The therapeutic potential of tuftsin-phosphorylcholine in giant cell arteritis.

Tuftsin-PhosphorylCholine (TPC) is a novel bi-specific molecule which links tuftsin and phosphorylcholine. TPC has shown immunomodulatory activities in experimental mouse models of autoimmune diseases. We studied herein the effects of TPC ex vivo on both peripheral blood mononuclear cells (PBMCs) and temporal artery biopsies (TABs) obtained from patients with giant cell arteritis (GCA) and age-matched disease controls. GCA is an immune-mediated disease affecting large vessels. Levels of 18 cytokines in supernatants, PBMC viability, T helper (Th) cell differentiation of PBMCs and gene expression in TABs were analyzed. Treatment ex vivo with TPC decreased the production of IL-1ß, IL-2, IL-5, IL-6, IL-9, IL-12(p70), IL-13, IL-17A, IL-18, IL-21, IL-22, IL-23, IFNγ, TNFα, GM-CSF by CD3/CD28 activated PBMCs whereas it negligibly affected cell viability. It reduced Th1 and Th17 differentiation while did not impact Th22 differentiation in PBMCs stimulated by phorbol 12-myristate 13-acetate plus ionomycin. In inflamed TABs, treatment with TPC down-regulated the production of IL-1ß, IL-6, IL-13, IL-17A and CD68 gene expression. The effects of TPC were comparable to the effects of dexamethasone, included as the standard of care, with the exception of a greater reduction of IL-2, IL-18, IFNγ in CD3/CD28 activated PBMCs and CD68 gene in inflamed TABs. In conclusion our results warrant further investigations regarding TPC as an immunotherapeutic agent in GCA and potentially other autoimmune and inflammatory diseases.

Tuftsin-phosphorylcholine (TPC) equally effective to methylprednisolone in ameliorating lupus nephritis in a mice model.

The role of helminth treatment in autoimmune diseases is growing constantly. Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease with challenging treatment options. Tuftsin-phosphorylcholine (TPC) is a novel helminth-based compound that modulates the host immune network. This study was conducted to evaluate the potential value of TPC in ameliorating lupus nephritis in a murine model and specifically to compare the efficacy of TPC to the existing first-line therapy for SLE: corticosteroids (methylprednisolone). Lupus-prone NZBxW/F1 mice were treated with TPC (5 µg/mouse), methylprednisolone (MP; 5 mg/body weight) or phosphate-buffered saline (PBS) (control) three times per week once glomerulonephritis, defined as proteinuria of grade > 100 mg/dl, was established. Levels of anti-dsDNA autoantibodies were evaluated by enzyme-linked immunosorbent assay (ELISA), splenic cytokines were measured in vitro and the kidney microscopy was analysed following staining. TPC and MP treatments improved lupus nephritis significantly and prolonged survival in NZBxW/F1 mice. TPC-treated mice showed a significantly decreased level of proteinuria (P < 0·001) and anti-dsDNA antibodies (P < 0·001) compared to PBS-treated mice. Moreover, TPC and MP inhibited the production of the proinflammatory cytokines interferon IFN-γ, interleukin IL-1ß and IL-6 (P < 0·001) and enhanced expression of the anti-inflammatory cytokine IL-10 (P < 0·001). Finally, microscopy analysis of the kidneys demonstrated that TPC-treated mice maintained normal structure equally to MP-treated mice. These data indicate that the small molecule named TPC hinders lupus development in genetically lupus-prone mice equally to methylprednisolone in most of the cases. Hence, TCP may be employed as a therapeutic potential for lupus nephritis.

Cloning, large-scale production and characterization of fusion protein (P-TUFT-ALT-2) of Brugian abundant larval transcript-2 with tuftsin in Pichia pastoris.

