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1.
Eur J Pharm Biopharm ; 174: 111-130, 2022 May.
Article in English | MEDLINE | ID: mdl-35378278

ABSTRACT

Mycobacterium tuberculosis is an intracellular pathogen and the uptake of the antimycobacterial compounds by host cells is limited. Novel antimycobacterials effective against intracellular bacteria are needed. New N-substituted derivatives of 4-aminosalicylic acid have been designed and evaluated. To achieve intracellular efficacy and selectivity, these compounds were conjugated to tuftsin peptides via oxime or amide bonds. These delivery peptides can target tuftsin- and neuropilin receptor-bearing cells, such as macrophages and various other cells of lung origin. We have demonstrated that the in vitro antimycobacterial activity of the 4-aminosalicylic derivatives against M. tuberculosis H37Rv was preserved in the peptide conjugates. The free drugs were ineffective on infected cells, but the conjugates were active against the intracellular bacteria and have the selectivity on various types of host cells. The intracellular distribution of the carrier peptides was assessed, and the peptides internalize and display mainly in the cytosol in a concentration-dependent manner. The penetration ability of the most promising carrier peptide OT5 was evaluated using Transwell-inserts and spheroids. The pentapeptide exhibited time- and concentration-dependent penetration across the non-contact monolayers. Also, the pentapeptide has a fair penetration rate towards the center of spheroids formed of EBC-1 cells.


Subject(s)
Aminosalicylic Acid , Mycobacterium tuberculosis , Tuftsin , Aminosalicylic Acid/pharmacology , Anti-Bacterial Agents/pharmacology , Antitubercular Agents/chemistry , Antitubercular Agents/pharmacology , Excipients/pharmacology , Microbial Sensitivity Tests , Peptides/chemistry , Tuftsin/chemistry , Tuftsin/pharmacology
2.
Acta Biochim Pol ; 68(3): 449-455, 2021 Aug 30.
Article in English | MEDLINE | ID: mdl-34460213

ABSTRACT

Search for new and efficient antibiotic is crucial because of microbial drug resistance and problems with side effects of the administered medication. In this study, we evaluate the in vitro microbiological activity of muramyl dipeptide derivatives, retro-tuftsin derivatives (i.e., tuftsin with reversed amino acid sequences), and combinations of retro-tuftsin derivatives with substituted anthraquinones. The potency of the investigated derivatives towards methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae ESBL (extended-spectrum ß-lactamases) was compared based on the spectroscopically-measured minimal inhibitory concentrations (MIC values). The bacterial growth have also been studied with different concentrations of compounds. Statistical analysis of the results revealed that certain modifications lead to promising activity against S. aureus (anthraquinone analogue - 3c and retro-tuftsin derivative - 2b), while other derivatives exhibit activity against P. aeruginosa (muramyl dipeptide derivative - 1d and retro-tuftsin derivative - 2b). The obtained results of microbiological activity indicate that the structure of the tested compounds may be the basis for further modifications.


Subject(s)
Acetylmuramyl-Alanyl-Isoglutamine/pharmacology , Anthraquinones/pharmacology , Anti-Bacterial Agents/pharmacology , Bacterial Infections/drug therapy , Tuftsin/pharmacology , Acetylmuramyl-Alanyl-Isoglutamine/analogs & derivatives , Animals , Bacteria/drug effects , Bacterial Infections/metabolism , Escherichia coli/drug effects , Humans , Klebsiella pneumoniae/drug effects , Methicillin-Resistant Staphylococcus aureus/drug effects , Microbial Sensitivity Tests , Oligopeptides/pharmacology , Pseudomonas aeruginosa/drug effects , Staphylococcus aureus/drug effects , Tuftsin/analogs & derivatives
3.
J Med Chem ; 64(6): 2982-3005, 2021 03 25.
Article in English | MEDLINE | ID: mdl-33719423

ABSTRACT

Most therapeutic agents used for treating brain malignancies face hindered transport through the blood-brain barrier (BBB) and poor tissue penetration. To overcome these problems, we developed peptide conjugates of conventional and experimental anticancer agents. SynB3 cell-penetrating peptide derivatives were applied that can cross the BBB. Tuftsin derivatives were used to target the neuropilin-1 transport system for selectivity and better tumor penetration. Moreover, SynB3-tuftsin tandem compounds were synthesized to combine the beneficial properties of these peptides. Most of the conjugates showed high and selective efficacy against glioblastoma cells. SynB3 and tandem derivatives demonstrated superior cellular internalization. The penetration profile of the conjugates was determined on a lipid monolayer and Transwell co-culture system with noncontact HUVEC-U87 monolayers as simple ex vivo and in vitro BBB models. Importantly, in 3D spheroids, daunomycin-peptide conjugates possessed a better tumor penetration ability than daunomycin. These conjugates are promising tools for the delivery systems with tunable features.


