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Background: Wound healing is a complex and dynamic process of restoring cellular structures and tissue layers. The aim of the present investigation was formulation and optimization of instant film forming thermoreversible chitosan hydrogel for wound healing. Materials and Methods: A novel hydrogel composing of chitosan, Ethanol, Sodium α-β Glycerophosphate and combination of three drugs (Ascorbic acid, Tuftsin, Zn gluconate) were prepared. Ethanol and α-β Glycerophosphate (α-β-GP) were selected based on gelling time and film formation time. Optimization of vehicle in formulation was done by using 3² full factorial design using Design Expert® 8.0.7.1 software. Optimization of drug loaded thermoreversible chitosan hydrogel was done with 0.2mg, 2mg, 20mg, ratio of Zn gluconate, Tuftsin (10 μg/mL), Ascorbic acid (0.1mg/ mL) by trial-and-error method. Results and Conclusion: The Gelling time, Film formation time, swelling ratio and Moisture retention capacity of the optimize drug loaded formulation (X2 ) were 160±19.5, 390±18.4, 296±7.58, 6.8±0.76 respectively. The in vitro release of optimize drug loaded hydrogel formulation was carried out by Franz diffusion cell. In vitro drug release data of optimize batch was 86.5+1.9%, 80.66±4.52, 85.55±2.95 in 24 hr for Ascorbic acid, Tuftsin, Zn gluconate respectively. Chitosan hydrogel showed powerful antibacterial efficacy up to 100% to Staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 87064) by hydrogel formulation with or without drug. Since the formulation contains thermosensitive drugs stability study of optimized batch was carried out as per ICH guidelines. The stability data shows that the optimized batch of thermoreversible chitosan hydrogel remains stable for 1 month-controlled room temperature (25°C±2°C/ 60%RH±5%RH) and refrigerated condition (5°C±3°C). In vivo studies also carried out. [ABSTRACT FROM AUTHOR]

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.

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.

In order to improve the release pattern of chemotherapy drug and reduce the possibility of drug resistance, poly(ethylene glycol amine) (PEG)-modified alginate microparticles (ALG-PEG MPs) were developed then two different mechanisms were employed to load doxorubicin (Dox): 1) forming Dox/ALG-PEG complex by electrostatic attractions between unsaturated functional groups in Dox and ALG-PEG; 2) forming Dox-ALG-PEG complex through EDC-reaction between the amino and carboxyl groups in Dox and ALG, respectively. Additionally, tuftsin (TFT), a natural immunomodulation peptide, was conjugated to MPs in order to enhance the efficiency of cellular uptake. It was found that the Dox-ALG-PEG-TFT MPs exhibited a significantly slower release of Dox than Dox/ALG-PEG-TFT MPs in neutral medium, suggesting the role of covalent bonding in prolonging Dox retention. Besides, the release of Dox from these MPs was pH-sensitive, and the release rate was observably increased at pH 6.5 compared to the case at pH 7.4. Compared with Dox/ALG-PEG MPs and Dox-ALG-PEG MPs, their counterparts further conjugated with TFT more efficiently inhibited the growth of HeLa cells over a period of 48 h, implying the effectiveness of TFT in enhancing cellular uptake of MPs. Over a period of 48 h, Dox-ALG-PEG-TFT MPs inhibited the growth of HeLa cells less efficiently than Dox/ALG-PEG-TFT MPs but the difference was not significant (p > 0.05). In consideration of the prolonged and sustained release of Dox, Dox-ALG-PEG-TFT MPs possess the advantages for long-term treatment.

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 anti-inflammatory 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. [ABSTRACT FROM AUTHOR]

Abstract 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-1β, 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-1β, 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. Highlights • TPC decreased the production of various pro-inflammatory cytokines by activated PBMCs. • TPC reduced Th1 and Th17 differentiation in activated PBMCs. • TPC decreased IL-1β, IL-6, IL-13, IL-17A production and CD68 gene expression by TABs. • TPC had effects on PBMCs and TABs mainly comparable to those of dexamethasone. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A tuftsin-modified peptide (pTUF-OA) was successfully synthesized. • Rifampicin nanostructured lipid carriers were functionalized with pTUF-OA (NP-pRIF). • NP-pRIF presented a controlled drug release profile and non-cytotoxicity potential. • NP-pRIF was significantly more taken up by cells than naked nanoparticles. • NP-pRIF was 2-fold more effective against M. tuberculosis than free rifampicin. Abstract 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. [ABSTRACT FROM AUTHOR]

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.

