Evaluation of collected outcome measures in axial spondyloarthritis in daily-care rheumatology settings: the experience of the RHEVER network.

OBJECTIVES: Nowadays, the recommended measures for optimal monitoring of axial Spondyloarthritis (ax-SpA) disease activity are either BASDAI and CRP, or ASDAS-CRP. However, there could be a gap between recommendations and daily practice. We aimed to determine the measures collected by rheumatologists in an ax-SpA follow-up visit, and to determine the impact of a meeting (where rheumatologists reached a consensus on the measures to be collected) on the collection of such measures. METHODS: A consensual meeting of a local network of 32 rheumatologists proposed, four months later, to report at least the BASDAI score in the medical file of every ax-SpA patient at every follow-up visit. An independent investigator reviewed the medical files of 10 consecutive patients per rheumatologist, seen twice during the year (e.g. before and after the meeting). The most frequently collected measures were assessed, and then, the frequency of collection before and after the meeting was compared. RESULTS: A total of 456 medical files from 228 patients were reviewed. Treatment (>60%), CRP (51.3%) and total BASDAI (28.5%) were the most reported measures in medical files. Before/After the meeting, the frequencies of collected measures in medical files were 28.5%/51.7%, 51.3%/52.2%, 16.7%/31.6% and 0.9%/6.1% for BASDAI, CRP, BASDAI + CRP and ASDAS, respectively reaching a statistically significance for BASDAI, ASDAS and BASDAI+CRP (p<0.05). CONCLUSIONS: This study revealed a low rate of systematic report of the recommended outcome measures in ax-SpA. However, it suggests that a consensual meeting involving practicing rheumatologists might be relevant to improve the implementation of such recommendations.

Modulation of polyfunctional HIV-specific CD8 T cells in patients responding differently to antiretroviral therapy.

Antiretroviral therapy allows a restoration of immune cell homeostasis associated with a normal immune competence. Our goal was to analyze the modulation of polyfunctional HIV-specific CD8+ T-cell responses during antiretroviral therapy. HIV-infected individuals were divided into four groups according to CD4+ cell count and viral load at the moment of recruitment. Whole blood was stimulated with a pool of CD8-specific HIV-antigens to assess cytokine/chemokine production and cytotoxicity activity by using flow cytometry. The groups show different modulation in HIV-specific CD8+ T-cell responses. In particular, immunological failure showed different distributions of polyfunctional HIVspecific CD8+ responses, mainly due to an increase of cells producing CD107alpha/IFNgamma/IL-2/MIP-1beta. Our results indicate that this particular 4+ functional subset is a possible correlate of immunological failure. Considering the complexity of interactions among HAART, immune system and HIV, work is in progress to find correlates of therapy efficacy.

HIV impairs CD34+-derived monocytic precursor differentiation into functional dendritic cells.

Dendritic cells (DCs) perform a basic role in the immune system by allowing the initiation of the primary T-cell-dependent immune response. Given previous indirect evidence that DC maturation and function are impaired by HIV, we have developed an in vitro culture system in order to verify the effect of HIV infection on DC function during the development from hematopoietic progenitors. Considering that monocytic (Mo) differentiating cells efficiently replicate monocytotropic HIV, we examined whether HIV-infected monocytic precursors (MoP) were able to generate functional DCs. CD34+ hematopoietic progenitor cells (HPCs) were induced along Mo differentiative pathway in liquid cultures and at an early stage of culture, MoP were infected with M-tropic BaL HIV strain, and after 2 days they were switched to DC differentiation with GM-CSF and IL-4. Derived DCs were actively infected, as detected by HIV-p24 production. HIV did not significantly affect cell viability, but induced a reduction in cell proliferation and an inefficient functional activity in terms of uptake capability and stimulation of allogenic T cells. These results indicate that HIV-infected MoP lost the capacity to generate functional DCs, and this may represent one of the many mechanisms of immunosuppression exploited by HIV.

Zoledronic acid enhances Vδ2 T-lymphocyte antitumor response to human glioma cell lines.

