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1.
Nat Chem ; 2021 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-33833446

RESUMO

The encoding of chemical compounds with amplifiable DNA tags facilitates the discovery of small-molecule ligands for proteins. To investigate the impact of stereo- and regiochemistry on ligand discovery, we synthesized a DNA-encoded library of 670,752 derivatives based on 2-azido-3-iodophenylpropionic acids. The library was selected against multiple proteins and yielded specific ligands. The selection fingerprints obtained for a set of protein targets of pharmaceutical relevance clearly showed the preferential enrichment of ortho-, meta- or para-regioisomers, which was experimentally verified by affinity measurements in the absence of DNA. The discovered ligands included novel selective enzyme inhibitors and binders to tumour-associated antigens, which enabled conditional chimeric antigen receptor T-cell activation and tumour targeting.

2.
Mol Cancer Ther ; 2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33632875

RESUMO

Interleukin-15 (IL15) is an immunostimulatory cytokine which holds promises for cancer therapy, but its performance (alone or as partner for fusion proteins) has often been limited by suboptimal accumulation in the tumor and very rapid clearance from circulation. Most recently, the Sushi Domain (SD, the shortest region of IL15 receptor alpha, capable of binding to IL15) has been fused to IL15-based anti-cancer products in order to increase its biological activity. Here, we describe two novel antibody fusion proteins (termed F8-F8-IL15 and F8-F8-SD-IL15), specific to the alternatively-spliced EDA domain of fibronectin (a marker of tumor neoangiogenisis, expressed in the majority of solid and hematological tumors, but absent in normal healthy tissues) and featuring the F8 antibody in single-chain diabody format (with a short linker between VH and VL, thus allowing the domains to pair with the complementary ones of another chain). Unlike previously described fusions of the F8 antibody with human IL15, F8-F8-IL15 and F8-F8-SD-IL15 exhibited a preferential uptake in solid tumors, as evidenced by quantitative biodistribution analysis with radioiodinated protein preparations. Both products were potently active in vivo against mouse metastatic colon carcinomas and in sarcoma lesion in combination with targeted tumor necrosis factor. The results may be of clinical significance, as F8-F8-IL15 and F8-F8-SD-IL15 are fully-human proteins, which recognize the cognate tumor-associated antigen with identical affinity in mouse and man.

3.
Head Neck ; 2021 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-33511711

RESUMO

BACKGROUND: The aim of this Italian multicenter study was to evaluate the diagnostic performance of a minimally invasive method for the detection of oral squamous cell carcinoma (OSCC) based on 13-gene DNA methylation analysis in oral brushing samples. METHODS: Oral brushing specimens were collected in 11 oral medicine centers across Italy. Twenty brushing specimens were collected by each center, 10 from patients with OSCC, and 10 from healthy volunteers. DNA methylation analysis was performed in blindness, and each sample was determined as positive or negative based on a predefined cutoff value. RESULTS: DNA amplification failed in 4 of 220 (1.8%) samples. Of the specimens derived from patients with OSCC, 93.6% (103/110) were detected as positive, and 84.9% (90/106) of the samples from healthy volunteers were negative. CONCLUSION: These data confirmed the diagnostic performance of our novel procedure in a large cohort of brushing specimens collected from 11 different centers and analyzed in blindness.