Human lymphatic filariasis, a mosquito-borne neglected tropical parasitic disease, needs an early development of prophylactic agents such as a vaccine for its successful elimination. Our earlier study suggested the enhanced immunological response by fusion protein (P-TUFT-ALT-2) of Tuftsin and ALT-2 in a mice model. We cultured human peripheral blood mononuclear cells (PBMCs) and treated cells with Escherichia coli-expressed ALT-2 (E-ALT-2) and P-TUFT-ALT-2. Real-time polymerase chain reaction was performed to identify the mRNA copy number of various cytokine and transcription factor genes. The recombinant vaccine candidate was also validated for humans by immunoreactivity with human sera samples of natural infection. In this study, P-TUFT-ALT-2 stimulated 12% higher PBMC proliferation in endemic normal (EN) individuals than E-ALT-2 alone. There was enhanced production of IFN γ, IL-2, IL-5 and IL-12, indicating a balanced Th1/Th2 response. However, higher expression of IL-5 and lower IL-4 validate the humoral response through an IL-5-dependent manner. Also, high level of IL-17 indicates a strong Th/Treg regulation over T-cell activation. The upregulated T-bet might have enhanced IFN-γ production, whereas GATA-3 was supposed to enhance IL-5 expression. The fusion protein also exhibited 15–16% higher reactivity with EN clinical sera, exposing the upregulation of IgG1 and IgM in natural infection. The higher reactivity of P-TUFT-ALT-2 with sera of natural infection (EN) was validated indirectly by B-cell activation through various cytokines and regulatory genes produced from different T cells. Thus, these findings endorse P-TUFT-ALT-2 as a potential vaccine candidate for human lymphatic filariasis.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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-β, 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. [ABSTRACT FROM AUTHOR]

Summary: 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/F1mice 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‐1β 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. [ABSTRACT FROM AUTHOR]

The series of 16 novel amino acid and peptide mycophenolic acid (MPA) derivatives was obtained as potential antibacterial agents. Coupling of MPA with respective amines was optimized with condensing reagents such as EDCI/DMAP and T3P/TEA. Amino acid analogs were received both as methyl esters and also with the free carboxylic group. The biological activity of the products was tested on five references bacterial strains: Klebsiella pneumoniae ATCC 700603 (ESBL), Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus MRSA ATCC 43300, Staphylococcus aureus MSSA ATCC 25923. Peptide derivatives proved to be the most versatile ones, their MIC values relative to most strains was lower than MPA alone. It has been noted that the activity of amino acid derivatives depends on the configuration at the chiral center in the amino acid unit and methyl esters indicated better antimicrobial activity than analogs with free carboxylic group. [ABSTRACT FROM AUTHOR]

Epitopes from different proteins expressed by Mycobacterium tuberculosis (Rv1886c, Rv0341, Rv3873) were selected based on previously reported antigenic properties. Relatively short linear T-cell epitope peptides generally have unordered structure, limited immunogenicity, and low in vivo stability. Therefore, they rely on proper formulation and on the addition of adjuvants. Here we report a convenient synthetic route to induce a more potent immune response by the formation of a trivalent conjugate in spatial arrangement. Chemical and structural characterization of the vaccine conjugates was followed by the study of cellular uptake and localization. Immune response was assayed by the measurement of splenocyte proliferation and cytokine production, while vaccine efficacy was studied in a murine model of tuberculosis. The conjugate showed higher tendency to fold and increased internalization rate into professional antigen presenting cells compared to free epitopes. Cellular uptake was further improved by the incorporation of a palmitoyl group to the conjugate and the resulted pal-A(P)I derivative possessed an internalization rate 10 times higher than the free epitope peptides. Vaccination of CB6F1 mice with free peptides resulted in low T-cell response. In contrast, significantly higher T-cell proliferation with prominent expression of IFN-γ, IL-2, and IL-10 cytokines was measured for the palmitoylated conjugate. Furthermore, the pal-A(P)I conjugate showed relevant vaccine efficacy against Mycobacterium tuberculosis infection.

Rheumatoid arthritis (RA) is characterized by chronic autoinflammation of the joints, with a prevalence of about 1% in Western populations. Evidence in recent years has linked RA to changes in the gut microbiota (dysbiosis). Interestingly, helminths have been shown to have therapeutic activity in RA. Specifically, a glycoprotein containing phosphorylcholine (PC) extracted from helminths was found to have immunomodulatory activity. We have previously developed a novel chimeric compound composed of tuftsin-PC (TPC) that attenuates the joint destruction in mice with collagen-induced arthritis (CIA). Here, we address the interrelationship between TPC immunomodulatory activity and the gut microbiota in CIA mice. Preventive therapy with TPC in mice with arthritis maintained a physiological arthritis score as well as a steady gut microbial environment, similar to that of healthy controls, in contrast to CIA mice with severe disease. The microbial composition differed significantly between healthy and phosphate-buffered saline-treated CIA mice, enabling classifying test samples by machine learning based on levels of a small number of bacterial species. Using these bacterial biomarkers, all TPC-treated CIA mice were classified as healthy. Thus, we describe a clear correlation between TPC treatment, healthy gut microbial communities, and prevention of arthritis. This is the first study to demonstrate the immunomodulatory effect of helminth derivatives in autoimmune diseases and the link to gut microbiota. [ABSTRACT FROM AUTHOR]