Subject(s)
Antineoplastic Agents/pharmacokinetics , Blood-Brain Barrier/metabolism , Brain Neoplasms/drug therapy , Cell-Penetrating Peptides/pharmacokinetics , Glioblastoma/drug therapy , Oligopeptides/pharmacokinetics , Tuftsin/pharmacokinetics , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Brain Neoplasms/metabolism , Cell Line, Tumor , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/pharmacology , Drug Carriers/chemistry , Drug Carriers/pharmacokinetics , Drug Carriers/pharmacology , Drug Delivery Systems , Glioblastoma/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Neuropilin-1/metabolism , Oligopeptides/chemistry , Oligopeptides/pharmacology , Rats , Spheroids, Cellular/drug effects , Spheroids, Cellular/metabolism , Tuftsin/analogs & derivatives , Tuftsin/pharmacology , Tumor Cells, Cultured
4.
Curr Drug Targets ; 22(7): 770-778, 2021.
Article in English | MEDLINE | ID: mdl-33243117

ABSTRACT

Tuftsin, a tetrapeptide (Thr-Lys-Pro-Arg), acts as an immunopotentiating molecule with its ability to bind and activate many immune cells, including macrophages or monocytes, neutrophils and dendritic cells. The specific targeting activity of tuftsin has been further increased by its palmitoylation followed by its incorporation into the lipid bilayer of liposomes. Tuftsin-bearing liposomes (Tuft-liposomes) possess several characteristics that enable them to act as a potential drug and vaccine carriers. Tuft-liposomes-loaded anti-microbial drugs have been shown to be highly effective against many infectious diseases, including tuberculosis, leishmaniasis, malaria, candidiasis and cryptococosis. Moreover, Tuft-liposomes also increased the activity of anticancer drug etoposide against fibrosarcoma in mice. Tuft-liposomes showed the immune-potentiating effect and rejuvenated the immune cells in the leukopenic mice. In addition, antigens encapsulated in Tuftsin-bearing liposomes demonstrated greater immunogenicity by increasing the T cell proliferation and antibody secretion. Keeping into consideration their specific targeting and immunopotentiating effects, Tuft-liposomes may potentially be used as promising drug and vaccine delivery systems.


Subject(s)
Communicable Diseases , Neoplasms , Tuftsin , Animals , Communicable Diseases/drug therapy , Drug Delivery Systems , Liposomes , Mice , Neoplasms/drug therapy , Tuftsin/pharmacology
5.
Nanoscale ; 12(3): 2111-2117, 2020 Jan 23.
Article in English | MEDLINE | ID: mdl-31913398

ABSTRACT

The development of molecules with immune stimulatory properties is crucial for cancer immunotherapy. In this work, we combined two peptide-based molecules, tuftsin (TKPR) and Nap-GDFDFDY, to develop a novel self-assembling molecule Nap-GDFDFDYTKPR (Comp.3), which has strong CD8+ T cell stimulatory properties. Comp.3 could self-assemble into nanofibers and hydrogels, which significantly improved the stability of tuftsin against enzyme digestion. The nanofibers of Comp.3 enhanced the phagocytic activity of macrophages, promoted the maturation of DCs, and stimulated the expression of cytokines. In addition, it demonstrated an excellent anti-tumor efficacy in vivo by eliciting a strong CD8+ T immune response. Taken together, our observations revealed a powerful immune stimulating nanomaterial that is a promising compound for cancer immunotherapy.


Subject(s)
CD8-Positive T-Lymphocytes , Hydrogels , Immunity, Cellular/drug effects , Mammary Neoplasms, Animal , Nanofibers , Tuftsin , Animals , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Cell Line, Tumor , Female , Hydrogels/chemistry , Hydrogels/pharmacology , Mammary Neoplasms, Animal/drug therapy , Mammary Neoplasms, Animal/immunology , Mammary Neoplasms, Animal/pathology , Mice , Mice, Inbred BALB C , Nanofibers/chemistry , Nanofibers/therapeutic use , RAW 264.7 Cells , Tuftsin/chemistry , Tuftsin/pharmacology
6.
Int J Nanomedicine ; 15: 10547-10559, 2020.
Article in English | MEDLINE | ID: mdl-33414637