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain tumor in humans, responds modestly to treatment: most patients survive less than one year after diagnosis, despite both classical and innovative treatment approaches. A recent paper focused on γδ T-cell response in GBM patients, suggesting the application of an immunomodulating strategy based on γδ T-cells which is already in clinical trials for other tumors. Human Vγ2 T-cells recognize changes in the mevalonate metabolic pathway of transformed cells by activating cytotoxic response, and by cytokine and chemokine release. Interestingly, this activation may also be induced in vivo by drugs, such as zoledronic acid, that induce the accumulation of Vγ2 T-cell ligand Isopentenyl-pyrophosphate by blocking the farnesyl pyrophosphate synthase enzyme. The aim of our work is to confirm whether bisphosphonate treatment would make glioma cell lines more susceptible to lysis by in vitro expanded γδ T-cells, improving their antitumor activity. We expanded in vitro human Vγ2 T-cells by phosphoantigen stimulation and tested their activity against glioma cell lines. Co-culture with glioma cells induced Vγ2 T-cell differentiation in effector/memory cells, killing glioma cells by the release of perforin. Interestingly, glioma cells were directly affected by zoledronic acid; moreover, treatment increased their activating ability on Vγ2 T-cells, inducing an effective antitumor cytotoxic response. Taken together, our results show that aminobisphosphonate drugs may play a dual role against GBM, by directly affecting tumor cells, and by enhancing the antitumor response of Vγ2 T-cells. Our results confirm the practicability of this approach as a new immunotherapeutic strategy for GBM treatment.

p53 regulates myogenesis by triggering the differentiation activity of pRb.

The p53 oncosuppressor protein regulates cell cycle checkpoints and apoptosis, but increasing evidence also indicates its involvement in differentiation and development. We had previously demonstrated that in the presence of differentiation-promoting stimuli, p53-defective myoblasts exit from the cell cycle but do not differentiate into myocytes and myotubes. To identify the pathways through which p53 contributes to skeletal muscle differentiation, we have analyzed the expression of a series of genes regulated during myogenesis in parental and dominant-negative p53 (dnp53)-expressing C2C12 myoblasts. We found that in dnp53-expressing C2C12 cells, as well as in p53(-/-) primary myoblasts, pRb is hypophosphorylated and proliferation stops. However, these cells do not upregulate pRb and have reduced MyoD activity. The transduction of exogenous TP53 or Rb genes in p53-defective myoblasts rescues MyoD activity and differentiation potential. Additionally, in vivo studies on the Rb promoter demonstrate that p53 regulates the Rb gene expression at transcriptional level through a p53-binding site. Therefore, here we show that p53 regulates myoblast differentiation by means of pRb without affecting its cell cycle-related functions.

Interference with p53 protein inhibits hematopoietic and muscle differentiation.

The involvement of p53 protein in cell differentiation has been recently suggested by some observations made with tumor cells and the correlation found between differentiation and increased levels of p53. However, the effect of p53 on differentiation is in apparent contrast with the normal development of p53-null mice. To test directly whether p53 has a function in cell differentiation, we interfered with the endogenous wt-p53 protein of nontransformed cells of two different murine histotypes: 32D myeloid progenitors, and C2C12 myoblasts. A drastic inhibition of terminal differentiation into granulocytes or myotubes, respectively, was observed upon expression of dominant-negative p53 proteins. This inhibition did not alter the cell cycle withdrawal typical of terminal differentiation, nor p21(WAF1/CIP1) upregulation, indicating that interference with endogenous p53 directly affects cell differentiation, independently of the p53 activity on the cell cycle. We also found that the endogenous wt-p53 protein of C2C12 cells becomes transcriptionally active during myogenesis, and this activity is inhibited by p53 dominant-negative expression. Moreover, we found that p53 DNA-binding and transcriptional activities are both required to induce differentiation in p53-negative K562 cells. Taken together, these data strongly indicate that p53 is a regulator of cell differentiation and it exerts this role, at least in part, through its transcriptional activity.

Mutant p53: an oncogenic transcription factor.

Inactivation of tumor-suppressor genes is one of the key hallmarks of a tumor. Unlike other tumor-suppressor genes, p53 is inactivated by missense mutations in half of all human cancers. It has become increasingly clear that the resulting mutant p53 proteins do not represent only the mere loss of wild-type p53 tumor suppressor activity, but gain new oncogenic properties favoring the insurgence, the maintenance, the spreading and the chemoresistance of malignant tumors. The actual challenge is the fine deciphering of the molecular mechanisms underlying the gain of function of mutant p53 proteins. In this review, we will focus mainly on the transcriptional activity of mutant p53 proteins as one of the potential molecular mechanisms. To date, the related knowledge is still quite scarce and many of the raised questions of this review are yet unanswered.

Activation of interferon response genes and of plasmacytoid dendritic cells in HIV-1 positive subjects with GB virus C co-infection.