Lymphatic filariasis is a "disease of poor people" due to a large section of affected people with economic backwardness. Therefore, successful elimination of this disease requires a cost-effective prophylactic agent such as vaccine along with conventional drugs. The Abundant Larval Transcript-2 (BmALT-2) protein of Brugia malayi has been recognized as the most potential vaccine candidate. Tuftsin, a tetra-peptide immunopotentiator has already shown the enhanced immunogenicity of various vaccine antigens in earlier studies. This study deals with the development of tuft-alt-2 fusion construct and a suitable culture condition for its large-scale production in Pichia pastoris. The recombinant P. pastoris/tuft-alt-2 with 9-11 copies of the gene construct exhibited the highest expression level. The molecular weight of P-TUFT-ALT-2 was determined as 28 kDa in SDS-PAGE including 3 kDa due to glycosylation. The dry cell biomass was 57.4 gL-1 in the bioreactor. The P-TUFT-ALT-2 expression was measured as about 35 mg L-1, which was 102% higher than flask culture. The P-TUFT-ALT-2 produced the highest 65,000 IgG peak titer in Balb/c mice. Moreover, P-TUFT-ALT-2 exhibited about 9.46% higher splenocyte proliferation than E. coli expressed E-ALT-2 alone. The enhanced secreted production of P-TUFT-ALT-2 in bioreactor would step up its commercialization as an inexpensive commercial vaccine for human lymphatic filariasis.

Novel therapeutic compound tuftsin-phosphorylcholine attenuates collagen-induced arthritis.

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.

Successful modulation of murine lupus nephritis with tuftsin-phosphorylcholine.

In areas where helminths infections are common, autoimmune diseases are rare. Treatment with helminths and ova from helminths, improved clinical findings of inflammatory bowel disease, multiple-sclerosis and rheumatoid-arthritis. The immunomodulatory functions of some helminths were attributed to the phosphorylcholine (PC) moiety. We aimed to decipher the tolerogenic potential of Tuftsin-PC (TPC) compound in mice genetically prone to develop lupus. Lupus prone NZBXW/F1 mice received subcutaneously TPC (5 µg/1 ml), 3 times a week starting at 14 weeks age. Autoantibodies were tested by ELISA, T-regulatory-cells by FACS, cytokines profile by RT-PCR and cytokines protein levels by DuoSet ELISA. Glomerulonephritis was addressed by detection of proteinuria, and immunoglobulin complex deposition in the mesangium of the kidneys of the mice by immunofluorescence. Our results show that TPC attenuated the development of glomerulonephritis in lupus prone mice, in particular, it ameliorated proteinuria (p < 0.02), and reduced immunoglobulin deposition in the kidney mesangium. TPC also enhanced the expression of TGFß and IL-10 (p < 0.001), and inhibited the production of IFNγ and IL-17 (p < 0.03). TPC Significantly enhanced the expansion of CD4+CD25+FOXP3+ T-regulatory cells (Tregs) phenotype in the treated mice. These data indicate that TPC hampered lupus development in genetically lupus prone mice which was exemplified by moderate glomerulonephritis, attenuation of pro-inflammatory cytokines and enhancement of anti-inflammatory cytokines expression, as well as Tregs expansion. Our results propose harnessing novel natural therapy for lupus patients.

Phosphorylcholine-tuftsin compound prevents development of dextransulfate-sodium-salt induced murine colitis: implications for the treatment of human inflammatory bowel disease.

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.

Helminth derivative tuftsin-phopshorylcholine to treat autoimmunity.

