A comparative study of [Leu1]Tuftsin and tuftsin, a natural phagocytosis-stimulating peptide

1. [Leu1]tuftsin was reported to have greater phagocytosis-stimulating activity than tuftsin (Thr-Lys-Pro-Arg). 2. However, a study on inactivation of tuftsin by polymorphonuclear leukocytes (PMNs) demonstrated that leucine aminopeptidase, an ecto-enzyme, located on PMN surface was responsible for this mechanism. 3. Since leucine aminopeptidase is known to cleave Leu more easily than Thr at the N-terminal position of peptides, this suggested to us that [Leu1]tuftsin might then be inactivated by PMNs more easily than tuftsin, and thus this analog might be less active than tuftsin. 4. In addition, many tuftsin preparations used in earlier studies were not fully active, as high-performance liquid chromatography was not available to separate out many contaminating diastereomers. 5. In view of this, we have synthesized and purified [Leu1]tuftsin and compared its phagocytosis-stimulating activity with tuftsin. 6. Our results indicate that [Leu1]tuftsin is not as active as tuftsin in stimulating phagocytosis.     

Co-administration of immunomodulator tuftsin and liposomised nystatin can combat less susceptible Candida albicans infection in temporarily neutropenic mice

In order to develop a prospective chemotherapeutic agent against opportunistic infections, it is important to know that host factors such as degree of immunological debility as well as recovery of immune functions to normality may contribute significantly to a successful elimination of the pathogens. We demonstrated previously that concomitant delivery of antimicrobial agents and immunomodulators to the pathogen harbouring-host contributes to the complete elimination of the deep-seated fungal infections (aspergillosis and candidiasis) in animals with normal immune status. Considering that neutropenic hosts are the main targets of such infections, it can be argued about the potential of the immunomodulator-based therapy in subjects with non-functional immune system. To resolve the hypothesis, we studied the role of immunomodulator tuftsin against experimental murine candidiasis in temporarily neutropenic Balb/c mice. The neutropenic mice were challenged with an isolate of Candida albicans that was showing less susceptibility to both free and liposomised-amphotericin B. The co-administration of tuftsin increased the efficiency of liposomised-polyene antibiotics (nystatin and amphotericin B) against experimental murine candidiasis in immunocompromised Balb/c mice. Pretreatment with liposomised tuftsin prior to C. albicans infection clearly enhanced protection against candidiasis, suggesting a prophylactic role of tuftsin in normal and temporarily neutropenic animals.     

Tuftsin,Thr-Lys-Pro-Arg

Summary Tuftsin, a natural occurring tetrapeptide, has been found to exhibit several biological activities connected with immune system function. Although little is known about tuftsin's biogenesis, much information has been gleaned about its structure-function relationships, which have shown that several features of the molecule are essential for expression of full biological activity. Furthermore, specific receptor sites for tuftsin have been found to exist exclusively on phagocytic cells. Research indicates that tuftsin binding to target cells effect intracellular calcium and cyclic nucleotide levels. Implication of these facts on tuftsin's mode of action are discussed.Basic peptidic segments resembling tuftsin are found in a variety of regulatory peptides. Questions are, therefore, raised as to the biospecificity and cross-reactivity of these sequences. Substance P, one such peptide, which binds with and activates tuftsin receptors, is described.In light of tuftsin's therapeutic potential, assays for its determination have been introduced. When applied to analyze human blood serum of normal as well as of various pathological origins, direct correlation was found between tuftsin levels and susceptibility to bacterial infections.     

Decreased tuftsin concentrations in patients who have undergone splenectomy.

Serum tuftsin concentrations were measured, using a radioimmunoassay developed in Israel, in normal subjects and in patients who had undergone splenectomy. Concentrations in those who had undergone traumatic and elective splenectomy were much lower. The tuftsin concentration in 38 patients with Hodgkin''s disease who had undergone splenectomy during staging laparotomy was not significantly different from the mean concentration in other patients who had had elective splenectomy. In four patients who underwent splenectomy for non-malignant haematological disorders measurements made before and after operation showed that tuftsin concentrations fell significantly in the days after operation. The increased susceptibility to overwhelming infections of patients with Hodgkin''s disease and others who have undergone splenectomy may be related to the low tuftsin concentrations. As pre-splenectomy tuftsin concentrations in patients with Hodgkin''s disease were normal, the practice of performing staging laparotomy and splenectomy in patients with Hodgkin''s disease should perhaps be reconsidered.     