GB virus C (GBV-C) coinfection has a protective role in Human Immunodeficiency Virus (HIV) infection, and increases the duration of suppression of HIV-1 viremia in patients under Highly Active Anti-Retroviral Therapy (HAART). Since innate antiviral response may be involved in the protection, we analyzed the possible role of GBV-C as activator of innate immunity. To this aim, we measured the extent of activation of the interferon (IFN) system and of circulating Dendritic Cells (DC) in vivo, and the ability of GBV-C to activate these functions in vitro. Activation of IFN system and of circulating DC was compared in GBV-positive and -negative HIV-1 co-infected patients with HAART-driven suppression of HIV-1 viremia. Endogenous levels of IFN-gamma and RNA-dependent protein kinase (PKR) mRNA were significantly higher in peripheral blood mononuclear cells (PBMC) from GBV-C-positive when compared to GBV-C-negative patients. IFN-gamma expression was correlated with all the Interferon response genes (IRGs) and with GBV-C viremia. The frequency of circulating plasmacytoid DC (pDC) expressing the CD80 activation marker was increased in GBV-C-positive patients, and was correlated with GBV-C viral load. In vitro experiments indicated that GBV-C is able to induce IFN-gamma expression in PBMC. In addition, in PBMC cultures GBV-C induced an increase of CD80 expression by pDC, that was reduced by antibody to IFN-gamma. Our data indicate that in HIV-positive patients GBV-C coinfection promotes the activation of IFN-gamma and downstream IRG expression, as well as with the activation/maturation of circulating pDC. GBV-C-driven IFN-gamma activation is, at least in part, responsible for the increased maturation of pDC. This crosstalk may suggest a role for GBV-C coinfection in boosting the innate antiviral response to HIV infection.

Intrahepatic Vγ9Vδ2 T-cells from HCV-infected patients show an exhausted phenotype but can inhibit HCV replication.

Hepatitis C virus (HCV) persistence results from inefficiencies of both innate and adaptive immune responses to eradicate the infection. A functional impairment of circulating Vγ9Vδ2 T-cells was described but few data are available on Vγ9Vδ2 T-cells in the liver that, however, represents the battlefield in the HCV/host interaction. Aim of this work was to compare circulating and intrahepatic Vγ9Vδ2 T-cells in chronic HCV-infected patients (HCVpos) and in HCV-negative (HCVneg) subjects. Phenotypic and functional analysis was performed by flow cytometry. Anti-HCV activity was analyzed by using an in vitro autologous liver culture system. Independently from HCV infection, the liver was enriched of Vγ9Vδ2 T-cells expressing an effector/activated phenotype. In contrast, an enrichment of PD-1 expressing Vγ9Vδ2 T-cells was observed both in the peripheral blood and in the liver of HCVpos patients, probably due to a persistent antigenic stimulation. Moreover, a lower frequency of IFN-γ producing Vγ9Vδ2 T-cells was observed in the liver of HCVpos patients, suggesting a functional impairment in the cytokine production in HCVpos liver. Despite this hypo-responsiveness, intrahepatic Vγ9Vδ2 T-cells are able to exert an anti-HCV activity after specific stimulation. Altogether, our data show that HCV infection induced a dysregulation of intrahepatic Vγ9Vδ2 T cells that maintain their anti-HCV activity after specific stimulation. A study aimed to evaluate the mechanisms of the antiviral activity may be useful to identify new pathways able to improve Vγ9Vδ2 T-cells intrahepatic function during HCV infection.

Mutant p53 gain of function: reduction of tumor malignancy of human cancer cell lines through abrogation of mutant p53 expression.

Mutations in the TP53 tumor suppressor gene are the most frequent genetic alteration in human cancers. These alterations are mostly missense point mutations that cluster in the DNA binding domain. There is growing evidence that many of these mutations generate mutant p53 proteins that have acquired new biochemical and biological properties. Through this gain of function activity, mutant p53 is believed to contribute to tumor malignancy. The purpose of our study was to explore mutant p53 as a target for novel anticancer treatments. To this aim, we inhibited mutant p53 expression by RNA interference in three different cancer cell lines endogenously expressing mutant p53 proteins, and evaluated the effects on the biological activities through which mutant p53 exerts gain of function. We found that depletion of mutant p53 reduces cell proliferation, in vitro and in vivo tumorigenicity, and resistance to anticancer drugs. Our results demonstrate that mutant p53 knocking down weakens the aggressiveness of human cancer cells, and provides further insight into the comprehension of mutant p53 gain of function activity in human tumor.

