PTX3 binds MD-2 and promotes TRIF-dependent immune protection in aspergillosis.

The long pentraxin 3 (PTX3) modulates different effector pathways involved in innate resistance to Aspergillus fumigatus, including complement activation or promotion of phagocytosis by interacting with FcγRs. However, whether and how TLRs modulate PTX3 mediates antifungal resistance is not known. In this study, we demonstrate that PTX3 binds myeloid differentiation protein 2 (MD-2) in vitro and exerts its protective antifungal activity in vivo through TLR4/MD-2-mediated signaling. Similar to Tlr4(-/-) mice, Md2(-/-) mice displayed high susceptibility to pulmonary aspergillosis, a phenotype associated with a proinflammatory cytokine profile and impaired antifungal activity of polymorphonuclear neutrophils. Treating Md2(-/-) mice with PTX3 failed to confer immune protection against the fungus, whereas adoptive transfer of MD-2-competent polymorphonuclear neutrophils restored it. Mechanistically, engagement of MD-2 by PTX3-opsonized Aspergillus conidia activated the TLR4/Toll/IL-1R domain-containing adapter inducing IFN-ß-dependent signaling pathway converging on IL-10. Thus, we have identified a novel receptor mechanism, involving the TLR4/MD-2/Toll/IL-1R domain-containing adapter inducing IFN-ß-mediated signaling, whereby PTX3 elicits antifungal resistance with limited immunopathology in A. fumigatus infection.

Efficacy of PTX3 and posaconazole combination in a rat model of invasive pulmonary aspergillosis.

Posaconazole is currently used for the prophylaxis of invasive pulmonary aspergillosis (IPA). Limitations to posaconazole usage are drug-drug interactions and side effects. PTX3 is an innate immunity glycoprotein with opsonic activity, proven to be protective in IPA animal models. This study investigated the combination of posaconazole with PTX3. The results indicate synergy between PTX3 and posaconazole against aspergillosis, suggesting that a combination of reduced doses of posaconazole with the immune response enhancer PTX3 might represent a treatment option with a higher therapeutic index than posaconazole.

Anti-proliferative effect of a triazole derivative (ST1959) on LNCaP human prostate cancer cells through down-regulation of cyclin and androgen receptor expression.

BACKGROUND: Previous studies demonstrated that ST1959, a triazole derivative endowed with immunomodulatory activities, also exerts inhibitory effects on proliferation and survival of a panel of tumor cells. In this study, we sought to ascertain the effects of ST1959 on the growth of androgen-dependent and androgen-independent prostate cancer (PCa) cells. METHODS: The growth of androgen-dependent (LNCaP) and androgen-independent (PC3, DU-145) cells was analyzed in vitro both in the presence and absence of ST1959. Modulation of cyclin and androgen receptor (AR) expression following treatment with ST1959 was analyzed by Western blot and cytofluorimetric analysis. RESULTS: We observed that ST1959 causes a significant growth inhibition of LNCaP cells without affecting proliferation of androgen-insensitive DU-145 and PC3 cell lines. These effects were associated with G0/G1 cell cycle arrest and down-regulation of cyclin D1, A and B and AR expression. CONCLUSIONS: Our present findings indicate that the anti-proliferative activity of ST1959 on cell growth of androgen-dependent LNCaP PCa cells may be brought about by decreasing expression of functional AR and selected cyclins, ultimately leading to cell growth inhibition.

Efficacy of a nanocochleate-encapsulated 3,5-diaryl-s-triazole derivative in a murine model of graft-versus-host disease.

