Helminth-Related Tuftsin-Phosphorylcholine Compound and its Interplay with Autoimmune Diseases.

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.

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.

Tuftsin-based, EGFR-targeting fusion protein and its enediyne-energized analog show high antitumor efficacy associated with CD47 down-regulation.

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.

Up-regulation of MDP and tuftsin gene expression in Th1 and Th17 cells as an adjuvant for an oral Lactobacillus casei vaccine against anti-transmissible gastroenteritis virus.

The role of muramyl dipeptide (MDP) and tuftsin in oral immune adjustment remains unclear, particularly in a Lactobacillus casei (L. casei) vaccine. To address this, we investigated the effects of different repetitive peptides expressed by L. casei, specifically the MDP and tuftsin fusion protein (MT) repeated 20 and 40 times (20MT and 40MT), in mice also expressing the D antigenic site of the spike (S) protein of transmissible gastroenteritis virus (TGEV) on intestinal and systemic immune responses and confirmed the immunoregulation of these peptides. Treatment of mice with a different vaccine consisting of L. casei expressing MDP and tuftsin stimulated humoral and cellular immune responses. Both 20MT and 40MT induced an increase in IgG and IgA levels against TGEV, as determined using enzyme-linked immunosorbent assay. Increased IgG and IgA resulted in the activation of TGEV-neutralising antibody activity in vitro. In addition, 20MT and 40MT stimulated the differentiation of innate immune cells, including T helper cell subclasses and regulatory T (Treg) cells, which induced robust T helper type 1 and T helper type 17 (Th17) responses and reduced Treg T cell immune responses in the 20MT and 40MT groups, respectively. Notably, treatment of mice with L. casei expressing 20MT and 40MT enhanced the anti-TGEV antibody immune responses of both the humoral and mucosal immune systems. These findings suggest that L. casei expressing MDP and tuftsin possesses substantial immunopotentiating properties, as it can induce humoral and T cell-mediated immune responses upon oral administration, and it may be useful in oral vaccines against TGEV challenge.

[Tuftsin--new analogues and properties]. / Tuftsyna--nowe analogi i wlasciwosci.

Tuftsin, a natural tetrapeptide of sequence TKPR, occuring in the blood of humans and other mammals, capable of stimulating certain white blood cells (monocytes, macrophages, and neutrophils), was isolated at Tufts University in 1970 by Najjar and Nishioka. Tuftsin is a compound with a wide spectrum of biological activities, notable enhances phagocytosis, immune response, bactericidal, tumoricidal and antifungal activities. This article concerns new analogues and properties of tuftsin.

Effect of tuftsin on embryo vaccination with Newcastle disease virus vaccine.

The effect of tuftsin of embryo and post-hatch vaccination with NDV-F was studied. The embryo vaccination with NDV-F resulted in more number of dead-in-shell embryos. To overcome this problem, the vaccine was treated separately with ethyl methane sulfate (EMS) and 5-fluorouracil (5-FU) and administered. Treating the vaccine with 5-FU resulted in better hatchability as compared to EMS treatment. In embryo, NDV antibody titres increased upto 2 weeks of age and declined thereafter, whereas in post-hatch vaccination, the antibody titre increased from second to fourth week of age and declined thereafter. The seroconversion was better when the vaccine was given along with tuftsin either to embryos or chicks (post-hatch vaccination) as compared to those vaccinated without tuftsin. Moreover, the percentage of hatchability was more in tuftsin administered groups. It was found that embryo vaccination can ensure definite protection during the early life of the chicks despite the presence of maternal antibodies. In cases where breeder vaccinations do not result in concomitant transfer of antibody to progeny chicks, embryo vaccination would give only neonatal resistance. During the later stages, embryo vaccination did not confer any advantage over post-hatch vaccination.

Enhanced mucosal immune responses induced by a combined candidate mucosal vaccine based on Hepatitis A virus and Hepatitis E virus structural proteins linked to tuftsin.

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.

Drug targeting in Leishmania donovani infections using tuftsin-bearing liposomes as drug vehicles.