S100A2 gene is a direct transcriptional target of p53 homologues during keratinocyte differentiation.

The p53 paralogues p73, p63 and their respective truncated isoforms have been shown to be critical regulators of developmental and differentiation processes. Indeed, both p73- and p63-deficient mice exhibit severe developmental defects. Here, we show that S100A2 gene, whose transcript and protein are induced during keratinocyte differentiation of HaCaT cells, is a direct transcriptional target of p73beta and DeltaNp63alpha and is required for proper keratinocyte differentiation. Transactivation assays reveal that p73beta and DeltaNp63alpha exert opposite transcriptional effects on S100A2 gene. While DeltaNp63alpha is found in vivo onto S100A2 regulatory regions predominantly in proliferating cells, p73beta is recruited in differentiating cells. Silencing of p73 impairs the induction of S100A2 during the differentiation of HaCaT cells. Moreover, silencing of p73 or S100A2 impairs the proper expression of keratinocyte differentiation markers. Of note, p53 family members do not trigger S100A2 gene expression in response to apoptotic doses of cisplatin and doxorubicin.

Lonidamine induces apoptosis in drug-resistant cells independently of the p53 gene.

Lonidamine, a dichlorinated derivative of indazole-3-carboxylic acid, was shown to play a significant role in reversing or overcoming multidrug resistance. Here, we show that exposure to 50 microg/ml of lonidamine induces apoptosis in adriamycin and nitrosourea-resistant cells (MCF-7 ADR(r) human breast cancer cell line, and LB9 glioblastoma multiform cell line), as demonstrated by sub-G1 peaks in DNA content histograms, condensation of nuclear chromatin, and internucleosomal DNA fragmentation. Moreover, we find that apoptosis is preceded by accumulation of the cells in the G0/G1 phase of the cell cycle. Interestingly, lonidamine fails to activate the apoptotic program in the corresponding sensitive parental cell lines (ADR-sensitive MCF-7 WT, and nitrosourea-sensitive LI cells) even after long exposure times. The evaluation of bcl-2 protein expression suggests that this different effect of lonidamine treatment in drug-resistant and -sensitive cell lines might not simply be due to dissimilar expression levels of bcl-2 protein. To determine whether the lonidamine-induced apoptosis is mediated by p53 protein, we used cells lacking endogenous p53 and overexpressing either wild-type p53 or dominant-negative p53 mutant. We find that apoptosis by lonidamine is independent of the p53 gene.

Expression of E1A in terminally differentiated muscle cells reactivates the cell cycle and suppresses tissue-specific genes by separable mechanisms.

Terminally differentiated cells are characterized by permanent withdrawal from the cell cycle; they do not enter S phase even when stimulated by growth factors or retroviral oncogenes. We have shown, however, that the adenovirus E1A oncogene can reactivate the cell cycle in terminally differentiated cells. In this report, we describe the molecular events triggered by E1A in terminally differentiated skeletal muscle cells. We found that in myotubes infected with the adenovirus mutant dl520, 12S E1A bypasses the early G1 phase and activates the expression of late-G1 genes, such as the cyclin E and cyclin A genes, cdk2, PCNA, and B-myb. Of these, the cyclin E gene and cdk2 were significantly overexpressed in comparison with levels in proliferating, undifferentiated myoblasts. p130 and pRb were phosphorylated before the infected myotubes entered S phase, despite the high expression of the cyclin-dependent kinase inhibitor p21, and E2F was released. Our results suggest that one of the mechanisms that E1A uses to overcome the proliferative block of terminally differentiated cells involves coordinated overexpression of cyclin E and cdk2. Following E1A expression, the myogenic transcription factors MyoD and myogenin and the muscle-specific structural genes encoding muscle creatine kinase and myosin heavy chain were downregulated. The muscle regulatory factors were also silenced in myotubes infected with adenovirus E1A mutants incapable of reactivating the cell cycle in terminally differentiated muscle cells. Thus, the suppression of the differentiation program is not a consequence of cell cycle reactivation in myotubes, and it is induced by an independent mechanism. Our results show that E1A reactivates the cell cycle and suppresses tissue-specific gene expression in terminally differentiated muscle cells, thus causing dedifferentiation.

The insulin-like growth factor I receptor as a physiologically relevant target of p53 in apoptosis caused by interleukin-3 withdrawal.