We have previously demonstrated that the compound 3-(2-ethylphenyl)-5-(3-methoxyphenyl)-1H-1,2,4-triazole exerts immunosuppressive effects in several experimental models of autoimmunity. These results were achieved by subcutaneously administering ST1959 after dissolution in an oily vehicle, because of its poor water solubility. To circumvent this problem, we sought to determine whether nanocochleate technology could be successfully exploited to deliver ST1959 and protect mice undergoing lethal acute graft-versus-host disease (GVHD). Orally-administered encochleated ST1959 significantly protected animals from lethality, resulting in survival rates of 57% and 100% at doses of 2 and 10 mg/kg, respectively, whereas oral administration of 2 mg/kg ST1959, mixed with empty nanocochleates, was completely inactive. Increased survival was associated with diminished serum chemokine levels and donor CD8+ T cells in the spleen of ST1959-treated mice. Moreover, ST1959 treatment significantly counteracted GVHD-induced normocitic anemia by increasing hemoglobin, hematocrit, platelet, and red and white blood cell counts. Overall, these data show that orally-administered encochleated ST1959 significantly protects mice from GVHD.

Pivotal Advance: Inhibition of MyD88 dimerization and recruitment of IRAK1 and IRAK4 by a novel peptidomimetic compound.

MyD88 is an adaptor protein, which plays an essential role in the intracellular signaling elicited by IL-1R and several TLRs. Central to its function is the ability of its Toll/IL-1R translation initiation region (TIR) domain to heterodimerize with the receptor and to homodimerize with another MyD88 molecule to favor the recruitment of downstream signaling molecules such as the serine/threonine kinases IL-1R-associated kinase 1 (IRAK1) and IRAK4. Herein, we have synthesized and tested the activity of a synthetic peptido-mimetic compound (ST2825) modeled after the structure of a heptapeptide in the BB-loop of the MyD88-TIR domain, which interferes with MyD88 signaling. ST2825 inhibited MyD88 dimerization in coimmunoprecipitation experiments. This effect was specific for homodimerization of the TIR domains and did not affect homodimerization of the death domains. Moreover, ST2825 interfered with recruitment of IRAK1 and IRAK4 by MyD88, causing inhibition of IL-1beta-mediated activation of NF-kappaB transcriptional activity. After oral administration, ST2825 dose-dependently inhibited IL-1beta-induced production of IL-6 in treated mice. Finally, we observed that ST2825 suppressed B cell proliferation and differentiation into plasma cells in response to CpG-induced activation of TLR9, a receptor that requires MyD88 for intracellular signaling. Our results indicate that ST2825 blocks IL-1R/TLR signaling by interfering with MyD88 homodimerization and suggest that it may have therapeutic potential in treatment of chronic inflammatory diseases.

Low and high tenascin-expressing tumors are efficiently targeted by ST2146 monoclonal antibody.

ST2146biot is a biotinylated anti-tenascin monoclonal antibody (mAb) to be used for Pretargeted Antibody Guided Radioimmunotherapy (PAGRIT) of solid tumors. In vivo biodistribution studies of (125)I-labeled ST2146biot were done in nude mice transplanted with human HT-29 colon carcinoma and/or human U-118MG glioblastoma cells characterized for low and high tenascin expression, respectively. In vitro results show that ST2146 retains immunoreactivity upon biotinylation, in contrast to other anti-tenascin mAbs. In vivo biodistribution of ST2146 shows specific tumor accumulation up to 10 days after the i.v. injection, with no relevant differences between biotinylated and nonbiotinylated ST2146. A dose of 4 microg/mouse saturates the low tenascin-expressing human colon carcinoma HT-29, whereas the high tenascin-expressing human glioblastoma U-118MG seems to be saturated at a ST2146biot dose between 320 and 640 microg/mouse. The percentage of injected dose per gram of tumor ranges from 10% to 30%, corresponding to an amount of ST2146biot/g of tumor of approximately 400 ng/g and >200 microg/g for HT-29 and U-118MG, respectively. Tumor to normal organs uptake ratios are between 15 and 60, confirming high tumor selectivity of ST2146biot despite its cross-reactivity with the tenascin expressed at low level in the normal mouse organs. The ST2146biot localization data are substantially confirmed even when both low and high tenascin-expressing tumors are implanted in the same animal. To our knowledge, the absolute amount of ST2146biot, specifically localized in xenotransplanted human tumors, is the highest thus far described and supports the clinical use of this mAb in PAGRIT(R).