Neutrophils play an important role in the innate immune response to infection with Streptococcus pneumoniae, the pneumococcus. Pneumococci are phagocytosed by neutrophils and undergo killing after ingestion. Other cellular processes may also be induced, including autophagy and the formation of neutrophil extracellular traps (NETs), which may play a role in bacterial eradication. We set out to determine how these different processes interacted following pneumococcal infection of neutrophils, and the role of the major pneumococcal toxin pneumolysin in these various pathways. We found that pneumococci induced autophagy in neutrophils in a type III phosphatidylinositol-3 kinase dependent fashion that also required the autophagy gene Atg5. Pneumolysin did not affect this process. Phagocytosis was inhibited by pneumolysin but enhanced by autophagy, while killing was accelerated by pneumolysin but inhibited by autophagy. Pneumococci induced extensive NET formation in neutrophils that was not influenced by pneumolysin but was critically dependent on autophagy. While pneumolysin did not affect NET formation, it had a potent inhibitory effect on bacterial trapping within NETs. These findings show a complex interaction between phagocytosis, killing, autophagy and NET formation in neutrophils following pneumococcal infection that contribute to host defence against this pathogen. [ABSTRACT FROM AUTHOR]

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 H 37 Rv 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. [ABSTRACT FROM AUTHOR]

Cucurbit[7]uril (CB7) is an uncharged and water-soluble macrocyclic host. CB7 binds to doubly protonated tuftsin, which is the tetrapeptide Thr-Lys-Pro-Arg, with moderate affinity (K a = 2.1 × 10 3 M − 1 ). In this study, the host–guest complexation was confirmed by fluorescence titration. This affinity would allow for easy release of the peptide under physiological conditions. According to density functional theory calculations, the structural binding motif involves hydrogen bonding. The most energetically stable form had the Arg side chain inside the CB7 cavity. The effects of the tuftsin–CB7 complex on the proliferation and cytokine activity of immune cells were studied. The complex had broader spectrum immunomodulation than free peptides, and caused statistically significant (p < 0,05) changes in cytokine production (tumor necrosis factor-α, interleukin-2, interferon-γ, and interleukin-10) by mononuclear cells. By contrast, the free peptide only activated tumor necrosis factor-α production. [ABSTRACT FROM AUTHOR]

Novel peptide conjugates of two antitubercular drug candidates were synthesised and characterised using new tuftsin peptide derivative (OT14) as carrier moiety. As antitubercular drug candidates two pyridopyrimidine derivatives, TB803 (2-allylamino-4-oxopyrido[1,2 -a ]pyrimidine-3-carbaldehyde) and TB820 (4-oxo-2-(pyrrolidin-1-yl)-pyrido[1,2 -a ]pyrimidin-3-carbaldehyde) inhibiting vital enzyme of Mycobacterium tuberculosis were applied. Membrane affinity of the compounds TB803 and TB820 and their peptide conjugates was evaluated using experimental lipid mono- and bilayer models. Penetration ability was assessed tensiometrically from Langmuir monolayer study and applying quartz crystal microbalance for the supported lipid bilayer (SLB) system. Minimal inhibitory concentration (MIC) values remained in a similar micromolar range for both of the conjugates while their cellular uptake rate was improved significantly compared to the drug candidates. A correlation was found between membrane affinity properties and results of in vitro biological investigations. Analysis of physical/structural properties of SLB in contact with bioactive components and visualization of the structural change by atomic-force microscopy (AFM) provided information on the type and route of molecular interaction of drug construction with lipid layers. The possible role of electrostatic interactions between lipid layer and drug candidates was tested in Langmuir-balance experiments using negatively charged lipid mixture (DPPC + DPPG). Especially the peptide conjugates presented increased membrane affinity due to cationic character of the peptide sequence selected for the conjugate formation. That is supposed to be one reason for the enhanced cellular uptake observed in vitro on MonoMac6 cell line. The conjugation of antitubercular agents to a peptidic carrier is a promising approach to enhance membrane affinity, cellular uptake rate and in vitro selectivity. [ABSTRACT FROM AUTHOR]