The efficacy of sodium stibogluconate against Leishmania donovani infections was markedly enhanced by encapsulating this drug in tuftsin-bearing liposomes. Also, pretreatment of the animals with these liposomes (free of drug) rendered them resistant to this infection, possibly by activating the host's macrophages. These results demonstrate that tuftsin-bearing liposomes besides delivering the drug to the target cells could also enhance the nonspecific resistance against infections, thus offering an additional advantage over the use of tuftsin-free liposomes as drug carriers in leishmania therapy.

Increase in the immunogenicity of HIV peptide antigens by chemical linkage to polytuftsin (TKPR40).

The use of synthetic peptide antigens in human prophylaxis still suffers from the very important problem of finding suitable carriers devoid of side effects. A desirable carrier for use in humans would be poorly immunogenic by itself, yet it would enhance the immune response to the peptide antigen. In the study reported herein, we examined the role of polytuftsin (TKPR40), a synthetic polymer of the natural immunomodulator tuftsin, as a carrier for synthetic peptides of HIV derived from the gp41 and gp120 proteins. Chimeric immunogens were constructed by chemical linkage between synthetic peptides of HIV and polytuftsin. These were employed for immunization of mice of different MHC haplotypes, and the humoral and cellular immune responses developed against the peptides were assessed by measuring total IgG, IgG, subclasses, T-cell proliferation, and in vitro cytokine release. A significantly stronger immune response was observed in mice immunized with the peptide-polytuftsin conjugates than in mice receiving the peptide dimers (peptide-peptide). Peptide-polytuftsin conjugates induced IgG2a and IgG2b isotype switching after both primary and secondary immunization. In addition, there was a positive correlation between the amounts of cytokines and the shift in the IgG isotypes. These data suggest that the use of polytuftsin as a carrier may increase the immune response against poorly immunogenic synthetic peptides.

The similarity between tuftsin (Thr-Lys-Pro-Arg) receptors and tuftsin antibody: a case of induced molecular mimicry.

The characteristics of binding of tuftsin (Thr-Lys-Pro-Arg) and several of its synthetic analogues, to specific membrane receptors on rabbit polymorphonuclear granulocytes, were compared with the binding characteristics of rabbit specific antibody to tuftsin. [3H-Arg4]-tuftsin was used as the principal ligand. Six analogues were studied. Two of these, Thr-Lys-Pro and Ala-Lys-tuftsin-Glu-Ala3, showed no binding affinity either to receptor or antibody. Ala-Lys-tuftsin showed less binding than tuftsin to both acceptors. Three showed stronger binding than tuftsin. The order of binding among these was tuftsin ( [Glu]2-tuftsin ( [Pro-Pro3]-tuftsin (tuftsinyltuftsin. This same order of binding was found with both receptor and antibody.

Tuftsin, a natural activator of phagocyte cells: an overview.

The recognition that a small oligopeptide was responsible for the full stimulation effect of specific cytophilic gamma-globulin on blood neutrophils arose from a study of the kinetics of phagocytosis. These were unusual in that the stimulation was short lived and that preincubation of the phagocyte with the gamma-globulin rendered the latter inactive. The oligopeptide was isolated, its structure determined (Thr-Lys-Pro-Arg) and synthesized. The discovery of human mutants with tuftsin deficiency exhibiting signs and symptoms of frequent severe infection further emphasized the specific biological function of the tetrapeptide. The mutant peptide was isolated, sequenced (Thr-Glu-Pro-Arg), and synthesized. Further studies showed that tuftsin requires two enzymes for its liberation from the parent carrier gamma-globulin. One enzyme is in the spleen that cleaves distal to the arginine end, and the other, on the outer side of the plasma membrane, cleaves proximal to the threonine residue. The tetrapeptide tuftsin stimulates all functions of phagocytic cells: phagocytosis, pinocytosis, motility, immunogenic activity including processing of the antigen and augmentation of the number of antibody-forming cells, bactericidal activity, and, above all, tumoricidal activity. The latter has been shown by several laboratories.

Tuftsin: on the 30-year anniversary of Victor Najjar's discovery.

After a short description of the results of Victor Najjar's research on tuftsin and of the discoveries done by other authors in the early stage of tuftsin investigation, the current state of work on tuftsin is presented, based mainly on the literature published in the years 1984-1997. The presentation follows this order: the occurrence of tuftsin and retro-tuftsin sequences in proteins, their synthesis and biology, the antigenic properties of tuftsin, its influence on phagocytic cells, and other biologic activities of tuftsin, including antimicrobial, antiviral, antitumor and central effects, and the search for tuftsin superactive analogs.