4.
Exp Biol Med (Maywood) ; 246(8): 940-951, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33475433

RESUMO

Interleukin-9 is a cytokine with multiple functions, including the ability to activate group 2 innate lymphoid cells, which has been postulated to be therapeutically active in mouse models of arthritis. Similarly, interleukin-9 has been suggested to play an important role in tumor immunity. Here, we describe the cloning, expression, and characterization of three fusion proteins based on murine interleukin-9 and the F8 antibody, specific to the alternatively spliced EDA domain of fibronectin. EDA is strongly expressed in cancer and in various arthritic conditions, while being undetectable in the majority of healthy organs. Interleukin-9-based fusion proteins with an irrelevant antibody specific to hen egg lysozyme served as negative control in our study. The fusion proteins were characterized by quantitative biodistribution analysis in tumor-bearing mice using radioiodinated protein preparations. The highest tumor uptake and best tumor:organ ratios were observed for a format, in which the interleukin-9 moiety was flanked by two units of the F8 antibody in single-chain Fv format. Biological activity of interleukin-9 was retained when the payload was fused to antibodies. However, the targeted delivery of interleukin-9 to the disease site resulted in a modest anti-tumor activity in three different murine models of cancer (K1735M2, CT26, and F9), while no therapeutic benefit was observed in a collagen induced model of arthritis. Collectively, these results confirm the possibility to deliver interleukin-9 to the site of disease but cast doubts about the alleged therapeutic activity of this cytokine in cancer and arthritis, which has been postulated in previous publications.

5.
MAbs ; 12(1): 1836713, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33136526

RESUMO

Antibody-based delivery of bioactive molecules represents a promising strategy for the improvement of cancer immunotherapy. Here, we describe the generation and characterization of R6N, a novel fully human antibody specific to the alternatively spliced domain D of Tenascin C, which is highly expressed in the stroma of primary tumors and metastasis. The R6N antibody recognized its cognate tumor-associated antigen with identical specificity in mouse and human specimens. Moreover, the antibody was able to selectively localize to solid tumors in vivo as evidenced by immunofluorescence-based biodistribution analysis. Encouraged by these results, we developed a novel fusion protein (termed mIL12-R6N) consisting of the murine interleukin 12 fused to the R6N antibody in homodimeric tandem single-chain variable fragment arrangement. mIL12-R6N exhibited potent antitumor activity in immunodeficient mice bearing SKRC52 renal cell carcinoma, as well as in immunocompetent mice bearing SMA-497 glioma. The experiments presented in this work provide a rationale for possible future applications for the R6N antibody for the treatment of cancer patients.

6.
Nat Commun ; 11(1): 4410, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32879310

RESUMO

The hypothalamic suprachiasmatic (SCN) clock contains several neurochemically defined cell groups that contribute to the genesis of circadian rhythms. Using cell-specific and genetically targeted approaches we have confirmed an indispensable role for vasoactive intestinal polypeptide-expressing SCN (SCNVIP) neurons, including their molecular clock, in generating the mammalian locomotor activity (LMA) circadian rhythm. Optogenetic-assisted circuit mapping revealed functional, di-synaptic connectivity between SCNVIP neurons and dorsomedial hypothalamic neurons, providing a circuit substrate by which SCNVIP neurons may regulate LMA rhythms. In vivo photometry revealed that while SCNVIP neurons are acutely responsive to light, their activity is otherwise behavioral state invariant. Single-nuclei RNA-sequencing revealed that SCNVIP neurons comprise two transcriptionally distinct subtypes, including putative pacemaker and non-pacemaker populations. Altogether, our work establishes necessity of SCNVIP neurons for the LMA circadian rhythm, elucidates organization of circadian outflow from and modulatory input to SCNVIP cells, and demonstrates a subpopulation-level molecular heterogeneity that suggests distinct functions for specific SCNVIP subtypes.


Assuntos
Ritmo Circadiano/fisiologia , Neurônios/metabolismo , Núcleo Supraquiasmático , Animais , Mapeamento Encefálico , Relógios Circadianos/fisiologia , Locomoção/fisiologia , Camundongos , Optogenética/métodos , Núcleo Supraquiasmático/citologia , Núcleo Supraquiasmático/metabolismo
7.
Nat Commun ; 11(1): 2769, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32488015

RESUMO

During obstructive sleep apnea, elevation of CO2 during apneas contributes to awakening and restoring airway patency. We previously found that glutamatergic neurons in the external lateral parabrachial nucleus (PBel) containing calcitonin gene related peptide (PBelCGRP neurons) are critical for causing arousal during hypercapnia. However, others found that genetic deletion of serotonin (5HT) neurons in the brainstem also prevented arousal from hypercapnia. To examine interactions between the two systems, we showed that dorsal raphe (DR) 5HT neurons selectively targeted the PBel. Either genetically directed deletion or acute optogenetic silencing of DRSert neurons dramatically increased the latency of mice to arouse during hypercapnia, as did silencing DRSert terminals in the PBel. This effect was mediated by 5HT2a receptors which are expressed by PBelCGRP neurons. Our results indicate that the serotonergic input from the DR to the PBel via 5HT2a receptors is critical for modulating the sensitivity of the PBelCGRP neurons that cause arousal to rising levels of blood CO2.