The wild-type p53 protein is known to modulate apoptosis induced in 32D murine hemopoietic cells by interleukin-3 withdrawal. In 32D cells and in 32D cells constitutively expressing a temperature-sensitive mutant of p53 (32Dtsp53), overexpression of a wild-type (but not a mutant) insulin-like growth factor I receptor (IGF-IR) protects these cells from apoptosis. A tsp53 in its wild-type conformation causes a decrease in the levels of IGF-IRs, and this decrease is accompanied by increased sensitivity of these cells to apoptosis. However, when the expression of the IGF-IR cDNA is regulated by a viral promoter, IGF-IR levels are not decreased by a wild-type p53, and apoptosis does not occur. These findings show that, in 32Dtsp53 cells, the IGF-IR is a physiologically relevant target of p53 in the process of apoptosis.

The transcriptional repressor ZEB regulates p73 expression at the crossroad between proliferation and differentiation.

The newly discovered p73 gene encodes a nuclear protein that has high homology with p53. Furthermore, ectopic expression of p73 in p53(+/+) and p53(-/-) cancer cells recapitulates some of the biological activities of p53 such as growth arrest, apoptosis, and differentiation. p73(-/-)-deficient mice exhibit severe defects in proper development of the central nervous system and pheromone sensory pathway. They also suffer from inflammation and infections. Here we studied the transcriptional regulation of p73 at the crossroad between proliferation and differentiation. p73 mRNA is undetectable in proliferating C2C12 cells and is expressed at very low levels in undifferentiated P19 and HL60 cells. Conversely, it is upregulated during muscle and neuronal differentiation as well as in response to tetradecanoyl phorbol acetate-induced monocytic differentiation of HL60 cells. We identified a 1-kb regulatory fragment located within the first intron of p73, which is positioned immediately upstream to the ATG codon of the second exon. This fragment exerts silencer activity on p73 as well as on heterologous promoters. The p73 intronic fragment contains six consensus binding sites for transcriptional repressor ZEB, which binds these sites in vitro and in vivo. Ectopic expression of dominant-negative ZEB (ZEB-DB) restores p73 expression in proliferating C2C12 and P19 cells. Thus, transcriptional repression of p73 expression by ZEB binding may contribute to the modulation of p73 expression during differentiation.

Wild-type p53 induces diverse effects in 32D cells expressing different oncogenes.

Expression of exogenous wild-type (wt) p53 in different leukemia cell lines can induce growth arrest, apoptotic cell death, or cell differentiation. The hematopoietic cell lines that have been used so far to study wt p53 functions have in common the characteristic of not expressing endogenous p53. However, the mechanisms involved in the transformation of these cells are different, and the cells are at different stages of tumor progression. It can be postulated that each type of neoplastic cell offers a particular environment in which p53 might generate different effects. To test this hypothesis, we introduced individual oncogenes into untransformed, interleukin-3 (IL-3)-dependent myeloid precursor 32D cells to have a single transforming agent at a time. The effects induced by wt p53 overexpression were subsequently evaluated in each oncogene-expressing 32D derivative. We found that in not fully transformed, v-ras-expressing 32D cells, as already shown for the parental 32D cells, overexpression of the wt p53 gene caused no phenotypic changes and no reduction of the proliferative rate as long as the cells were maintained in their normal culture conditions (presence of IL-3 and serum). An accelerated rate of apoptosis was observed after IL-3 withdrawal. In contrast, in transformed, IL-3-independent 32D cells, wt p53 overexpression induced different effects. The v-abl-transformed cells manifested a reduction in growth rate, while the v-src-transformed cells underwent monocytic differentiation. These results show that the phenotype effects of wt p53 action(s) can vary as a function of the cellular environment.

Reconstitution of cyclin D1-associated kinase activity drives terminally differentiated cells into the cell cycle.

Terminal cell differentiation entails definitive withdrawal from the cell cycle. Although most of the cells of an adult mammal are terminally differentiated, the molecular mechanisms preserving the postmitotic state are insufficiently understood. Terminally differentiated skeletal muscle cells, or myotubes, are a prototypic terminally differentiated system. We previously identified a mid-G(1) block preventing myotubes from progressing beyond this point in the cell cycle. In this work, we set out to define the molecular basis of such a block. It is shown here that overexpression of highly active cyclin E and cdk2 in myotubes induces phosphorylation of pRb but cannot reactivate DNA synthesis, underscoring the tightness of cell cycle control in postmitotic cells. In contrast, forced expression of cyclin D1 and wild-type or dominant-negative cdk4 in myotubes restores physiological levels of cdk4 kinase activity, allowing progression through the cell cycle. Such reactivation occurs in myotubes derived from primary, as well as established, C2C12 myoblasts and is accompanied by impairment of muscle-specific gene expression. Other terminally differentiated systems as diverse as adipocytes and nerve cells are similarly reactivated. Thus, the present results indicate that the suppression of cyclin D1-associated kinase activity is of crucial importance for the maintenance of the postmitotic state in widely divergent terminally differentiated cell types.