Insect tachykinin-like peptide: distribution of leucokinin immunoreactive neurons in the cockroach and blowfly brains.

Antisera were raised against leucokinin I, a cockroach myotropic neuropeptide with some resemblance to vertebrate tachykinins. These antisera were used for immunocytochemical mapping of neurons and neurosecretory cells in the brains of a cockroach and a blowfly species. The leucokinin immunoreactive cells are distinct from neurons that can be labeled with antisera against vertebrate type tachykinins. It is suggested that leucokinin-like peptides may have roles as neurohormones and neuromodulators in the insect nervous system.

[Autologous spleen transplantation]. / Lép autotransplantatio.

Splenectomy is known to increase the risk of overwhelming bacterial infection. There is a decrease in immunoglobulin IgM, T-lymphocytes, impaired primary antibody response to antigen challenge, an altered opsonic function and a tuftsin deficiency. Splenic autotransplantation has been suggested as a method of preserving function and this concept is supported by experiments in animals (dogs). The study describes autotransplantation of the traumatized spleen in human beings for the preservation of splenic function. Eleven patients operated on for abdominal trauma in the Kenézy Hospital in Debrecen, required total splenectomy, than splenic autotransplantation. In these patients splenic slides were implanted in between two layers of omental pouch (Furka's "spleen chip"). In 10 patients the follow-up radionuclid imaging, the IgM level, and the tuftsin level unambiguously confirmed the functioning of the splenic tissue.

Enhanced immune response induced by a potential influenza A vaccine based on branched M2e polypeptides linked to tuftsin.

Vaccination is the most effective means for preventing influenza-associated morbidity and mortality. Since the influenza virus mutates frequently, the virus strains for new vaccine production should be changed according to predicted epidemic strains. The extracellular domain of matrix protein 2 (M2e) is 24 amino acids long, which is highly conserved and therefore a good target for the development of a universal vaccine which may protect against a much wider range of influenza A virus strains. However its low antigenicity and immunogenicity, which are related to its small size, poses a big challenge for vaccine development. Multiple antigen peptide system (MAP) is based on an inert core molecule of radially branching lysine dendrites onto which a number of peptide antigens are anchored. Tuftsin is an immuno-stimulant molecule peptide. Here we developed a novel peptide vaccine by connecting a tuftsin to a branched, four-copy M2e. Not only did this increase the molecular mass, but also potentiate the immunogenicity. Two branched peptides, (M2e)4-tuftsin and (M2e)4-G4(tuftsin was replaced with four glycines), and a M2e monomer were synthesized using standard solid-phase methods. In vitro and in vivo studies were performed to compare their antigenicity and immunogenicity. Experiments in BALB/c mice demonstrated that the branched M2e could induce stronger humoral and cellular immune responses than the M2e monomer, and (M2e)4-tuftsin induced stronger humoral and cellular immune response than (M2e)4-G4. After lethal challenge with influenza virus PR8 strain, up to 80% of the animals in the (M2e)4-tuftsin vaccinated group still survived, in contrast to 44% in the (M2e)4-G4 group and 30% in the M2e monomer group. The combination of branched polypeptides and tuftsin in vaccine design is presented here for the first time, and the results show that the new construct is a promising candidate for a universal vaccine against the influenza A virus.

Anti-idiotypic single chain mimicking CA125 linked with tuftsin provides protective immunity against ovarian cancer in mice.

The activation of effector cells by bifunctional proteins to kill target cells has great potential in the treatment of cancer. In this study, a recombinant fusion protein composed of an anti­idiotypic single chain mimicking CA125 connected with tuftsin by an artificial linker was constructed. The fusion protein was found to manifest a number of biological activities, including activation of macrophages and stimulation of the T-cell response against cancer. Compared with single­chain Fv without tuftsin, the fusion protein showed stronger immunogenicity triggering humoral and cellular immune responses in mice. Fusion of tuftsin to scFv resulted in enhanced production of anti-anti-idiotypic antibodies and T-cell response. Protection against tumor challenges may be achieved in animals immunized with fusion protein. These results raise the possibility of employing cancer immunotherapy by administration of fusion proteins composed of anti­idiotypic antibodies and tuftsin.