Autoimmune diseases (AIDs) affect 5 to 10% of the population. There are more than ∼100 different autoimmune diseases. The AIDs are one of the top 10 causes of death in women under 65; 2nd highest cause of chronic illness; top cause of morbidity in women in the US. The NIH estimates annual direct healthcare costs for autoimmune diseases about $100 billion, in comparison, with cancers investment of $57 billion, heart and stroke cost of $200 billion. The current treatments for autoimmune diseases encompasses: steroids, chemotherapy, immunosuppressants, biological drugs, disease specific drugs (like acethylcholine-estherase for myasthenia gravis). The treatments for autooimmune diseases supress the patient immune network, which leads the patients to be more susceptible to infections. Hence, there is a need to develop immunomodulatory small molecules with minimal side effects to treat autoimmune diseases. The helminths developed secreting compounds which modulate the human defense pathways in order to develop tolerance and survive in the host environment. We have imitated the immunomodulatory activity of the helminth by using a derivative of the helminth secretory molecule. A bi-functional small molecule -tuftsin (T)-phosphorylcholine (PC), coined as TPC, was constructed. This chimeric molecule showed its immunomodulatory activity in 4 murine models of autoimmune diseases, attenuating the clinical score and the inflammatory response by immunomodutating the host immune system. Ex-vivo in human peripheral blood mononuclear cells (PBMCs) and biopsies originated from arteries of patients with giant cell arteritis. This paper decipher the mode of action of TPC immunomodulatory activity. Our data propose the potential for this small molecule to be a novel therapy for patients with autoimmune diseases.

Tuftsin signals through its receptor neuropilin-1 via the transforming growth factor beta pathway.

Tuftsin (Thr-Lys-Pro-Arg) is a natural immunomodulating peptide found to stimulate phagocytosis in macrophages/microglia. Tuftsin binds to the receptor neuropilin-1 (Nrp1) on the surface of cells. Nrp1 is a single-pass transmembrane protein, but its intracellular C-terminal domain is too small to signal independently. Instead, it associates with a variety of coreceptors. Despite its long history, the pathway through which tuftsin signals has not been described. To investigate this question, we employed various inhibitors to Nrp1's coreceptors to determine which route is responsible for tuftsin signaling. We use the inhibitor EG00229, which prevents tuftsin binding to Nrp1 on the surface of microglia and reverses the anti-inflammatory M2 shift induced by tuftsin. Furthermore, we demonstrate that blockade of transforming growth factor beta (TGFß) signaling via TßR1 disrupts the M2 shift similar to EG00229. We report that tuftsin promotes Smad3 phosphorylation and reduces Akt phosphorylation. Taken together, our data show that tuftsin signals through Nrp1 and the canonical TGFß signaling pathway. Despite the 40-year history of the tetrapeptide tuftsin (TKPR), a macrophage and microglial activator, its mechanism of action has not been defined. Here, we report that the tuftsin-mediated anti-inflammatory M2 shift in microglia is caused specifically by tuftsin binding to the receptor neuropilin-1 (Nrp1) and signaling through TGFß receptor-1, a coreceptor of Nrp1. We further show that tuftsin signals via the canonical TGFß pathway and promotes TGFß release from target cells.

Tuftsin-tailored fusion protein inhibits the growth of circulating gastric tumor cells associated with macrophage phagocytosis.

Circulating tumor cells (CTCs) are a major cause of tumor metastasis and resistance to anticancer therapies. To date, no effective low-toxicity chemotherapeutic agents or antibodies have exhibited significant clinical activity against CTCs. Macrophages are important mediators of antitumor immunity. Tuftsin (TF), a tetrapeptide located at residues 289-292 of the CH2 domain of the Fc region of the IgG heavy chain, binds to Nrp-1, a receptor on the surface of macrophages that promotes phagocytosis and induces nonspecific activation of the immune system against tumors. Lidamycin (LDM) is an antitumor chemotherapy agent that is strongly cytotoxic to tumors and can dissociate into an apoprotein (LDP) and active enediyne (AE) in vitro. We previously constructed the fusion protein LDP-TF through genetic engineering and inserted the chromophore AE to produce LDM-TF, which can target macrophages to promote their phagocytic and cytotoxic activity against tumor cells. Preliminary experiments confirmed the anti-tumor activity of LDM-TFs. In this study, we found that LDM-TF effectively inhibited the growth of CTCs of gastric cancer origin and enhanced macrophage phagocytosis both in vivo and in vitro. Tumor cell expression of CD47, which helps to evade phagocytosis by macrophages, was substantially downregulated by LDM-TF. Notably, our in vitro experiments demonstrated that the combination of LDM-TF and anti-CD47 antibodies promoted phagocytosis more than either component alone. Our findings demonstrate the significant inhibitory effect of LDM-TF on the growth of CTCs of gastric cancer origin and suggest that the combination of LDM-TF and anti-CD47 antibodies may exhibit synergistic effects, thereby providing a new option for the clinical treatment of patients with advanced tumors that have metastasized.

