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.

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.

Immune stimulating and therapeutic potential of tuftsin-incorporated nystatin liposomes against Cryptococcus neoformans in leukopenic BALB/C mice.

Cryptococcus neoformans infection is a common fungal infection in persons infected with human immune deficiency virus (HIV) or those with defective cell-mediated immunity. Since treatment of cryptococcal meningitis poses a big challenge, the present study aimed to develop a novel liposomal therapeutic formulation against cryptococcosis. Treatment with tuftsin-incorporated liposomes increased the anti-cryptococcal activity of murine peritoneal macrophages. Prophylactic treatment of mice with tuftsin-incorporated liposomes reduced the dissemination of C. neoformans to brain tissues. Moreover, the co-administration of tuftsin with nystatin liposomes augmented the anti-cryptococcal activity of nystatin, as mice treated with tuftsin-incorporated nystatin liposomes showed the highest survival and least fungal burden in their brain tissues. The results of the present study favour the use of immune-stimulating molecules along with antifungal agents in the treatment of opportunistic fungal infections.

Tuftsin promotes an anti-inflammatory switch and attenuates symptoms in experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a demyelinating autoimmune disease mediated by infiltration of T cells into the central nervous system after compromise of the blood-brain barrier. We have previously shown that administration of tuftsin, a macrophage/microglial activator, dramatically improves the clinical course of experimental autoimmune encephalomyelitis (EAE), a well-established animal model for MS. Tuftsin administration correlates with upregulation of the immunosuppressive Helper-2 T cell (Th2) cytokine transcription factor GATA-3. We now show that tuftsin-mediated microglial activation results in shifting microglia to an anti-inflammatory phenotype. Moreover, the T cell phenotype is shifted towards immunoprotection after exposure to tuftsin-treated activated microglia; specifically, downregulation of pro-inflammatory Th1 responses is triggered in conjunction with upregulation of Th2-specific responses and expansion of immunosuppressive regulatory T cells (Tregs). Finally, tuftsin-shifted T cells, delivered into animals via adoptive transfer, reverse the pathology observed in mice with established EAE. Taken together, our findings demonstrate that tuftsin decreases the proinflammatory environment of EAE and may represent a therapeutic opportunity for treatment of MS.

Augmentation of antileishmanial efficacy of miltefosine in combination with tuftsin against experimental visceral leishmaniasis.

Current drugs for the treatment of visceral leishmaniasis are inadequate, and their efficacies are also compromised due to suppression of immune function during the course of infection. Miltefosine is the only promising orally active antileishmanial drug, but due to its long half-life, there is risk of development of resistance. To overcome these problems, efforts are needed to develop combination therapy of miltefosine with effective immunostimulating agents where a decrease of parasitic burden and simultaneous enhancement of adaptive immunity can be achieved. In the present study, we have explored the antileishmanial efficacy of a subcurative dose of miltefosine in combination with free as well as liposomal palmitoyl tuftsin (p-tuftsin) using a Leishmania donovani/BALB/c mouse model. When miltefosine (2.5 mg/kg for 5 days) was given with free p-tuftsin, the inhibitory effect was significantly increased from 49.6% to 66% (P < 0.01), which was further enhanced up to 81% (P < 0.001) when given after liposomal encapsulation of p-tuftsin. Significant enhancement in parasitic inhibition (93%, P < 0.01) was witnessed when animals were co-administered with liposomal p-tuftsin + 5 mg/kg × 5 days dose of miltefosine (72.1%). Enhancement in the production of Th1 cytokines (IL-12, TNF-α, and IFN-γ), reactive oxygen, and nitrogen metabolites was witnessed in the combination group. A remarkable increase in phagocytosis index was also observed indicating overall immunological enhancement to antileishmanial activity of miltefosine by p-tuftsin.

Protective effect of liposomal formulation of tuftsin (a naturally occurring tetrapeptide) against cyclophosphamide-induced genotoxicity and oxidative stress in mice.