ABSTRACT

BACKGROUND: Targeted multidrug-loaded delivery systems have emerged as an advanced strategy for cancer treatment. In this context, antibodies, hormones, and small peptides have been coupled to the surface of drug carriers, such as liposomes, polymeric and metallic nanoparticles loaded with drugs, as tumor-specific ligands. In the present study, we have grafted a natural macrophage stimulating peptide, tuftsin, on the surface of the liposomes (LPs) that were loaded with doxorubicin (DOX) and/or curcumin (CUR), by attaching to its C-terminus a palmitoyl residue (Thr-Lys-Pro-Arg-CO-NH-(CH2)2-NH-COC15H31, P.Tuft) to enable its grafting within the liposome's bilayer. METHODS: The prepared drug-loaded liposomes (DOX LPs, CUR LPs, DOX-CUR LPs, P.Tuft-LPs, P.Tuft-DOX LPs, P.Tuft-CUR LPs, P.Tuft-DOX-CUR LPs) were thoroughly characterised in terms of particle size, drug content, encapsulation efficiency and structural properties using UV-visible spectroscopy, dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FTIR). The anti-cancer activity and drug toxicity of the liposomal formulations were examined on Ehrlich ascites carcinoma (EAC) tumor-induced mice model. RESULTS: A significant reduction in the tumor weight and volume was observed upon treating the tumor-bearing mice with palmitoyl tuftsin-grafted dual drug-loaded liposomes (P.Tuft-DOX-CUR LPs), as compared to the single drug/peptide-loaded formulation (DOX LPs, CUR LPs, DOX-CUR LPs, P.Tuft- LPs, P.Tuft-DOX LPs, P.Tuft-CUR LPs). Western blot analysis revealed that the tumor inhibition was associated with p53-mediated apoptotic pathway. Further, the biochemical and histological analysis revealed that the various liposomal preparation used in this study were non-toxic to the animals at the specified dose (10mg/kg). CONCLUSION: In conclusion, we have developed a targeted liposomal formulation of P.Tuftsin-bearing liposomes co-encapsulated with effective anti-cancer drugs such as doxorubicin and curcumin. In experimental animals, tumor inhibition by P.Tuft-DOX-CUR LPs indicates the synergistic therapeutic effect of the peptide and the dual drug.


Subject(s)
Carcinoma, Ehrlich Tumor/pathology , Curcumin/pharmacology , Doxorubicin/analogs & derivatives , Tuftsin/pharmacology , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Proliferation/drug effects , Curcumin/adverse effects , Doxorubicin/adverse effects , Doxorubicin/pharmacology , Drug Carriers/chemistry , Drug Compounding , Drug Liberation , Endocytosis/drug effects , HeLa Cells , Humans , Kinetics , Mice , Particle Size , Polyethylene Glycols/adverse effects , Polyethylene Glycols/pharmacology , Spectroscopy, Fourier Transform Infrared , Tuftsin/adverse effects
7.
Mol Med Rep ; 20(6): 5190-5196, 2019 Dec.
Article in English | MEDLINE | ID: mdl-31702807

ABSTRACT

Microglia were once thought to serve a pathogenic role in demyelinating diseases, particularly in multiple sclerosis (MS). However, it has recently been shown that in the experimental autoimmune encephalomyelitis (EAE) model of MS, microglia could serve a protective role by promoting remyelination via the efficient removal of apoptotic cells, the phagocytosis of debris and the support of myelinating oligodendrocytes. The aim of the present study was to determine if the effect of microglia could promote the recovery of EAE and attenuate symptoms in EAE. The severity of EAE was assessed by clinical scores, pathologic changes revealed by luxol fast blue staining and immunohistochemical techniques. The results suggested that microglia reduced clinical scores in mice, suppressed ongoing severe EAE and promoted remyelination and recovery in EAE mice. In addition, following induction with tuftsin, the M1/M2 cytokine balance was shifted, downregulating the proinflammatory M1 response and upregulating the anti­inflammatory M2 response. Generally, microglia can stimulate remyelination, which serves a protective role in different phases of EAE and may represent a potential therapeutic strategy for the treatment of MS.


Subject(s)
Central Nervous System Stimulants/pharmacology , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Microglia/drug effects , Phagocytosis/drug effects , Remyelination/drug effects , Tuftsin/pharmacology , Animals , Calcium-Binding Proteins/metabolism , Cytokines/metabolism , Disease Models, Animal , Encephalomyelitis/drug therapy , Female , Mice , Mice, Inbred C57BL , Microfilament Proteins/metabolism , Multiple Sclerosis/drug therapy , Multiple Sclerosis/pathology , Myelin Sheath/pathology , Oligodendroglia/drug effects
8.
Med Sci Monit ; 25: 5465-5472, 2019 Jul 23.
Article in English | MEDLINE | ID: mdl-31333222

ABSTRACT

BACKGROUND The aim of this study was to investigate the effect of antigenic peptides on dendritic cell maturation and activation as well as the role of dendritic cell induced cell function. The tumor-specific cytotoxic T lymphocytes induced by activation of the dendritic cells were also evaluated. MATERIAL AND METHODS SW-480 cell lysate and peptide antigens were selected as adjuvants in dendritic cell sensitization, and tuftsin was used to induce the phagocytosis of dendritic cells. Immature dendritic cells were stimulated with the antigen and adjuvant as follows: group A was negative control; group B was SW-480 (20 µg/mL); group C was SW-480 (20 µg/mL)+tumor necrosis factor (TNF)-alpha (10 µg/mL); group D was SW-480 (20 µg/mL)+tuftsin (20 µg/mL); group E was antigen peptide (2 µg/mL); group F was antigen peptide (2 µg/mL)+TNF-alpha (10 µg/mL); group G was antigen peptide (2 µg/mL)+tuftsin (20 µg/mL). Cytotoxic T lymphocytes activation and in vitro anti-tumor effects were examined by detecting the maturation marks of dendritic cells as well as interleukin (IL)-10 and IL-12 levels secreted by dendritic cells. Cells with the strongest immunizing effects were injected into nude mice and tumor suppression status was evaluated. RESULTS Group D (SW-480+tuftsin), group E (antigen peptides), group F (antigen peptide+TNF-alpha), and group G (antigen peptides+tuftsin) displayed significant differences compared to the control group (P<0.05). Group G (antigen peptides+tuftsin) could also promote the secretion of cytokines IL-12, as well as inhibit cytokine IL-10 secretion, compared to the other experimental groups (P<0.05). In the in vivo experiments of tumor inhibitions, antigenic polypeptide+tuftsin was the most effective (P<0.05). CONCLUSIONS Combination of cytotoxic T lymphocytes and T peptide therapy in treating human colorectal cancer might be used as a new treatment strategy based on adoptive cellular immunotherapy.