13C nuclear magnetic resonance and circular dichroism studies of the tuftsin conformation in water

13C-NMR and circular dichroic (CD) spectra of tuftsin and its analogues are discussed in connection with our hypothesis that the beta-turn is the biologically active conformation of tuftsin. The changes in CD spectra evoked by an increase in pH are interpreted as a demonstration of the increasing amount of beta-turn conformers in solution. Configurational changes in successive residues of tuftsin showed that residues 2 and 3 of the peptide chain are important for the tuftsin conformation.     

Interaction on the antinociceptive effect between neurotensin and enkephalins or tuftsin

The aim of this paper was to study the interaction between neurotensin and both enkephalins or its synthetic analogue D-Ala2-metenkephalinamide, or tuftsin, on the antinonciceptive effect of these peptides in mice after intracisternal injection. Antinociception was measured by the hot-plate method. It was shown that neurotensin antagonized evidently the antinociceptive effect of enkephalins and their analogue. On the contrary, neurotensin and tuftsin were agonists in induction of analgesia. It is concluded that neurotensin modulates in an opposite way the function of the enkephalinergic neurons and the central action of tuftsin.     

The immunomodulatory effects of tuftsin on the non-specific immune system of Indian Major carp, Labeo rohita

The purpose of this study was to determine if injections of different dosages of tuftsin would enhance the immune response and disease resistance against the infections due to the opportunistic pathogens Aeromonas hydrophila and Edwardsiella tarda in Labeo rohita fingerlings. Hence, four different dosages of tuftsin in PBS suspension at the rate of 0, 5, 10, 15 mg kg(-1) body weight of fish were injected intraperitoneally to the fingerlings of L. rohita at 2-week intervals for four times. After every 2-week interval, different serum biochemical, haematological and immunological parameters of fish were evaluated. Biochemical and haematological parameters including serum total protein content, albumin content, globulin content, albulin:globulin ratio, glucose content, leucocyte counts etc.; cellular immune parameters including superoxide anion production, phagocytic activities, lymphokine production index etc.; humoral immune parameters including lysozyme activity, complement activity, serum bactericidal activity etc., in the fish were evaluated after every 2-week interval. After 56 days, fish were divided into two subgroups under each major treatment group for challenge with two pathogens A. hydrophila and E. tarda. The mortality (%) and agglutinating antibody titre was recorded on 28th day post challenge. Most of the immune parameters including leucocyte count, phagocytic ratio, phagocytic index, lysozyme activity, complement activity, and serum bactericidal activity were significantly (p相似文献   

Receptor-mediated endocytosis of tuftsin by macrophage cells

A fluorescent analog of the phagocytosis stimulating peptide tuftsin was prepared by coupling tetramethyl rhodamine isothiocyanate to a C-terminal elongated derivative of tuftsin. This analog, Thr-Lys-Pro-Arg-Gly-Lys(N epsilon-tetramethyl rhodamine)-OH, was used to visualize tuftsin receptors on mice macrophage cells by fluorescent image intensification. Fluorescent labelling was carried out at 37 degrees C, using a concentration of 200 nM and 2 microM of the fluorescent tuftsin derivative. The formation of peptide-receptor clusters and their subsequent internalization, as discerned by image intensification, were rapid processes, 5 min and 5-30 min, respectively. Preincubation of macrophages with tuftsin for various time intervals, followed by quantification of the tuftsin receptor using radiolabelled tuftsin, suggest that tuftsin receptors are initially increased in amount (5-7 min) and subsequently reduced (after 10-15 min) as judged by sites available for tritiated tuftsin. The binding studies are rather complementary to the fluorescence observations and support the assumption that the tuftsin receptor on the membrane of the mice macrophage cell is rapidly mobilized.     