Induction of hTERT expression and telomerase activity by estrogens in human ovary epithelium cells.

In mammals, molecular mechanisms and factors involved in the tight regulation of telomerase expression and activity are still largely undefined. In this study, we provide evidence for a role of estrogens and their receptors in the transcriptional regulation of hTERT, the catalytic subunit of human telomerase and, consequently, in the activation of the enzyme. Through a computer analysis of the hTERT 5'-flanking sequences, we identified a putative estrogen response element (ERE) which was capable of binding in vitro human estrogen receptor alpha (ERalpha). In vivo DNA footprinting revealed specific modifications of the ERE region in ERalpha-positive but not ERalpha-negative cells upon treatment with 17beta-estradiol (E2), indicative of estrogen-dependent chromatin remodelling. In the presence of E2, transient expression of ERalpha but not ERbeta remarkably increased hTERT promoter activity, and mutation of the ERE significantly reduced this effect. No telomerase activity was detected in human ovary epithelial cells grown in the absence of E2, but the addition of the hormone induced the enzyme within 3 h of treatment. The expression of hTERT mRNA and protein was induced in parallel with enzymatic activity. This prompt estrogen modulation of telomerase activity substantiates estrogen-dependent transcriptional regulation of the hTERT gene. The identification of hTERT as a target of estrogens represents a novel finding which advances the understanding of telomerase regulation in hormone-dependent cells and has implications for a potential role of hormones in their senescence and malignant conversion.

An IL-15 dependent CD8 T cell response to selected HIV epitopes is related to viral control in early-treated HIV-infected subjects.

In some early-treated HIV-positive patients, Structured Treatment Interruption (STI) is associated to spontaneous control of viral rebound. Thus, in this clinical setting, we analyzed the immunological parameters associated to viral control. Two groups of early treated patients who underwent STI were retrospectively defined, according to the ability to spontaneously control HIV replication (Controller and Non-controller). Plasma cytokine levels were analyzed by multiplex analysis. CD8 T cell differentiation was determined by polychromatic flow cytometry. Antigen-specific IFN-gamma production was analyzed by ELISpot and intracellular staining after stimulation with HIV-peptides. Long-term Elispot assays were performed in the presence or absence of IL-15. Plasma IL-15 was found decreased over a period of time in Non-Controller patients, whereas a restricted response to Gag (aa.167-202 and 265-279) and Nef (aa.86-100 and 111-138) immunodominant epitopes was more frequently observed in Controller patients. Interestingly, in two Non-Controller patients the CD8-mediated T cells response to immunodominant epitopes could be restored in vitro by IL-15, suggesting a major role of cytokine homeostasis on the generation of protective immunity. In early-treated HIV+ patients undergoing STI, HIV replication control was associated to CD8 T cell maturation and sustained IL-15 levels, leading to HIV-specific CD8 T cell responses against selected Gag and Nef epitopes.

Enhancement of tumor necrosis factor alpha antitumor immunotherapeutic properties by targeted delivery to aminopeptidase N (CD13).

The clinical use of tumor necrosis factor alpha (TNF) as an anticancer drug is limited to local treatments because of its dose-limiting systemic toxicity. We show here that murine TNF fused with CNGRC peptide (NGR-TNF), an aminopeptidase N (CD13) ligand that targets activated blood vessels in tumors, is 12-15 times more efficient than murine TNF in decreasing the tumor burden in lymphoma and melanoma animal models, whereas its toxicity is similar. Similarly, human NGR-TNF induced stronger antitumor effects than human TNF, even with 30 times lower doses. Coadministration of murine NGR-TNF with a CNGRC peptide or an anti-CD13 antibody markedly decreased its antitumor effects. Tumor regression, induced by doses of murine NGR-TNF lower than the LD50, was accompanied by protective immunity. In contrast, no cure was induced by TNF at any dose. These results suggest that targeted delivery of TNF to CD13 may enhance its immunotherapeutic properties. Moreover, these findings reveal the potential of tumor homing peptides to generate a new class of recombinant cytokines that compared to immunocytokines have a simpler structure, could be easier to produce and are potentially less immunogenic.