Tuftsin: A Natural Molecule Against SARS-CoV-2 Infection.

Coronavirus disease 2019 (COVID-19) continuously progresses despite the application of a variety of vaccines. Therefore, it is still imperative to find effective ways for treating COVID-19. Recent studies indicate that NRP1, an important receptor of the natural peptide tuftsin (released from IgG), facilitates SARS-CoV-2 infection. Here, we found 91 overlapping genes between tuftsin targets and COVID-19-associated genes. We have demonstrated that tuftsin could also target ACE2 and exert some immune-related functions. Molecular docking results revealed that tustin could combine with ACE2 and NRP1 in stable structures, and their interacted regions cover the binding surfaces of S1-protein with the two receptors. Using surface plasmon resonance (SPR) analysis, we confirmed that tuftsin can bind ACE2 and NRP1 directly. Importantly, using SPR-based competition assay we have shown here that tuftsin effectively prevented the binding of SARS-CoV-2 S1-protein to ACE2. Collectively, these data suggest that tuftsin is an attractive therapeutic candidate against COVID-19 and can be considered for translational as well as clinical studies.

Tuftsin ameliorates splenic inflammatory injury by promoting neuropilin-1 in severe acute pancreatitis.

Severe acute pancreatitis (SAP)-associated spleen injury causing immune disturbances aggravates organs injuries, which contributes to higher mortality rate. However, there are no effective drugs to cure SAP-induced spleen injury. Here, we found that Tuftsin (TN) is effective for ameliorating SAP-induced pathological damage and inflammation of spleen, mainly via alleviating mitochondrial dysfunction, oxidative stress, ATP depletion and the expression of pro-inflammatory factors. We further found that TN promoted anti-inflammatory macrophage phenotype M2 via up-regulating NRP1 on macrophage in spleen during SAP. Meanwhile, EG00229 (an inhibitor of NRP1 bound to TN) weakened TN's therapeutic effect in SAP-associated spleen injury. And EG00229 also inhibited M2 macrophage, leading to increasing inflammasome formation. Additionally, EG00229 reduced the protective efficiency of TN on mitochondrial dysfunction, and inflammation injury via NRP1 in spleen caused by SAP. Similarly, siRNA-Nrp1 into macrophage also prevented TN's inhibition on apoptosis. These findings reveal that TN alleviates SAP-induced spleen injury by promoting NRP1.

Legumain protease-sheddable PEGylated, tuftsin-modified nanoparticles for selective targeting to tumour-associated macrophages.

Tumour-associated macrophages (TAMs) represent an attractive cell target for anticancer therapy. However, selective and efficient targeting of TAMs remains difficult. Here, we constructed a novel dually functionalised nanoparticle platform (s-Tpep-NPs) by surface co-modification of nanoparticles (NPs) with tuftsin (Tpep) and legumain protease-sheddable polyethylene glycol 5k (PEG5k) to achieve selective targeted delivery to TAMs. The fluorescence resonance energy transfer experiment and in vitro cellular uptake assay confirmed that s-Tpep-NPs can responsively shed PEG5k and transform into active Tpep-NPs upon the cleavage of legumain that is overexpressed on TAM surfaces, which then promotes TAM phagocytosis through Fc receptor-mediated pathways. Owing to the shielding effect by legumain-sheddable PEG5k, s-Tpep-NPs can effectively decrease the Tpep-induced non-specific accumulation in mononuclear phagocyte system (MPS) organs during systemic circulation. Moreover, s-Tpep-NPs can significantly enhance the tumoural accumulation and improve the specificity and efficiency of targeting to TAMs, as compared with both controls of Tpep-NPs and non-sheddable ns-Tpep-NPs. Overall, this study provides a robust nanoplatform with a novel avenue for improved selectivity of targeted delivery to TAMs.