Assuntos
Nível de Alerta/fisiologia , Núcleo Dorsal da Rafe/metabolismo , Hipercapnia/metabolismo , Neurônios Serotoninérgicos/metabolismo , Animais , Tronco Encefálico/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Dióxido de Carbono , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Transgênicos , Optogenética , Núcleos Parabraquiais , Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética
8.
Dent J (Basel) ; 8(2)2020 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-32403226

RESUMO

The aim of this case report was to evaluate the use of Partsch I cystotomy in order to preserve a dental implant located in an odontogenic cyst extended from 3.2 to 4.4. A 50 year-old woman showed a circular, well-defined unilocular radiolucent area, Ø2.5 cm, in the right mandibular region with an oral implant intruding inside it. The overdenture in the mandibular right site showed no clinical mobility. The authors decided to perform a surgical treatment aimed to preserve the implant. The patient underwent Partsch I surgery followed by iodoform gauze insertion replaced weekly for one month, revision of the previous orthograde endodontic treatments, and an acrylic resin obturator prosthesis application for the following two months. The twelve month follow-up showed no clinical mobility of the right lateral mandibular implant prostheses. Radiographical analysis revealed cystic lesion healing and perimplant bone regeneration. This report highlights the opportunity to apply cystotomy when the cyst involves a dental implant and undermines its stability. This possibility is offered by the peculiar clinical scenario where the implant was stabilized by the presence of a previous prosthetic fixation. Our study led to the application of an operative protocol that allowed for the preservation of the implant.

9.
Anticancer Drugs ; 31(8): 799-805, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32304410

RESUMO

Antibody-cytokine fusion proteins (also called 'immunocytokines') represent an emerging class of biopharmaceutical products, which are being considered for cancer immunotherapy. When used as single agents, pro-inflammatory immunocytokines are rarely capable of inducing complete and durable cancer regression in mouse models and in patients. However, the combination treatment with conventional chemotherapy or with other immune-stimulatory agents typically increases the therapeutic efficacy of immunocytokines. In this article, we describe combination treatments of a tumor-targeting antibody-cytokine fusion protein based on the L19 antibody (specific to a splice isoform of fibronectin) fused to murine tumor necrosis factor with standard chemotherapy (dacarbazine, trabectedin or melphalan) or with an immune check-point inhibitor (anti-PD-1) in a BALB/c derived immunocompetent murine model of sarcoma (WEHI-164). All combination treatments led to improved tumor remission compared to single-agent treatments, suggesting that these combination partners may be suitable for further clinical development in sarcoma patients.

10.
J Control Release ; 317: 282-290, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31790729

RESUMO

Antibody-cytokine fusion proteins are being considered as biopharmaceuticals for cancer immunotherapy. Tumor-homing cytokine fusions typically display an improved therapeutic activity compared to the corresponding unmodified cytokine products, but toxicity profiles at equivalent doses are similar, since side effects are mainly driven by the cytokine concentration in blood. In order to explore avenues to harness the therapeutic potential of antibody-cytokine fusions while decreasing potential toxicity, we compared bolus and fractionated administration modalities for two tumor-targeting antibody-cytokine fusion proteins based on human interleukin-2 (IL2) and murine tumor necrosis factor (TNF) (i.e., L19-hIL2 and L19-mTNF) in two murine immunocompetent mouse models of cancer (F9 and C51). A comparative quantitative biodistribution analysis with radio-labeled protein preparations revealed that a fractionated administration of L19-hIL2 could deliver comparable product doses to the tumor with decreased product concentration in blood and normal organs, compared to bolus injection. By contrast, L19-mTNF (a product that causes a selective vascular shutdown in the tumor) accumulated most efficiently after bolus injection. Fractionated schedules allowed the safe administration of a cumulative dose of L19-mTNF, which was 2.5-times higher than the lethal dose for bolus injection. Dose fractionation led to a prolonged tumor growth inhibition for F9 teratocarcinomas, but not for C51 colorectal tumors, which responded best to bolus injection. Thus, dose fractionation may have different outcomes for the same antibody-cytokine product in different biological contexts.