Subject(s)
Colorectal Neoplasms/drug therapy , Dendritic Cells/immunology , Tuftsin/pharmacology , Animals , Cell Line, Tumor , Colonic Neoplasms/pathology , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Humans , Immunotherapy, Adoptive/methods , Lymphocyte Activation/immunology , Mice , Mice, Nude , Peptides/pharmacology , T-Lymphocytes, Cytotoxic , Tuftsin/metabolism , Tumor Necrosis Factor-alpha/metabolism
9.
Isr Med Assoc J ; 21(3): 158-162, 2019 Mar.
Article in English | MEDLINE | ID: mdl-30905098

ABSTRACT

BACKGROUND: The hygiene theory represents one of the environmental facets that modulate the risk for developing autoimmune diseases. There is a reverse correlation between the presence of helminthes and flares of autoimmune diseases, which explains the rise in incidence of certain autoimmune diseases in developed countries. The protective properties of certain helminthes are attributed to their secretory compounds which immunomodulate the host immune network in order to survive. Thus, the helminthes use an array of mechanisms. One of the major mechanisms enabling manipulation of the host-helminth interaction is by targeting the pattern recognition receptors (PRRs)-dependent and -independent mechanisms, which include toll-like receptors, C-type lectin receptors, and the inflammasome. The current review provides a glimpse of numerous helminth secreted products which have a role in the immunomodulation of the host immune network, focusing on bifunctional tuftsin-phosphorylcholine (TPC). TPC is a natural compound based on phosphorylcholine of helminth origin that was used in the past to cover stents and tuftsin, a self-peptide derived from the spleen. TPC was proven to be efficient in three murine experimental models (lupus, colitis, and arthritis) and ex vivo in giant cell arteritis.


Subject(s)
Autoimmune Diseases/prevention & control , Helminthiasis/immunology , Helminths/immunology , Immunologic Factors/immunology , Immunologic Factors/pharmacology , Immunomodulation/immunology , Phosphorylcholine/immunology , Phosphorylcholine/pharmacology , Tuftsin/immunology , Tuftsin/pharmacology , Animals , Disease Models, Animal , Humans , Mice
10.
Immunol Res ; 66(6): 637-641, 2018 12.
Article in English | MEDLINE | ID: mdl-30554380

ABSTRACT

The distinction that in areas where helminthic infections are common, autoimmune diseases are less prevalent, led to the investigation of immune modulatory properties of helminths and their derivatives. Such are phosphorylcholine (PC) moieties which are a component of secreted products of helminths. PC has been broadly studied for its attenuating effects on the human immune system. In an attempt to develop a novel therapeutic small molecule for the treatment of autoimmune conditions, we have conjugated PC with tuftsin, a natural immunomodulatory tetrapeptide, to create TPC. Herein, we review our findings regarding the effects of TPC in murine models of three autoimmune diseases-systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), and rheumatic arthritis (RA), as well as ex-vivo samples from giant cell arteritis (GCA) patients. In all four disease models examined, TPC was shown to attenuate the inflammatory response by reducing expression of pro-inflammatory cytokines and altering the phenotype of T cell expression. In murine models, TPC has further produced a significant improvement in clinical disease scores with no significant side effects noted. Our findings suggest TPC presents promising potential as a novel therapeutic agent for the effective treatment of various autoimmune conditions.


Subject(s)
Autoimmunity/drug effects , Helminths/drug effects , Phosphorylcholine/pharmacology , Phosphorylcholine/therapeutic use , Tuftsin/pharmacology , Tuftsin/therapeutic use , Animals , Autoimmune Diseases/drug therapy , Autoimmune Diseases/parasitology , Helminthiasis/drug therapy , Helminthiasis/parasitology , Humans , Inflammation/drug therapy , Inflammation/parasitology
11.
Front Immunol ; 9: 2784, 2018.
Article in English | MEDLINE | ID: mdl-30555470