Host cell targeting of novel antimycobacterial 4-aminosalicylic acid derivatives with tuftsin carrier peptides.

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.

Targeted Drug Delivery Using Tuftsin-bearing Liposomes: Implications in the Treatment of Infectious Diseases and Tumors.

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.

Legumain protease-activated tuftsin-functionalized nanoparticles for dual-targeting TAMs and cancer chemotherapy.

M2 tumor-associated macrophages (TAMs) play a pivotal role in cancer progression and therapy resistance. Inhibition of TAMs is of great significance to reshape the protumor environment to benefit therapeutic outcomes. In this work, we developed a novel TAMs and tumor cells dual-targeting nanoparticle (ATpep-NPs) system for cancer chemotherapy by integrating a docetaxel (DTX)-loaded nanocarrier and a multi-function peptide ATpep, which is composed of a phagocytosis-stimulating peptide-tuftsin (Tpep) fused with a substrate peptide-alanine-alanine-asparagine (AAN) of endoprotease legumain. In vitro protelytic and cellular uptake assays confirmed ATpep-NPs can be responsively activated into Tpep-NPs by cleavage of legumain that is overexpressed in both tumor cells and TAMs, which then promoted tumor cells internalization and TAMs phagocytosis through neuropilin-1/Fc receptor pathways. Due to AAN deactivation effect, ATpep-NPs can effectively decrease the Tpep-induced non-specific uptake by M1-polarized and normal macrophage during systemic circulation. Our results of in vivo experiments demonstrated ATpep-NPs outperformed Tpep-NPs in tumor and TAMs dual-targeting delivery efficiency with markedly enhanced efficacy against both tumor growth inhibition and TAMs depletion. Overall, this study offers a novel approach for development of multitargeted delivery vehicle for improved cancer chemotherapy.

Tuftsin-Bearing Liposomes Co-Encapsulated with Doxorubicin and Curcumin Efficiently Inhibit EAC Tumor Growth in Mice.

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.

Immunosuppressive properties of amino acid and peptide derivatives of mycophenolic acid.

Mycophenolic acid (MPA) was coupled with amino acids and biologically active peptides including derivatives of tuftsin to modify its immunosuppressive properties. Both amino acid unit in the case of simple MPA amides and modifications within peptide moiety of MPA - tuftsin conjugates influenced the observed activity. Antiproliferative potential of the obtained conjugates was investigated in vitro and MPA amides with threonine methyl ester and conjugate of MPA with retro-tuftisin occurred to be more selective against PBMC in comparison to parent MPA. Both amino acid and peptide derivatives of MPA acted as inosine-5'-monophosphate dehydrogenaze (IMPDH) inhibitors.

Synthesis and Biological Evaluation of Acridine/Acridone Analogs as Potential Anticancer Agents.