11.
Mol Ther Nucleic Acids ; 19: 267-277, 2020 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-31855835

RESUMO

MicroRNAs (miRNAs) have increasingly been shown to be involved in human cancer, and interest has grown about the potential use of miRNAs for cancer therapy. miRNA levels are known to be altered in cancer cells, including in non-small cell lung cancer (NSCLC), a subtype of lung cancer that is the most prevalent form of cancer worldwide and that lacks effective therapies. The let-7 miRNA is involved in the regulation of oncogene expression in cells and directly represses cancer growth in the lung. let-7 is therefore a potential molecular target for tumor therapy. However, applications of RNA interference for cancer research have been limited by a lack of simple and efficient methods to deliver oligonucleotides (ONs) to cancer cells. In this study, we have used in vitro and in vivo approaches to show that HCC827 cells internalize hydrophobically modified let-7b miRNAs (hmiRNAs) added directly to the culture medium without the need for lipid formulation. We identified functional let-7b hmiRNAs targeting the HMGA2 mRNA, one of the let-7 target genes upregulated in NSCLC, and show that direct uptake in HCC827 cells induced potent and specific gene silencing in vitro and in vivo. Thus, hmiRNAs constitute a novel class of ONs that enable functional studies of genes involved in cancer biology and are potentially therapeutic molecules.

12.
Front Oncol ; 9: 1228, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31799191

RESUMO

Certain cytokines synergize in activating anti-cancer immunity at the site of disease and it may be desirable to generate biopharmaceutical agents, capable of simultaneous delivery of cytokine pairs to the tumor. In this article, we have described the cloning, expression and characterization of IL2-XE114-TNFmut, a dual-cytokine biopharmaceutical featuring the sequential fusion of interleukin-2 (IL2) with the XE114 antibody in scFv format and a tumor necrosis factor mutant (TNFmut). The fusion protein recognized the cognate antigen (carbonic anhydrase IX, a marker of hypoxia and of renal cell carcinoma) with high affinity and specificity. IL2-XE114-TNFmut formed a stable non-covalent homotrimeric structure, displayed cytokine activity in in vitro tests and preferentially localized to solid tumors in vivo. The product exhibited a partial growth inhibition of murine CT26 tumors transfected for carbonic anhydrase IX. When administered to Cynomolgus monkey as intravenous injection, IL2-XE114-TNFmut showed the expected plasma concentration of ~1,500 ng/ml at early time points, indicating the absence of any in vivo trapping events, and a half-life of ~2 h. IL2-XE114-TNFmut may thus be considered as a promising biopharmaceutical for the treatment of metastatic clear-cell renal cell carcinoma, since these tumors are known to be sensitive to IL2 and to TNF.

13.
Curr Biol ; 29(24): 4155-4168.e5, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31761703

RESUMO

Among the neuronal populations implicated in sleep-wake control, the ventrolateral preoptic (VLPO) nucleus has emerged as a key sleep-promoting center. However, the synaptic drives that regulate the VLPO to control arousal levels in vivo have not to date been identified. Here, we show that sleep-promoting galaninergic neurons within the VLPO nucleus, defined pharmacologically and by single-cell transcript analysis, are postsynaptic targets of lateral hypothalamic GABAergic (LHGABA) neurons and that activation of this pathway in vivo rapidly drives wakefulness. Ca2+ imaging from LHGABA neurons indicate that they are both wake and rapid eye movement (REM)-sleep active. Consistent with the potent arousal-promoting property of the LHGABA → VLPO pathway, presynaptic inputs to LHGABA neurons originate from several canonical stress- and arousal-related network nodes. This work represents the first demonstration that direct synaptic inhibition of the VLPO area can suppress sleep-promoting neurons to rapidly promote arousal.