Tuftsin and some analogs: synthesis and interaction with human polymorphonuclear leukocytes

The phagocytosis-stimulating tetrapeptide tuftsin, L-threonyl-L-lysyl-L-prolyl-L-arginine, was synthesized by both conventional and polymeric-reagent approaches. Using a combination of the two methods several analogs were prepared, including: [Ala1]tuftsin, [Lys1]tuftsin, [Ser1]tuftsin, [Val1]tuftsin, acetyl-tuftsin, p-aminophenylacetyl-tuftsin and tyrosyl-tuftsin. [Des-Thr1]tuftsin and [omega-NO2(4)]tuftsin were synthesized using a conventional procedure. The effects of synthetic peptides on the phagocytosis of heat-killed yeasts and on the reduction of the dye nitroblue tetrazolium by normal human polymorphonuclear leukocytes were investigated. Tuftsin and to a lesser extent [Lys1]tuftsin and [Ser1]tuftsin were found to stimulate phagocytosis, whereas the other analogs synthesized as well as [Ser1]tuftsin exhibited inhibitory effects to tuftsin's action. Tuftsin alone has stimulated nitroblue tetrazolium reduction; [Des-Thr1]tuftsin and [Ala1]tuftsin repressed this stimulation, while the other peptides showed no effect.     

Taftsin correction of pharmacologically induced behavioral disorders in white rats

Tetrapeptide tuftsin in doses adapted to its physiological blood concentrations partially normalized locomotor activity and orientation behaviour of rats altered by drugs affecting aminergic brain systems. At the same time tuftsin had no effect when applied after the treatment by dopaminergic drugs (DTC, haloperidol, apomorphine). It can be concluded that the central effect observed in the first minutes after tuftsin administration is mediated through dopaminergic system. Elimination of some drug-induced behavioural disturbances by tuftsin opens new prospects for its therapeutic application.     

Tuftsin: a naturally occurring immunopotentiating factor. I. In vitro enhancement of murine natural cell-mediated cytotoxicity

Tuftsin is a physiologic tetrapeptide, which has recently been shown to possess immunoadjuvant properties including the stimulation of macrophage and granulocyte phagocytosis, migration, bactericidal, and tumoricidal activities. Tuftsin has also been reported to possess in vivo immunologically mediated anti-tumor potential. To determine the potential role of tuftsin as an antineoplastic immunoadjuvant, the in vitro effects of tuftsin on murine natural cell-mediated cytotoxicity were studied. We observed that in vitro treatment of mouse splenic effector cells with synthetic tuftsin induced a pronounced enhancement of natural killer cell (NKC) cytotoxicity against the T cell lymphoma Yac-1. The magnitude of NKC enhancement was directly dependent upon the concentration of tuftsin employed, with maximum NKC stimulation observed at tuftsin concentrations of 50 to 100 microgram/ml. The tuftsin induced enhancement of NKC activity was not strain specific, since equivalent stimulation was seen in CBA/J, C56BL/10, and DBA/2 mice. Elimination of macrophages, monocytes, T cells, and immunoglobulin-bearing cells had no effect on the dose-dependent tuftsin stimulation of natural cell-mediated cytotoxicity; thus the characteristics of the effector cells activated by tuftsin were consistent with those reported for NKC. We also observed that treatment of splenic effector cells with tuftsin prolonged the cytotoxic capabilities of these cells beyond 18 hr.     

Potential use of tuftsin in treatment of candida peritonitis in a murine model

A major complication of continuous ambulatory peritoneal dialysis (CAPD) is peritonitis caused by Candida albicans. Increasing the activity of the peritoneal macrophages, the predominant cell type found in the peritoneal cavity, may be a promising treatment for this infection. Tuftsin was found to increase thioglycollate-elicited mouse peritoneal macrophage activity. 2x10(-7) M tuftsin enhanced two-fold cell association with radiolabelled candida, superoxide aniom production, and killing activity. Thus, a model consisting of mice undergoing peritoneal dialysis was developed in order to study the use of tuftsin as a therapeutic drug against peritoneal candidiasis. Administration of tuftsin (50 micrograms/mouse) before candidiasis induction with a lethal dose of candida (7x10(8) candida per mouse) improved mouse survival up to 70%, compared with 10% in the control group. The potential of tuftsin as a treatment for candidiasis was shown when the infection was induced with a sublethal dose of candida. Daily intraperitoneal injections of tuftsin (50 micrograms) to the sublethally infected mice caused a significant decrease in the number of candida recovered from the peritoneal cavity and from the blood (from 700 +/- 190 to 110 +/- 26 CFU/ml and from 100 +/- 26 CFU/ml to 17 +/- 8 CFU/ml, respectively). In addition, a larger number of peritoneal macrophages with greater phagocytic and killing activity were found in the tuftsin-treated mice. The effect of tuftsin may promote its potential use in the therapy of peritonitis in patients undergoing chronic peritoneal dialysis.     