Tuftsin, a naturally occurring tetrapeptide with a sequence Thr-Lys-Pro-Arg was evaluated for its in vivo protective effect against cyclophosphamide-induced genotoxicity and oxidative stress in Swiss albino mice. The anticancer drug cyclophosphamide (CP) was administered intra-peritonially to induce mutagenic effect. The drug treatment caused significant increase in chromosomal aberrations, formation of micronucleated polychromatic erythrocytes (MNPCE's), as well as oxidative stress and decrease in lipid peroxidation in liver of the animals. The pretreatment with tuftsin abolished such effects in dose-dependent manner and also increased mitotic index in the experimental animals. Results of the present study validated chemo-preventive properties of tuftsin against CP-induced chromosomal mutations and cellular injury of liver by oxidative stress.

[Experimental hemorrhagic stroke: the study of neuropeptides (MIF, selank) in the intraperitoneal injection].

The aim of this study was to examine Taftsin derivates--macrophage inhibitory factor (MIF, Thr-Lys-Pro) and heptapeptide selank (Thr-Lys-Pro-Arg-Pro-Gly-Pro) in the model of experimental intracerebral hemorrhage in rats. The double autologous blood injection in the basal nucleus was used as a model of intracerebral hemorrhage. Animals ware randomly divided into three groups--the control group (n = 5) was treated with saline, the second group (n = 5) was injected with MIF in dose 150 mkg/ kg/day, the third group (n = 5) received Selank in dose 300 mkg/kg/day. Intraperitoneal injection of peptides was used. Body weight assessment, neurological examination and brain MRI were performed in 24, 72 hours and 10 days after the hematoma formation. The effect of neuropeptides on the functional restoration in animals, in the absence of the effect on hematoma volume and perifocal edema, was found. The significant reduction of perifocal edema and hematoma volume was observed in the 10th day after the hematoma formation in all experimental groups (p < 0.05). Only the control group of animals showed the significant (p < 0.05) weight loss in the 3rd day after the operation. The rate of neurological deficit was different: the significant improvement assessed with Menzes and limb placing test scales was seen only in the groups treated with neuropeptides in the 10th day.

Modulation of microglial/macrophage activation by macrophage inhibitory factor (TKP) or tuftsin (TKPR) attenuates the disease course of experimental autoimmune encephalomyelitis.

BACKGROUND: Myelin Oligodendrocyte Glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) is the most commonly used mouse model for multiple sclerosis (MS). During the of progression of EAE, microglia, the immunocompetent cells of the brain, become activated and accumulate around demyelinated lesions. Microglial activation is mediated by the extracellular protease tissue Plasminogen Activator (tPA), and mice lacking tPA display altered EAE progression. In this study, we have used pharmacological inhibitors and stimulators of microglial/macrophage activation to examine the temporal requirement for microglial activation in EAE progression and to determine whether such approaches might potentially be of therapeutic value. RESULTS: Intervention using the tripeptide macrophage/microglia inhibitory factor MIF (TKP) and the tetrapeptide macrophage/microglial stimulator tuftsin (TKPR) attenuated EAE symptoms and revealed that the timing of macrophage/microglial activation is critical for the clinical outcome of EAE. We show that the disease progression can potentially be manipulated favorably at early stages by altering the timing of microglial activation, which in turn alters the systemic immune response to favor upregulation of T helper cell 2 genes that promote recovery from EAE. CONCLUSION: Preventative and therapeutic modulation of macrophage/microglial activity significantly alters the outcome of EAE at symptomatic stages. Specific molecular targets have been identified that represent potential avenues of exploration for the treatment and prevention of MS.

Prophylactic use of liposomized tuftsin enhances the susceptibility of Candida albicans to fluconazole in leukopenic mice.