Assuntos
Área Pré-Óptica/metabolismo , Sono/fisiologia , Vigília/fisiologia , Animais , Nível de Alerta/fisiologia , Encéfalo/fisiologia , Eletroencefalografia/métodos , Feminino , Neurônios GABAérgicos/metabolismo , Região Hipotalâmica Lateral/fisiologia , Hipotálamo/fisiologia , Masculino , Camundongos , Neurônios/fisiologia , Área Pré-Óptica/fisiologia , Transtornos do Sono do Ritmo Circadiano/fisiopatologia
14.
J Neurosci ; 39(45): 8929-8939, 2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31548232

RESUMO

The histaminergic neurons of the tuberomammillary nucleus (TMNHDC) of the posterior hypothalamus have long been implicated in promoting arousal. More recently, a role for GABAergic signaling by the TMNHDC neurons in arousal control has been proposed. Here, we investigated the effects of selective chronic disruption of GABA synthesis (via genetic deletion of the GABA synthesis enzyme, glutamic acid decarboxylase 67) or GABAergic transmission (via genetic deletion of the vesicular GABA transporter (VGAT)) in the TMNHDC neurons on sleep-wake in male mice. We also examined the effects of acute chemogenetic activation and optogenetic inhibition of TMNHDC neurons upon arousal in male mice. Unexpectedly, we found that neither disruption of GABA synthesis nor GABAergic transmission altered hourly sleep-wake quantities, perhaps because very few TMNHDC neurons coexpressed VGAT. Acute chemogenetic activation of TMNHDC neurons did not increase arousal levels above baseline but did enhance vigilance when the mice were exposed to a behavioral cage change challenge. Similarly, acute optogenetic inhibition had little effect upon baseline levels of arousal. In conclusion, we could not identify a role for GABA release by TMNHDC neurons in arousal control. Further, if TMNHDC neurons do release GABA, the mechanism by which they do so remains unclear. Our findings support the view that TMNHDC neurons may be important for enhancing arousal under certain conditions, such as exposure to a novel environment, but play only a minor role in behavioral and EEG arousal under baseline conditions.SIGNIFICANCE STATEMENT The histaminergic neurons of the tuberomammillary nucleus of the hypothalamus (TMNHDC) have long been thought to promote arousal. Additionally, TMNHDC neurons may counter-regulate the wake-promoting effects of histamine through co-release of the inhibitory neurotransmitter, GABA. Here, we show that impairing GABA signaling from TMNHDC neurons does not impact sleep-wake amounts and that few TMNHDC neurons contain the vesicular GABA transporter, which is presumably required to release GABA. We further show that acute activation or inhibition of TMNHDC neurons has limited effects upon baseline arousal levels and that activation enhances vigilance during a behavioral challenge. Counter to general belief, our findings support the view that TMNHDC neurons are neither necessary nor sufficient for the initiation and maintenance of arousal under baseline conditions.


Assuntos
Nível de Alerta , Histamina/metabolismo , Região Hipotalâmica Lateral/fisiologia , Neurônios/metabolismo , Ácido gama-Aminobutírico/metabolismo , Potenciais de Ação , Animais , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Região Hipotalâmica Lateral/citologia , Região Hipotalâmica Lateral/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Sono , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo
15.
Oral Oncol ; 90: 141-144, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30638760

RESUMO

Solitary fibrous tumor is an uncommon neoplasm with unpredictable clinical behavior. Malignant solitary fibrous tumor is a rare morphological variant with more aggressive behavior and higher rates of local recurrences and distant metastasis, exceeding rare in oral cavity; our case occurred in the floor of the mouth in the sublingual gland.