BACKGROUND: The lack of efficacious therapy for advanced melanoma and neuroblastoma makes new approaches necessary. Therefore, many scientists seek new, more effective, more selective and less toxic anticancer drugs. OBJECTIVE: We propose the synthesis of the new functionalized analogs of 1-nitroacridine/4- nitroacridone connected to tuftsin/retro-tuftsin derivatives as potential anticancer agents. METHODS: Acridine and acridone analogues were prepared by Ullmann condensation and then cyclization reaction. As a result of nucleophilic substitution reaction 1-nitro-9-phenoxyacridine or 1- chloro-4-nitro-9(10H)-acridone with the corresponding peptides, the planned acridine derivatives (10a-c, 12, 17-a-d, 19) have been obtained. The cytotoxic activity of the newly obtained analogs were evaluated against melanotic (Ma) and amelanotic (Ab) melanoma cell lines and neuroblastoma SH-SY5Y by using the XTT method. Apoptosis and cell cycle were analyzed by flow cytometry. RESULTS: Among the investigated analogs compound 12 exhibited the highest potency comparable to dacarbazine action for amelanotic Ab melanoma cells. FLICA test (flurochrome-labeled inhibitors of caspases) showed that this analog significantly increased the content of cells with activated caspases (C+) among both neuroblastoma lines and only Ab melanoma line. Using phosphatidylserine (PS) externalization assay, 12 induced changes in the Ab melanoma plasma membrane structure as the externalization of phosphatidylserine (An+ cells). These changes in neuroblastoma cells were less pronounced. CONCLUSION: Analog 12 could be proposed as the new potential chemotherapeutic against amelanotic melanoma form especially.

Immunomodulation of Murine Chronic DSS-Induced Colitis by Tuftsin-Phosphorylcholine.

Helminths or their products can immunomodulate the host immune system, and this phenomenon may be applied as the basis of new anti-inflammatory treatments. Previously, we have shown the efficacy of tuftsin-phosphorylcholine (TPC), based on a helminth product, in four animal models of autoimmune diseases: arthritis, colitis, systemic lupus erythematosus, and experimental autoimmune encephalomyelitis. We demonstrated that TPC reduced inflammatory process ex vivo in peripheral blood lymphocytes (PBLs) and in biopsies from giant-cell arteritis. In the present study, we assessed the therapeutic potential of TPC treatment on a chronic colitis murine model. C57BL/6 mice with chronic colitis were treated with TPC after the third cycle of 2% dextran sodium sulfate (DSS). Oral TPC treatment resulted in amelioration of the colitis clinical manifestations exemplified by reduced disease activity index (DAI) score, expansion of mesenteric lymph nodes (MLN) T regulatory cells (shown by Fluorescence Activated Cell Sorting (FACS)), significant reduction in the expression of pro-inflammatory cytokines (IL-1ß, IL17, IL-6, TNFα), and elevation in the expression of anti-inflammatory cytokine IL-10 (shown by RT-PCR). This study demonstrated the potential immunomodulatory effects of oral administration of TPC in a chronic colitis murine model. Further clinical trials are needed in order to evaluate this novel approach for the treatment of patients with inflammatory bowel disease.

Functionalized rifampicin-loaded nanostructured lipid carriers enhance macrophages uptake and antimycobacterial activity.

Tuberculosis is an infectious bacterial disease that causes millions of deaths worldwide. Current treatment recommended by WHO is effective, however it is an extensive and arduous process associated to severe adverse effects, which induces a low patient compliance and the emerging of multidrug resistant tuberculosis. Thus, as a main goal of this study, rifampicin nanoparticles were surface functionalized with a tuftsin-modifed peptide to selectively recognize receptors located on infected alveolar macrophages, enhancing nanoparticles uptake by these cells and improving antimycobacterial activity. A tuftsin-based modified peptide was synthesized and successfully attached to nanoparticles interface (NP-pRIF). In parallel, nanoparticles without peptide were also developed for comparison (NP-RIF). Physicochemical characterization demonstrated that stable and monodisperse nanodelivery systems were obtained, with a controlled drug release profile and non-cytotoxic potential. Moreover, nanoparticles containing peptide were significantly more internalized by macrophages than nanoparticles without peptide over a wide range of time. Both nanoparticles were 2-fold more effective against M. tuberculosis than free rifampicin, suggesting NP-pRIF as a promising strategy for the management of tuberculosis treatment.