Biochemical aspects of tuftsin deficiency syndrome

From work reported so far it is possible to draw certain conclusions namely, that Tuftsin, Thr-Lys-Pro-Arg, is a biologically functional entity. The presence of congenital familial deficiency reinforces this conclusion. The fact that these patients suffer from repeated infections points at an in vivo system that parallels the in vitro studies showing tuftsin stimulation of the phagocytic activity of the tissue macrophage and blood granulocyte. Such stimulation occurs at hormonal concentrations; (half maximal at 100 M). Furthermore, tuftsin enchances pinocytosis, as it does phagocytosis, only in phagocytic cells. It stimulates the motility of these cells as well as their longevity. Tuftsin stimulates the hexosemonophosphate shunt and, presumably through the formation of active oxygen-derived compounds, augments the bactericidal as well as the tumoricidal activity of the macrophage. There are highly specific receptors on the cell membrane of phagocytic cells. The structure of tuftsin cannot be altered without producing inactive and/or inhibitory analogs, an exception being the interchange of lysine and arginine. The release of tuftsin from carrier leukokinin requires two enzymes, one of which is on the outer membrane of the phagocyte and the other in the spleen. The absence of the latter explains the deficiency observed after the abrogation of splenic function for whatever cause.     

Effect of tuftsin and oligotuftsins on chemotaxis and chemotactic selection in Tetrahymena pyriformis

The chemotactic properties of tuftsin (H-TKPR-OH), tuftsin derivatives (H-KPR-OH, H-TKPKG-NH(2), Ac-TKPKG-NH(2)) and TKPKG-based oligotuftsins (T20, T30, T40) were investigated in Tetrahymena pyriformis GL. In contrast to its effects on Mammalia, tuftsin elicited chemorepellent or neutral responses; truncation of the N-terminal part (KPR) led to similar results, though with more neutral effects. The significance of the C-terminal part of the molecule was revealed by the chemoattractant properties of TKPKG, which are nevertheless abolished by acylation. Among the oligotuftsins, T20 and T40 were chemoattractants at higher concentrations (10(-9)-10(-6) M), while T30 had a wide-ranging chemorepellent effect, indicating that chemotaxis is elicited in Tetrahymena only by ligands with optimal physicochemical characters (mass, conformation, etc.). The chemotactic selection data indicated that tuftsin-induced chemotaxis results from fairly short-term signalling in Tetrahymena.     

Flow cytometry and biochemical analysis of DNA degradation characteristic of two types of cell death

Interactions of tuftsin (Thr-Lys-Pro-Arg) with bovine serum albumin (BSA) were analysed by fluorescence spectroscopy and circular dichroism. The data show that tuftsin interacts weakly with BSA, but this interaction is considerably enhanced by introducing an apolar substituent at the C-terminus of the tetrapeptide. It is suggested that strong binding of tuftsin to albumin in blood may enhance its macrophage-stimulating activity in vivo.     

Functional tuftsin binding sites on macrophage-like tumor line P388DI and on bone marrow cells differentiated in vitro into mononuclear phagocytes

Summary Mouse bone marrow cells, differentiated in vitro into mononuclear phagocytes (BMDMP), possess functional tuftsin binding sites, i.e. both tuftsin binding capacity and augmented phagocytic response related to tuftsin binding. The binding capacity of BMDMP was shown to be higher by a factor of about three than that exhibited by mouse monocytes and by normal, thioglycollate and Corynebacterium parvum induced peritoneal macrophages. The relatively high binding capacity did not depend on experimental variations in the in vitro culture of these populations (i.e. length of in vitro cultivation, source of serum or presence of conditioned medium leading to cell proliferation).The macrophage-like line, P388D1, was also shown to possess functional tuftsin binding sites and its binding capacity was comparable to that of the peritoneal macrophage populations.     