The present approach enumerates the effectiveness of tuftsin tagged nano-liposome for the cytosolic transport of 2,6-di-isopropylphenol-linolenic acid conjugate against liver cancer in mice. Initially, the conjugate in its free form was examined for anticancer potential on HepG2 liver cancer cells. Induction of apoptosis and suppression of migration and adhesion of HepG2 cells confirmed the effectiveness of conjugate as an anticancer agent. After this, role of the conjugate entrapped in a nano-carrier was evaluated in animal model. The nano-formulation comprising of conjugate bearing tuftsin tagged liposome was firsly characterized and then its therapeutic effect was determined. The nano-formulation had 100–130 nm size nanoparticles and showed sustained release of the conjugate in the surrounding milieu. The nano-formulation distinctly reduced the expression of COX-2, an important molecule that is vastly expressed in hepatocellular carcinoma. The utilization of in-house engineered nano-formulation was also successful in significantly up-regulating Bax and down-regulating bcl-2 gene expression eventually helping in better survival of treated mice. Histopathological analysis also revealed positive recovery of the general architecture and the violent death of cancer cells by apoptosis at tumor specific site. The site specific delivery of conjugate entrapped in tuftsin tagged liposomes was highly safe as well as efficaceous. Nano-formulation based approach showed a visible chemotherapeutic effect on liver cancer progression in experimental mice thereby making it a potential candidate for treatment of liver cancer in clinical settings. [ABSTRACT FROM AUTHOR]

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-1β ( P < 0.0001). TPC significantly expanded the CD4+CD25+ forkhead box protein 3 (FoxP3+) Treg cells and CD19+IL-10+CD5highCD1dhighT 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. [ABSTRACT FROM AUTHOR]

It has been shown by the method of probe microfluorimetry of single cells that tuftsin at concentrations of 0.1 and 1.0 μg/mL, increasing the phagocytic activity of murine peritoneal macrophages, causes biphasic changes in the intracellular pH (pHi) over time. First there was a decrease in pHi, reaching the limit in 5 min of incubation. Then, the pHi value increased until reaching the maximum 30 min after the interaction between the cells and the agent. Afterwards, the observed parameter (pHi) did not vary up to minute 55. It was shown that there was no increase in intracellular pH on the addition of the Na/H-exchange blocker ethylisopropylamiloride to the incubation medium in the presence of tuftsin. This fact suggests that the observed increase in pHi during tuftsin treatment in the second phase of cell response is associated with the Na/H exchange system. [ABSTRACT FROM AUTHOR]

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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

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. [ABSTRACT FROM AUTHOR]

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 TGFβ 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. [ABSTRACT FROM AUTHOR]

Hepatitis A virus (HAV) and Hepatitis E virus (HEV) are the most common causes of infectious hepatitis. These viruses are spread largely by the fecal-oral route and lead to clinically important disease in developing countries. To evaluate the potential of targeting hepatitis A and E infection simultaneously, a combined mucosal candidate vaccine was developed with the partial open reading frame 2 (ORF2) sequence (aa 368–607) of HEV (HE-ORF2) and partial virus protein 1 (VP1) sequence (aa 1–198) of HAV (HA-VP1), which included the viral neutralization epitopes. Tuftsin is an immunostimulatory peptide which can enhance the immunogenicity of a protein by targeting it to macrophages and dendritic cells. Here, we developed a novel combined protein vaccine by conjugating tuftsin to HE-ORF2 and HA-VP1 and used synthetic CpG oligodeoxynucleotides (ODNs) as the adjuvant. Subsequent experiments in BALB/c mice demonstrated that tuftsin enhanced the serum-specific IgG and IgA antibodies against HEV and HAV at the intestinal, vaginal and pulmonary interface when delivered intranasally. Moreover, mice from the intranasally immunized tuftsin group (HE-ORF2-tuftsin + HA-VP1-tuftsin + CpG) showed higher levels of IFN-γ-secreting splenocytes (Th1 response) and ratio of CD4+/CD8+ T cells than those of the no-tuftsin group (HE-ORF2 + HA-VP1 + CpG). Thus, the tuftsin group generated stronger humoral and cellular immune responses compared with the no-tuftsin group. Moreover, enhanced responses to the combined protein vaccine were obtained by intranasal immunization compared with intramuscular injection. By integrating HE-ORF2, HA-VP1 and tuftsin in a vaccine, this study validated an important concept for further development of a combined mucosal vaccine against hepatitis A and E infection. [ABSTRACT FROM AUTHOR]

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-1β, 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. [ABSTRACT FROM AUTHOR]

Experimental data on the effect of the natural immunopeptide tuftsin and its synthetic counterpart on hemostasis parameters in vitro and in vivo when administered intranasally to animals (white laboratory rats) are given. It has been shown that in vitro both preparations induced a fibrin-depolymerization effect on fibrin that is not stabilized by factor XIIIa. For each preparation, it was noted that, when administered to rats, simultaneous antiplatelet, anticoagulant, and fibrin-depolymerization effects were observed in the blood plasma, with Selank having a more pronounced effect. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]