In the present study, we investigated the immunopotentiating activity of the immunomodulator tuftsin for the treatment of dose-dependent susceptible Candida albicans infection in a murine model. Our results demonstrated that tuftsin increases the susceptibility of C. albicans to phagocytosis by activating murine macrophages. Fluconazole used for the treatment of mice infected with C. albicans showed less in vivo efficacy and proved to be ineffective in the elimination of the infection from leukopenic mice even at higher doses. However, the antifungal activity of fluconazole against the same isolate of C. albicans significantly increased in tuftsin-pretreated mice and resulted in an improved survival rate in mice. The treated mice also showed less severity of infection as supported by a reduced fungal burden in their kidneys. This study indicates that the use of immunopotentiating substances can enhance the therapeutic efficacy of azole antifungal agents and thus can effectively combat azole-resistant fungal pathogens under conditions of immunosuppression.

Immunomodulator tuftsin increases the susceptibility of Cryptococcus neoformans to liposomal amphotericin B in immunocompetent BALB/c mice.

The co-administration of immunomodulators and antibiotics has been proved very successful for treatment of opportunistic infectious diseases. In the present study, we evaluated the combination of liposomal amphotericin B (lip-Amp B) and immunomodulator tuftsin to cure Cryptococcus neoformans infection in BALB/c mice. Mice infected with C. neoformans were treated with Amp B deoxycholate and tuftsin free or tuftsin-loaded Amp B liposomes. The results of the present study demonstrated higher efficacy of tuftsin-loaded Amp B liposomes against experimental murine cryptococcosis, in terms of enhanced survival rate and reduced fungal burden in organs (lungs and brain) of the treated mice. Interestingly, pre-treatment of mice with liposomal tuftsin before challenging them with the C. neoformans infection resulted in 100% survival of the treated animals followed by treatment with lip-Amp B. Immunomodulator-based therapy seems likely to be more beneficial for treatment of fungal infectious diseases.

Incorporation of amphotericin B in tuftsin-bearing liposomes showed enhanced efficacy against systemic cryptococcosis in leucopenic mice.

OBJECTIVES: The role of the immunomodulator tuftsin in enhancing the antifungal activity of liposomal amphotericin B against Cryptococcus neoformans in leucopenic mice was assessed. METHODS: In the present study, we investigated the antifungal activity of amphotericin B liposomes with tuftsin grafted on the surface. Mice were treated with free amphotericin B as well as liposomal formulations after C. neoformans infection. For prophylactic studies, mice were pre-treated with liposomal tuftsin (50 microg/mL) for three consecutive days prior to C. neoformans infection (7 x 10(5) cfu/mouse). Chemotherapy, with tuftsin-free and tuftsin-bearing amphotericin B liposomes, was started 24 h post C. neoformans infection. The role of tuftsin in immunoaugmentative therapy was assessed by survival and cfu of treated mice. RESULTS: Amphotericin B entrapped in tuftsin-bearing liposomes showed increased anticryptococcal activity in the murine model. Moreover, tuftsin pre-treatment further augmented the antifungal activity of liposomal amphotericin B in leucopenic mice. Incorporation of tuftsin in liposomes resulted in increased anticryptococcal activity of liposomal amphotericin B compared with amphotericin B deoxycholate and conventional liposomal amphotericin B formulations. CONCLUSIONS: The enhanced anticryptococcal activity of amphotericin B in tuftsin-liposomes can be attributed to the immune-stimulating property of tuftsin. Tuftsin activates the key immune cells, due to the presence of its receptors on macrophages and neutrophils, for a better fight against pathogens. Simultaneous liposome-mediated delivery of amphotericin B to the site of infection kills the pathogens more effectively.

[Evaluation of the effect of splenectomy with autologous spleen tissue implantation in some monocyte functions in children with hepatosplenic schistosomiasis mansoni]. / Avaliação do efeito da esplenectomia e auto-implante esplênico sobre algumas funções de monócitos em crianças com esquistossomose mansônica.

