2 on human multiple myeloma cells delineates a novel therapeutic target.

The interleukin-12 (IL-12) receptor (R) B2 gene acts as tumor suppressor in human acute and chronic B-cell leukemias/lymphomas and IL-12rb2-deficient mice develop spontaneously localized plasmacytomas. With this background, we investigated the role of IL-12R beta 2 in multiple myeloma (MM) pathogenesis. Here we show the following: (1) IL-12R beta 2 was expressed in primary MM cells but down-regulated compared with normal polyclonal plasmablastic cells and plasma cells (PCs). IL-6 dampened IL-12R beta 2 expression on polyclonal plasmablastic cells and MM cells. (2) IL-12 reduced the proangiogenic activity of primary MM cells in vitro and decreased significantly (P = .001) the tumorigenicity of the NCI-H929 cell line in SCID/NOD mice by inhibiting cell proliferation and angiogenesis. The latter phenomenon was found to depend on abolished expression of a wide panel of proangiogenic genes and up-regulated expression of the antiangiogenic genes IFN-gamma, IFN-alpha, platelet factor-4, and TIMP-2. Inhibition of the angiogenic potential of primary MM cells was related to down-regulated expression of the proangiogenic genes CCL11, vascular endothelial-cadherin, CD13, and AKT and to up-regulation of an IFN-gamma-related antiangiogenic pathway. Thus, IL-12R beta 2 directly restrains MM cell growth, and targeting of IL-12 to tumor cells holds promise as new therapeutic strategy.

Synthesis, in vitro activity and in vivo toxicity of the new 2,3-dinitrobutadiene derivative (1E,3E)-1,4-bis(2-naphthyl)-2,3-dinitro-1,3-butadiene.

Our interesting results on the antiproliferative (in vitro) and antitumour (in vivo) activities of (1E,3E)-1,4-bis(1-naphthyl)-2,3-dinitro-1,3-butadiene (1-Naph-DNB) have more recently induced us to design and synthesize some new 1,4-diaryl-2,3-dinitro-1,3-butadienes characterized by a common arylnitrobutadiene array but with different geometric and/or functional properties. This task was undertaken with the aim to obtain new compounds with an enhanced antiproliferative activity and, possibly, a different specificity with respect to the original (lead) compound. (1E,3E)-1,4-Bis(2-naphthyl)-2,3-dinitro-1,3-butadiene (2-Naph-DNB) is one of the molecules so obtained, a structural isomer of 1-Naph-DNB provided with a different spatial arrangement. When analyzed in vitro for its inhibition of cell proliferation 2-Naph-DNB showed a remarkable activity in the range of micromolar concentrations, with significant differences, with respect to 1-Naph-DNB, against some cell lines. Furthermore, it was able to significantly trigger apoptosis, to up-regulate p53, to block cells in the G2/M phase of the cell cycle and, finally, to slightly bind to DNA forming interstrand cross-links (ISCL). 2-Naph-DNB was then analyzed for its toxic activity in vivo in CD1 mice. This allowed the determination of toxicity parameters such as the lethal doses (LD) and the maximal tolerated dose (MTD) together with the definition of the spectrum of tissue alterations due to its administration i.v. Altogether our data suggest that the idea of modifying the geometry of the lead compound 1-Naph-DNB deserves further investigation aimed at synthesizing new molecules with similar chemical functionalities but with different spatial requirements, hopefully characterized by still enhanced activities in terms of inhibition of cell proliferation and apoptosis.

Naphthylnitrobutadienes as pharmacologically active molecules: evaluation of the in vivo antitumour activity.

On the basis of our previous interesting results in vitro on the antiproliferative activity of (1E,3E)-1,4-bis(1-naphthyl)-2,3-dinitro-1,3-butadiene (1-Naph-DNB) we have designed and synthesized the new molecule methyl (2Z,4E)-2-methylsulphanyl-5-(1-naphthyl)-4-nitro-2,4-pentadienoate (1-Naph-NMCB) characterized by the same naphthylnitrobutadiene array but with a different functional group at one end of the diene system. This new molecule showed an in vitro antiproliferative activity more significant than that found for the original 1-Naph-DNB. In order to verify in vivo our in vitro results we have tested the antitumour activity of 1-Naph-DNB and 1-Naph-NMCB in several murine tumour models, namely the myelomonocytic P388 and the Lewis lung carcinoma 3LL in BDF1 mice, the melanoma B16 in C57Bl mice, the fibrosarcoma WEHI 164 in nude mice and, finally, the C51 colon cancer in Balb/c mice. In the case of 1-Naph-NMCB the analysis of the antitumour activity has been preceded by toxicological experiments on CD-1 mice, in order to determine the lethal (LD) and the maximal tolerated (MTD) doses together with the spectrum of histological alterations caused by its iv administration. The results obtained show that the modification of the original structure of 1-Naph-DNB according to the molecular-simplification strategy has led to an asymmetric nitrobutadiene array, i.e. that of 1-Naph-NMCB, endowed with an antitumour activity which is in some cases even better than that showed by the parental compound itself, together with differences in tumour selectivity and negligible histological toxic effects.A promising, versatile route to new, more active and/or safe nitrobutadiene derivatives has thus been positively tested.

Human NK cell infusions prolong survival of metastatic human neuroblastoma-bearing NOD/scid mice.

AIM: Several lines of evidence suggest that NK cell immunotherapy may represent a successful approach in neuroblastoma (NB) patients refractory to conventional therapy. However, homing properties, safety and therapeutic efficacy of NK cell infusions need to be evaluated in a suitable preclinical murine NB model. MATERIALS AND METHODS: Here, the therapeutic efficacy of NK cell infusions in the presence or absence of NK-activating cytokines have been evaluated in a NB metastatic model set up in NOD/scid mice, that display reduced functional activity of endogenous NK cells. RESULTS: In NOD/scid mice the injected NB cells rapidly reached all the typical sites of metastatization, including bone marrow. Infusion of polyclonal IL2-activated NK cells was followed by dissemination of these cells into various tissues including those colonized by metastatic NB cells. The early repeated injection of IL2-activated NK cells in NB-bearing NOD/scid mice significantly increased the mean survival time, which was associated with a reduced bone marrow infiltration. The therapeutic effect was further enhanced by low doses of human recombinant IL2 or IL15. CONCLUSION: Our results indicate that NK-based adoptive immunotherapy can represent a valuable adjuvant in the treatment of properly selected NB patients presenting with metastatic disease, if performed in a minimal residual disease setting.