ABSTRACT

Though promoting remyelination in multiple sclerosis (MS) has emerged as a promising therapeutic strategy, it does not address inflammatory signals that continue to induce neuronal damage and inhibit effectiveness of repair mechanisms. Our lab has previously characterized the immunomodulatory tetrapeptide, tuftsin, which induces an anti-inflammatory shift in microglia and macrophages. This targeted anti-inflammatory agent improves physical deficits in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Here, we sought to determine whether tuftsin is also effective in combination with benztropine, an FDA-approved drug that stimulates remyelination, in both EAE and in the cuprizone model of demyelination. We show that combining these two agents to promote anti-inflammatory and remyelinating mechanisms alleviates symptoms in EAE and lessens pathological hallmarks in both MS models. Importantly, tuftsin is required to transform the inflammatory CNS environment normally present in EAE/MS into one of an anti-inflammatory nature, and benztropine is required in the cuprizone model to improve remyelination. Our data further support tuftsin's beneficial immunomodulatory activity in the context of EAE, and show that when studying remyelination in the absence of an autoimmune insult, tuftsin still activated microglia toward an anti-inflammatory fate, but benztropine was necessary for significant repair of the damaged myelin. Overall, tuftsin effectively combined with benztropine to significantly improve MS-like pathologies in both models.


Subject(s)
Benztropine/pharmacology , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Multiple Sclerosis/drug therapy , Myelin Sheath/immunology , Tuftsin/pharmacology , Animals , Cuprizone/adverse effects , Cuprizone/pharmacology , Disease Models, Animal , Drug Therapy, Combination , Encephalomyelitis, Autoimmune, Experimental/chemically induced , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/pathology , Mice , Multiple Sclerosis/chemically induced , Multiple Sclerosis/immunology , Multiple Sclerosis/pathology , Myelin Sheath/pathology
12.
PLoS One ; 13(8): e0200615, 2018.
Article in English | MEDLINE | ID: mdl-30089122

ABSTRACT

A novel small molecule named tuftsin-phosphorylcholine (TPC), which is linked to the biological activity of helminths, was constructed. The current study address the effect of TPC treatment in established collagen-induced arthritis (CIA) mice and propose TPC bi-functional activity. TPC treatment was initiated when clinical score was 2 to 4. Arthritis scores in TPC treated mice were lower compared to mice treated with vehicle (P < 0.001). Joint staining showed normal joint structure in TPC-treated mice compared to control groups treated with phosphate buffered saline (PBS), phosphorylcholine, or tuftsin, which exhibited severely inflamed joints. TPC enhanced anti-inflammatory response due to increased IL-10 secretion, and reduced pro-inflammatory cytokine secretion (IL-1-ß, IL-6, TNF-αP < 0.001). Furthermore, TPC therapy increased expansion of CD4+CD25+FOXP3+T regulatory cells and IL-10+CD5+CD1d+B regulatory cells. We propose that the immunomodulatory activity of TPC can be a result of a bi-specific activity of TPC: (a) The tuftsin part of the TPC shifts RAW macrophage cells from pro-inflammatory macrophages M1 to anti-inflammatory M2-secreting IL-10 (P < 0.001) through neuropilin-1 and (b) TPC significantly reduce mouse TLR4 expression via NFkB pathway by HEKTM cells (P < 0.02) via the phosphorylcholine site of the molecule. Our results indicate that TPC, significantly ameliorated established CIA by its immunomodulatory activity. These data could lead to a novel self bi-functional small molecule for treating patients with progressive RA.


Subject(s)
Arthritis, Experimental/drug therapy , Helminths/metabolism , Phosphorylcholine/therapeutic use , Tuftsin/therapeutic use , Animals , Arthritis, Experimental/pathology , B-Lymphocytes/cytology , B-Lymphocytes/drug effects , B-Lymphocytes/metabolism , Disease Models, Animal , HEK293 Cells , Humans , Interleukin-10/metabolism , Interleukin-1beta/metabolism , Joints/pathology , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Male , Mice , Mice, Inbred DBA , NF-kappa B/metabolism , Neuropilin-1/metabolism , Phosphorylcholine/pharmacology , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/metabolism , Toll-Like Receptor 4/drug effects , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism , Tuftsin/pharmacology
13.
Eur J Med Chem ; 133: 152-173, 2017 Jun 16.
Article in English | MEDLINE | ID: mdl-28384546

ABSTRACT

Tuberculosis is caused by Mycobacterium tuberculosis, an intracellular pathogen that can survive in host cells, mainly in macrophages. An increase of multidrug-resistant tuberculosis qualifies this infectious disease as a major public health problem worldwide. The cellular uptake of the antimycobacterial agents by infected host cells is limited. Our approach is to enhance the cellular uptake of the antituberculars by target cell-directed delivery using drug-peptide conjugates to achieve an increased intracellular efficacy. In this study, salicylanilide derivatives (2-hydroxy-N-phenylbenzamides) with remarkable antimycobacterial activity were conjugated to macrophage receptor specific tuftsin based peptide carriers through oxime bond directly or by insertion of a GFLG tetrapeptide spacer. We have found that the in vitro antimycobacterial activity of the salicylanilides against M. tuberculosis H37Rv is preserved in the conjugates. While the free drug was ineffective on infected macrophage model, the conjugates were active against the intracellular bacteria. The fluorescently labelled peptide carriers that were modified with different fatty acid side chains showed outstanding cellular uptake rate to the macrophage model cells. The conjugation of the salicylanilides to tuftsin based carriers reduced or abolished the in vitro cytostatic activity of the free drugs with the exception of the palmitoylated conjugates. The conjugates degraded in the presence of rat liver lysosomal homogenate leading to the formation of an oxime bond-linked salicylanilide-amino acid fragment as the smallest active metabolite.