Assuntos
Tumores Fibrosos Solitários/diagnóstico , Tumores Fibrosos Solitários/cirurgia , Neoplasias da Glândula Sublingual/diagnóstico , Neoplasias da Glândula Sublingual/cirurgia , Antígeno 12E7/metabolismo , Adulto , Antígenos CD34/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Margens de Excisão , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Retinal Desidrogenase/metabolismo , Resultado do Tratamento
16.
J Biotechnol ; 291: 17-25, 2019 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-30586544

RESUMO

Antibody-cytokine fusion proteins are a class of biopharmaceuticals, with the potential to modulate the activity of the immune system at the site of disease. The molecular format used to connect antibody moiety and cytokine payload can have a profound influence on biological activity and pharmacokinetic properties. The optimization of fusion protein format is particularly challenging for heterodimeric cytokines, since various molecular arrangements can be considered. In this article, we have explored the role of linker in a tumor-targeting IL12 fusion protein, based on the L19 antibody, specific to the extra-domain B of fibronectin. In biodistribution studies performed in tumor-bearing mice using radioiodinated protein preparations, fusion of human IL12 at the N-terminus of the L19 antibody in tandem-diabody format led to higher tumor uptake and improved tumor-to-organ ratios, compared to a similar fusion protein featuring L19 in IgG1 format. Moreover, optimization of the amino acid composition in eight variants of the linker connecting the IL12 moiety to the tandem-diabody revealed that a 15-amino acid linker (GSADGGSSAGGSDAG) displayed the best tumor targeting characteristics, with a long residence time at the tumor site and a rapid clearance from blood and normal organs. The product is being developed for industrial and clinical applications.


Assuntos
Anticorpos/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Interleucina-12/uso terapêutico , Neoplasias/tratamento farmacológico , Peptídeos/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Anticorpos/genética , Células CHO , Linhagem Celular Tumoral , Cricetulus , Feminino , Interleucina-12/genética , Camundongos , Peptídeos/genética
17.
Neuropharmacology ; 143: 327-338, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30219501

RESUMO

Histaminergic (HA) neurons located in the tuberomamillary nucleus (TMN) of the posterior hypothalamus fire exclusively during waking and support many physiological functions. We investigated the role of the endovanilloid N-oleoyldopamine (OLDA) in TMN, where dopamine synthesis and its conjugation with oleic acid likely occur. We show that several known targets of OLDA including TRPV1 and cannabinoid receptors are expressed in HA neurons. In contrast to capsaicin, which failed to increase firing of HA neurons in TRPV1 knockout mice (TRPVI KO), OLDA was still able to induce excitation. This excitation was not sensitive to the blockade of cannabinoid receptors 1 and 2 and could result from OLDA interaction with GPR119, as the ligand of GPR119, oleoylethanolamide (OEA), also increased the firing of HA neurons. However, we ruled out this possibility as OEA- (but not OLDA-) excitation was abolished by the PPAR (peroxisome proliferator activated receptor) alpha antagonist MK886. The dopamine uptake blocker nomifensine blanked OLDA-excitation and dopamine receptor antagonists abolished the OLDA action in TRPV1 KO mice. Therefore OLDA excites HA neurons through multiple targets suggesting a central role of the histaminergic system in the behavioral stimulation seen after systemic OLDA application.


Assuntos
Dopamina/análogos & derivados , Histamina/metabolismo , Região Hipotalâmica Lateral/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurotransmissores/farmacologia , Animais , Dopamina/farmacologia , Região Hipotalâmica Lateral/citologia , Região Hipotalâmica Lateral/crescimento & desenvolvimento , Região Hipotalâmica Lateral/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/citologia , Neurônios/metabolismo , Técnicas de Patch-Clamp , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Técnicas de Cultura de Tecidos
18.
Cancer Immunol Immunother ; 67(9): 1381-1391, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29971465