Polytuftsin: a potential precursor for slow release of the phagocytosis stimulating peptide tuftsin

1. Polytuftsin (Thr-Lys-Pro-Arg)n, was synthesized through polycondensation of an amino-free and carboxyl-activated derivative of tuftsin, H2N-Thr-Lys(Z)-Pro-Arg(Tos)-OSu, following suitable deprotection and fractionation steps. 2. Digestion of polytuftsin by trypsin, as well as by normal human serum, at 37 degrees C, yielded free tuftsin. 3. Polytuftsin affected the decreased formation of lung-metastasis, in B16 melanoma treated mice and prolonged the survival of animals more efficiently than tuftsin. 4. Tuftsin was found to be totally degraded by serum enzymes within approximately 60 min at 37 degrees C.     

Tuftsin-macrophage interaction: specific binding and augmentation of phagocytosis.

The binding of [3H]tuftsin to normal and in vivo stimulated mouse peritoneal macrophage populations was studied at 22 degrees C. The [3H]tuftsin binding to thioglycollate-stimulated macrophages was shown to be rapid and saturable, with an equilibrium dissociation constant (K(D)) (calculated from a Scatchard plot) of 5.3 X 10(-8) M. The calculated number of binding sites per macrophage amounts to approximately 72,000. Binding competition studies with unlabelled tuftsin yielded a K(D) of 5.0 X 10(-8) M. [3H] [N-Acetyl-Thr1]tuftsin, an inactive analog of tuftsin, failed to bind specifically to thioglycollate-stimulated macrophages. [N-Acetyl-Thr1]tuftsin and the tripeptide [Des-Arg4]tuftsin failed to compete for tuftsin binding sites, while [D-Arg4]tuftsin, an analog with small tuftsin-like activity, exhibited a low degree of inhibition of [3H]tuftsin binding. Thus a rather high degree of specificity is involved in the binding of the tetrapeptide. Normal as well as six different macrophage populations induced by stimulation with thioglycollate, concanavalin-A, starch, mineral oil, glucan and Bacillus Calmette Guerrin (BCG), exhibited a similar degree of binding of [3H]tuftsin. Corynebacterium parvum (CP)-stimulated macrophages, on the other hand, showed a 6- to 10-fold-lower capacity for tuftsin binding. Under similar experimental conditions, mouse fibroblast and lymphocyte preparations revealed no detectable specific binding. Tuftsin augmented the phagocytic response of normal and stimulated macrophages assessed both for phagocytosis mediated via the Fc-receptor and via non-specific receptors. CP-stimulated macrophages did not exhibit an increased phagocytic response upon treatment with tuftsin.     

Non-histone chromatin proteins during various stages of activity of immunocompetent cells

Summary Some of the properties of the tetrapeptide tuftsin, Thr-Lys-Pro-Arg, are discussed. We describe three phases of tuftsin activation of the macrophage. Tuftsinyltuftsin, the octapeptide Thr-Lys-Pro-Arg-Thr-LysPro-Arg, was synthesized with a view of minimizing the formation of Lys-Pro-Arg, from tuftsin by tissue aminopeptidases. The tripeptide is a tuftsin inhibitor. The octapeptide proved to be quite effective in prolonging the life of syngeneic mice injected with L1210 leukemia cells. Its effect in our laboratory, was considerably better than we could obtain with tuftsin. A simple method for purifying tuftsin by high performance liquid chromatography is described using 0.75% trifluoroacetic acid in water.The tuftsin sequence Thr-Lys-Pro-Arg is present in P 12 protein of Rausher murine leukemia virus. A close analog Thr-Arg-Pro-Lys appears in yet another virus protein the haemagglutinin of influenza virus. A second close analog Thr-Arg-Pro-Arg forms the penultimate carboxyterminal of a pancreatic polypeptide found in human and several animals.