Subject(s)
Antitubercular Agents/chemistry , Antitubercular Agents/pharmacology , Mycobacterium/drug effects , Salicylanilides/chemistry , Salicylanilides/pharmacology , Tuftsin/analogs & derivatives , Tuftsin/pharmacology , Animals , Antitubercular Agents/pharmacokinetics , Cell Line , Humans , Mycobacterium Infections/drug therapy , Mycobacterium tuberculosis/drug effects , Rats , Salicylanilides/pharmacokinetics , Tuberculosis/drug therapy , Tuberculosis, Multidrug-Resistant/drug therapy , Tuftsin/pharmacokinetics
14.
Oncotarget ; 7(49): 81791-81805, 2016 Dec 06.
Article in English | MEDLINE | ID: mdl-27835904

ABSTRACT

Our previous research showed that neuropilin (Nrp) -1highCD4+CD25+Regulatory T cells (Tregs) exhibited primary negative immunoregulation in sepsis induced immune dysfunction. Tuftsin is the typical ligand of Nrp-1. Herein, we investigated the potential therapeutic value and mechanisms of tuftsin in sepsis. Sepsis per se markedly decreased the serum concentration of tuftsin, administration of tuftsin improved the survival rate of septic mice with cecal ligation and puncture (CLP). In vitro study, tuftsin prevented the negative immunoregulation of Nrp-1highCD4+CD25+Tregs, including weakening the expression of forkhead/winged helix transcription factor (Foxp)- 3/cytotoxic T lymphocyte associated antigen (CTLA)-4, inhibiting the secretion of transforming growth factor (TGF)-ß, and weakening the immunosuppressive function of Nrp-1highCD4+CD25+Tregs to conventional CD4+CD25-T cells. Tuftsin markedly inhibited the demethylation of Foxp3-Tregs specific demethylated region (TSDR) of Nrp-1highCD4+CD25+Tregs. Tuftsin could represent a new potential therapeutic agentia to improve the outcome of septic mice, and associate with preventing the negative immunoregulation of Tregs via Nrp-1.


Subject(s)
Immunologic Factors/pharmacology , Interleukin-2 Receptor alpha Subunit/metabolism , Neuropilin-1/metabolism , Sepsis/drug therapy , T-Lymphocytes, Regulatory/drug effects , Tuftsin/pharmacology , Animals , CTLA-4 Antigen/metabolism , Cells, Cultured , DNA Methylation , Disease Models, Animal , Dose-Response Relationship, Drug , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Interleukin-2 Receptor alpha Subunit/immunology , Male , Mice, Inbred BALB C , Neuropilin-1/immunology , Sepsis/genetics , Sepsis/immunology , Sepsis/metabolism , Signal Transduction/drug effects , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Time Factors , Transforming Growth Factor beta/metabolism
16.
Clin Exp Immunol ; 184(1): 19-28, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26618631

ABSTRACT

Treatment with helminthes and helminthes ova improved the clinical symptoms of several autoimmune diseases in patients and in animal models. Phosphorylcholine (PC) proved to be the immunomodulatory molecule. We aimed to decipher the tolerogenic potential of tuftsin-PC (TPC), a novel helminth-based compound in collagen-induced arthritis (CIA) a mouse model of rheumatoid arthritis (RA). CIA DBA/1 mice were treated with TPC subcutaneously (5 µg/0.1 ml) or orally (250 µg/0.1 ml), starting prior to disease induction. The control groups were treated with PBS. Collagen antibodies were tested by enzyme-linked immunosorbent assay (ELISA), cytokine protein levels by ELISA kits and regulatory T (Treg ) and regulatory B (Breg ) cell phenotypes by fluorescence-activated cell sorter (FACS). TPC-treated mice had a significantly lower arthritis score of 1.5 in comparison with control mice 11.8 (P < 0.0001) in both subcutaneous and orally treated groups at day 31. Moreover, histology analysis demonstrated highly inflamed joints in control mice, whereas TPC-treated mice maintained normal joint structure. Furthermore, TPC decreased the titres of circulating collagen II antibodies in mice sera (P < 0.0001), enhanced expression of IL-10 (P < 0.0001) and inhibited production of tumour necrosis factor (TNF)-α, interleukin (IL)-17 and IL-1ß (P < 0.0001). TPC significantly expanded the CD4(+) CD25(+) forkhead box protein 3 (FoxP3(+) ) Treg cells and CD19(+) IL-10(+) CD5(high) CD1d(high) T cell immunoglobulin mucin-1 (TIM-1(+) ) Breg cell phenotypes (P < 0.0001) in treated mice. Our data indicate that treatment with TPC attenuates CIA in mice demonstrated by low arthritic score and normal joints histology. TPC treatment reduced proinflammatory cytokines and increased anti-inflammatory cytokine expression, as well as expansion of Treg and Breg cells. Our results may lead to a new approach for a natural therapy for early rheumatoid arthritis onset.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antibodies/blood , Arthritis, Experimental/drug therapy , Phosphorylcholine/pharmacology , Tuftsin/pharmacology , Administration, Oral , Animals , Arthritis, Experimental/immunology , Arthritis, Experimental/pathology , B-Lymphocytes, Regulatory/drug effects , B-Lymphocytes, Regulatory/immunology , B-Lymphocytes, Regulatory/pathology , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/pathology , Collagen Type II/blood , Collagen Type II/immunology , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/immunology , Gene Expression , Hepatitis A Virus Cellular Receptor 1 , Immunophenotyping , Injections, Subcutaneous , Interleukin-10/genetics , Interleukin-10/immunology , Interleukin-17/genetics , Interleukin-17/immunology , Interleukin-1beta/genetics , Interleukin-1beta/immunology , Joints/drug effects , Joints/immunology , Joints/pathology , Male , Membrane Proteins/genetics , Membrane Proteins/immunology , Mice , Mice, Inbred DBA , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/pathology , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology
17.
Sci Rep ; 5: 16725, 2015 Nov 18.
Article in English | MEDLINE | ID: mdl-26577833