Dissecting drug and vehicle metabolic effects in rats by a metabonomic approach.

A combined application of high resolution (1)H NMR spectroscopy and multivariate statistical techniques focused on establishing a consistent statistical approach to metabonomic studies was tested. The data reduction, which is preliminary to the application of multivariate analysis to NMR spectra, was carried out by means of two complementary methods: pure Pattern Recognition (PR) and Assigned Signal Analysis (ASA). The simultaneous use of both approaches allowed us to obtain additional information in the analysis of metabonomic data, compared to the use of PR alone. This additional information consists in the possibility of a biochemical interpretation of the effects induced by treatment with xenobiotics, such as drugs or drug vehicles, on the metabolic networks of the systems under investigation. This approach allowed us to ascertain that a single-dose treatment with ST1959 vehicled by Sesame oil affects the production of hepatic glucose associated to an increment of the amino acid ketogenic process.

Efficacy of aerosol therapy of lung cancer correlates with EGFR paralysis induced by AvidinOX-anchored biotinylated Cetuximab.

Lung cancer, as well as lung metastases from distal primary tumors, could benefit from aerosol treatment. Unfortunately, because of lung physiology, clearance of nebulized drugs is fast, paralleled by unwanted systemic exposure. Here we report that nebulized AvidinOX can act as an artificial receptor for biotinylated drugs. In nude and SCID mice with advanced human KRAS-mutated A549 metastatic lung cancer, pre-nebulization with AvidinOX enables biotinylated Cetuximab to control tumor growth at a dose lower than 1/25,000 the intravenous effective dose. This result correlates with a striking, specific and unpredictable effect of AvidinOX-anchored biotinylated Cetuximab, as well as Panitumumab, observed on a panel of tumor cell lines, leading to inhibition of dimerization and signalling, blockade of endocytosis, induction of massive lysosomal degradation and abrogation of nuclear translocation of EGFR. Excellent tolerability, together with availability of pharmaceutical-grade AvidinOX and antibodies, will allow rapid clinical translation of the proposed therapy.

Comparative activity of Sant7 and anti-IL-6, IL-6R monoclonal antibodies in a murine model of B-cell lymphoma.

Interleukin-6 (IL-6) plays a central role in the pathogenesis of several autoimmune and inflammatory diseases as well as B-cell lymphoproliferative disorders. This work describes the effects of the recombinant or adenovirally-delivered IL-6 superantagonist Sant7, anti-IL-6 and IL-6 receptor monoclonal antibodies in a severe murine model of human B-cell lymphoma induced in SCID mice by transplantation of an LCL-41 cell line variant (isotype-switched IgM>IgG). Survival of 60% of the animals treated with anti-gp130 was observed up to day 33, while about 20% of the animals survived with anti-gp80 and Sant7 treatment. No survival was observed with the anti-IL-6 monoclonal antibody treatment. No significant change in serum and peritoneal levels of human IL-6 (hIL-6) and soluble human IL-6 receptor (shIL-6R) was observed in the recombinant Sant7-treated group towards the control group. The anti-gp80 monoclonal antibody induced significant increase of both hIL-6R and hIL-6 in serum and peritoneum. The anti-gp130 monoclonal antibody treatment determined a reduction of the seric shIL-6R and a significant increase of the seric hIL-6. Anti-IL-6 monoclonal antibody administration resulted in a reduction of serum and in an increase of peritoneal hIL-6. Treatment with adenoviral Sant7 was associated with a reduction of circulating shIL-6R, hIgG and mSAP. However, only marginal anti-tumor efficacy of the adenoviral Sant7 was observed. Overall, the present data suggest a potential for anti-hIL-6 therapy in B-cell lymphomas. Less severe animal models might be useful to better evaluate Sant7 efficacy alone or in combination with other anti-IL-6 therapeutics.