RESUMO

We have recently described a novel therapeutic antibody product (IL2-F8-TNFmut), featuring the simultaneous fusion of murine IL2 and of a TNF mutant with scFv(F8), an antibody specific to the alternatively-spliced extra domain A of fibronectin (EDA). Here, we report on the in vivo characterization of the anti-cancer activity of IL2-F8-TNFmut in four immunocompetent murine models of cancer, CT26, WEHI-164, F9 teratocarcinoma and Lewis lung carcinoma (LLC), using the product alone or in combination with a monoclonal antibody specific to murine PD-L1. All four models exhibited a strong expression of EDA-fibronectin, which was confined to vascular structures for F9 tumors, while the other three malignancies exhibited a more stromal pattern of staining. A complete and long-lasting tumor eradication of CT26 and WEHI-164 tumors was observed in BALB/c mice when IL2-F8-TNFmut was used in combination with PD-L1 blockade. The combination treatment led to improved tumor growth inhibition in 129/SvEv mice bearing murine teratocarcinoma or in C57BL/6 mice bearing murine LLC, but those cancer cures were difficult to achieve in those models. A microscopic analysis of tumor sections, obtained 24 h after pharmacological treatment, revealed that the PD-L1 antibody had homogenously reached tumor cells in vivo and that the combination of PD-L1 blockade with IL2-F8-TNFmut stimulated an influx of NK cells and of T cells into the neoplastic mass. These data indicate that potency-matched dual-cytokine fusion proteins may be ideally suited to potentiate the therapeutic activity of immune check-point inhibitors.


Assuntos
Antígeno B7-H1/antagonistas & inibidores , Imunotoxinas/farmacologia , Interleucina-2/farmacologia , Neoplasias Experimentais/terapia , Proteínas Recombinantes de Fusão/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Antígeno B7-H1/imunologia , Feminino , Fibronectinas/imunologia , Imunotoxinas/genética , Imunotoxinas/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Cadeia Única/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
19.
Protein Eng Des Sel ; 31(5): 173-179, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29982719

RESUMO

A novel dual-cytokine-antibody fusion protein, consisting of an antibody directed against CD38 [a tumor-associated antigen mainly expressed on the surface of multiple myeloma (MM) cells], simultaneously fused to both tumor necrosis factor ligand superfamily member 10 (TRAIL) and interleukin-2 (IL2), was designed, expressed and purified to homogeneity. The novel fusion protein, termed IL2-αCD38-αCD38-scTRAIL, was able to selectively recognize its cognate antigen expressed on the surface of MM and lymphoma cell lines, as evidenced by flow cytometry analysis. Moreover, the targeted version of TRAIL was able to induce cancer cell death in vitro, both with MM cell lines and with fresh isolates from the bone marrow of MM patients. The experiments provide a rationale for possible future applications of IL2-αCD38-αCD38-scTRAIL for the treatment of patients with MM or other CD38-positive malignancies.


Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Citocinas/metabolismo , Imunoconjugados/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , ADP-Ribosil Ciclase 1/imunologia , Linhagem Celular Tumoral , Humanos , Imunoconjugados/genética , Imunoconjugados/imunologia , Imunoconjugados/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo
20.
Ann Ist Super Sanita ; 54(2): 90-95, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29916412

RESUMO

INTRODUCTION: Ethanol is the most widely used drug worldwide. Its consumption has been increasing, and it is reported even during childbearing. Prenatal exposure to ethanol can lead to irreversible damages of the fetus. Knowledge about this risk could prevent these damages. There is no information about knowledge of the Italian students on this issue. METHODS: Therefore the aim of this study was to describe the awareness of the Italian students attending the last year of secondary school about risk of gestational alcohol drinking for the delivering mother and the fetus. An online multiple-choice anonymous survey for students was used and e-mailed to the all Italian secondary schools. RESULTS: The respondents were 9.921 and the obtained results evidenced that that young females are more informed than males, and students in Northern and Central Italy are better informed than those in the South, especially on general aspects. The most of respondents knows that alcohol consumption during pregnancy can damage the fetus. However, many youngsters failed to translate this belief into the practice. CONCLUSIONS: In conclusion, interventions are needed to enhance knowledge and prevent these damages, and health professionals, with nurses in the first row are entitled to provide education on this topic.


Assuntos
Consumo de Bebidas Alcoólicas/efeitos adversos , Gravidez , Adolescente , Estudos Transversais , Feminino , Transtornos do Espectro Alcoólico Fetal , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Itália , Masculino , Assunção de Riscos , Instituições Acadêmicas , Fatores Sexuais , Estudantes , Inquéritos e Questionários
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