ABSTRACT

The primary mechanisms of sepsis induced cellular immunosuppression involve immune dysfunction of T lymphocytes and negative immunoregulation of regulatory T cells (Tregs). It has been found that tuftsin is an immune modulating peptide derived from IgG in spleen. T-peptide is one of tuftsin analogs. Herein, we examined the effect of T-peptide on cell-mediated immunity in the presence of lipopolysaccharide (LPS) and the survival rate in septic mice. T-peptide regulated the proliferative ability of CD4(+)CD25(-) T cells in dual responses. Meanwhile, 10 and 100 µg/ml T-peptides were able to enhance the apoptotic rate of CD4(+)CD25(-) T cells compared with 1 µg/ml T-peptide, but markedly lowered interleukin (IL)-2 levels. When CD4(+)CD25(+) Tregs were treated with T-peptide for 24 hours, and co-cultured with normal CD4(+)CD25(-) T cells, the suppressive ability of CD4(+)CD25(+) Tregs on CD4(+)CD25(-) T cells was significantly lowered, along with decreased expression in forkhead/winged helix transcription factor p-3 (Foxp-3) as well as cytotoxic T lymphocyte-associated antigen (CTLA)-4, and secretion of transforming growth factor (TGF)-ß. Moreover, T-peptide has the ability to improve outcome of septic mice in a dose- and time- dependent manner, and associated with improvement in the microenvironment of cellular immunosuppression in septic mice.


Subject(s)
Immunity, Cellular/drug effects , Immunologic Factors/pharmacology , Immunomodulation/drug effects , Sepsis/immunology , Sepsis/mortality , Tuftsin/pharmacology , Animals , Antigens, Surface/metabolism , Apoptosis/drug effects , Apoptosis/immunology , CTLA-4 Antigen/genetics , CTLA-4 Antigen/metabolism , Cytokines/metabolism , Disease Models, Animal , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Gene Expression , Immunophenotyping , Interleukin-2/biosynthesis , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Mice , Phenotype , Sepsis/drug therapy , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism
18.
Eur J Med Chem ; 106: 85-94, 2015 Dec 01.
Article in English | MEDLINE | ID: mdl-26520842

ABSTRACT

New batracylin conjugates with tuftsin/retro-tuftsin derivatives were designed and synthesized using T3P as a coupling agent. The conjugates possess an amide bond formed between the carboxyl group of heterocyclic molecule and the N-termini of the tuftsin/retro-tuftsin chain. The in vitro cytotoxic activity of the new analogues and their precursors was evaluated using a series of human and murine tumor cells. BAT conjugates containing retro-tuftsin with branched side aminoacid chain, in particular with leucine or isoleucine, were about 10-fold more cytotoxic toward two human tumor cell lines (lung adenocarcinoma (A549) and myeloblastic leukemia (HL-60)). These compounds showed about 10-fold increased cytotoxicity against the two types of tumor cells compared to parent BAT. We have not observed important differences in the mechanism of action between BAT and its cytotoxic tuftsin/retro-tuftsin conjugates. We propose that high biological activity of the most active BAT conjugates is a result of their greatly increased intracellular accumulation.


Subject(s)
Antineoplastic Agents/pharmacology , Quinazolines/pharmacology , Topoisomerase Inhibitors/pharmacology , Tuftsin/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Cycle/drug effects , Cell Line , Cell Proliferation/drug effects , Cell Survival/drug effects , DNA Damage , DNA Topoisomerases/metabolism , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , HL-60 Cells , Humans , Mice , Molecular Structure , NIH 3T3 Cells , Quinazolines/chemistry , Structure-Activity Relationship , Topoisomerase Inhibitors/chemical synthesis , Topoisomerase Inhibitors/chemistry , Tuftsin/analogs & derivatives , Tuftsin/chemistry
19.
J Autoimmun ; 56: 111-7, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25479760

ABSTRACT

Improved clinical findings of inflammatory bowel disease (IBD) upon treatment with helminthes and their ova were proven in animal models of IBD and in human clinical studies. The immunomodulatory properties of several helminthes were attributed to the phosphorylcholine (PC) molecule. We assessed the therapeutic potential of tuftsin-PC conjugate (TPC) to attenuate murine colitis. Colitis was induced by Dextransulfate-Sodium-Salt (DSS) in drinking water. TPC was given by daily oral ingestion (50 µg/0.1 ml/mouse or PBS) starting at day -2. Disease activity index (DAI) score was followed daily and histology of the colon was performed by H&E staining. Analysis of the cytokines profile in distal colon lysates was performed by immunoblot. Treatment of DSS induced colitis with TPC prevented the severity of colitis, including a reduction in the DAI score, less shortening of the colon and less inflammatory activity in histology. The immunoblot showed that the colitis preventive activity of TPC was associated with downregulation of colon pro-inflammatory IL-1ß, TNFα and IL-17 cytokines expression, and enhancement of anti-inflammatory IL-10 cytokine expression. In the current study, we demonstrated that TPC treatment can prevent significantly experimental colitis induction in naïve mice. We propose the TPC as a novel potential small synthetic molecule to treat colitis.


Subject(s)
Colitis/pathology , Immunologic Factors/pharmacology , Phosphorylcholine , Tuftsin/pharmacology , Animals , Colitis/chemically induced , Colitis/drug therapy , Colitis/immunology , Cytokines/metabolism , Dextran Sulfate/adverse effects , Disease Models, Animal , Humans , Immunologic Factors/administration & dosage , Immunologic Factors/chemistry , Inflammatory Bowel Diseases/drug therapy , Inflammatory Bowel Diseases/immunology , Inflammatory Bowel Diseases/pathology , Intestinal Mucosa/immunology , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Male , Mice , Severity of Illness Index , Tuftsin/administration & dosage , Tuftsin/chemistry
20.
Cancer Immunol Immunother ; 63(12): 1261-72, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25164878

ABSTRACT

Tuftsin (TF) is an immunomodulator tetrapeptide (Thr-Lys-Pro-Arg) that binds to the receptor neuropilin-1 (Nrp1) on the surface of cells. Many reports have described anti-tumor activity of tuftsin to relate with nonspecific activation of the host immune system. Lidamycin (LDM) that displays extremely potent cytotoxicity to cancer cells is composed of an apoprotein (LDP) and an enediyne chromophore (AE). In addition, Ec is an EGFR-targeting oligopeptide. In the present study, LDP was used as protein scaffold and the specific carrier for the highly potent AE. Genetically engineered fusion proteins LDP-TF and Ec-LDP-TF were prepared; then, the enediyne-energized fusion protein Ec-LDM-TF was generated by integration of AE into Ec-LDP-TF. The tuftsin-based fusion proteins LDP-TF and Ec-LDP-TF significantly enhanced the phagocytotic activity of macrophages as compared with LDP (P < 0.05). Ec-LDP-TF effectively bound to tumor cells and macrophages; furthermore, it markedly suppressed the growth of human epidermoid carcinoma A431 xenograft in athymic mice by 84.2 % (P < 0.05) with up-regulated expression of TNF-α and IFN-γ. Ec-LDM-TF further augmented the therapeutic efficacy, inhibiting the growth of A431 xenograft by 90.9 % (P < 0.05); notably, the Ec-LDM-TF caused marked down-regulation of CD47 in A431 cells. Moreover, the best therapeutic effect was recorded in the group of animals treated with the combination of Ec-LDP-TF with Ec-LDM-TF. The results suggest that tuftsin-based, enediyne-energized, and EGFR-targeting fusion proteins exert highly antitumor efficacy with CD47 modulation. Tuftsin-based fusion proteins are potentially useful for treatment of EGFR- and CD47-overexpressing cancers.


Subject(s)
CD47 Antigen/immunology , Enediynes/pharmacology , ErbB Receptors/immunology , Immunotoxins/pharmacology , Recombinant Fusion Proteins/pharmacology , Tuftsin/pharmacology , Animals , Carcinoma, Squamous Cell/immunology , Carcinoma, Squamous Cell/therapy , Cell Communication , Cell Line, Tumor , Down-Regulation , Enediynes/chemistry , Female , Humans , Immunotoxins/chemistry , Immunotoxins/immunology , Mice , Mice, Inbred BALB C , Murine pneumonia virus , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/immunology , Tuftsin/chemistry , Tuftsin/immunology , Xenograft Model Antitumor Assays
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