Adherence index, superoxide and TNF-alpha production in monocytes, with or without tuftsin treatment, were investigated in hepatosplenic schistosomiasis mansoni bearers with splenectomy with or without autologous implantation of spleen tissue. Three groups were evaluated: Healthy volunteers control group (CG) (n=12); Splenectomy with seft auto-transplant AG (n=18) and Splenectomy without auto-transplant WAG (n=9). Adherence index and TNF-alpha did not differ among the groups. Superoxide production was similar in CG and AG, in the 1st hour after cell stimulation. SP was larger in each hour time in CG and AG groups as compared WAS group. TT recovered normal pattern of SP in AG comparable with levels found in CG, with increase from the 1st to 2nd hour. However, TT did not alter SP in WAG, which remained reduced in all time points. Autologous implantation of spleen tissue seems to contribute for recovery and maintenance of the evaluated immunological reactions, which might be important in response to infections.

Tuftsin-bearing liposomes as antibiotic carriers in treatment of macrophage infections.

Tuftsin is a tetrapeptide (Thr-Lys-Pro-Arg) that specifically binds monocytes, macrophages, and polymorphonuclear leukocytes and potentiates their natural killer activity against tumors and pathogens. The antimicrobial activity of this peptide is significantly increased by attaching at the C-terminus a fatty acyl residue through the ethylenediamine spacer arm. This activity is further augmented by incorporating the modified tuftsin in the liposomes. The tuftsin-bearing liposomes not only enhance the host's resistance against a variety of infections but also serve as useful vehicles for the site-specific delivery of drugs in a variety of macrophage-based infections, such as tuberculosis and leishmaniasis.

Avaliação do efeito da esplenectomia e auto-implante esplênico sobre algumas funções de monócitos em crianças com esquistossomose mansônica / Evaluation of the effect of splenectomy with autologous spleen tissue implantation in some monocyte functions in children with hepatosplenic schistosomiasis mansoni

Investigamos em portadores de esquistossomose hepatoesplênica após esplenectomia com ou sem auto-implante esplênico: índice de aderência, produção de superóxido (SP) e de TNF-alfa em monócitos, tratados ou não com tuftsina. Avaliamos três grupos: voluntários sadios CG (grupo controle) (n=12); esplenectomizados com auto-implante AG (n=18) e esplenectomizados sem auto-implante WAG (n=9). índice de aderência e TNF-alfa não diferiram entre os grupos. SP foi semelhante em CG e AG na 1ª hora após estimulação celular. SP foi maior em todos intervalos de tempo nos grupos CG e AG, comparados ao WAG. O tratamento com tuftsina recuperou o padrão de normalidade de SP em AG, com aumento da 1ª para a 2ª hora nos níveis do CG. O tratamento com tuftsina não alterou SP em WAG, permanecendo reduzida em todos intervalos. O auto-implante esplênico parece recuperar e manter os parâmetros imunológicos avaliados, que têm participação importante na resposta do hospedeiro às infecções.

Prophylactic role of immunomodulators in treatment of systemic candidiasis in leukopenic mice.

In the present study, we have evaluated prophylactic role of various immunomodulators viz. lipopolysachharide, protein A and tuftsin to impart protection against experimental candidiasis in leukopenic mice. Both free as well as liposomised form of nystatin was not effective enough in offering complete cure against less susceptible isolate of Candida albicans (JNMCR) infection in immunodebilitant mice. Interestingly, the pretreatment of leukopenic mice with immunomodulators before challenging them with C. albicans increased therapeutic efficacy of the nystatin against systemic candidiasis. Efficacy of the treatment was evaluated on the basis of survival of the animals as well as fungal load in systemic circulation and various organs viz. liver, kidney, spleen and lungs of the treated animals.

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.

Use of tuftsin bearing nystatin liposomes against an isolate of Candida albicans showing less in vivo susceptibility to amphotericin B.

In the present study, we evaluated tuftsin bearing nystatin liposomes for their potential against an isolate of Candida albicans (C. albicans) showing less in vivo susceptibility to amphotericin B (Amp B). The liposomised-Amp B in higher doses was found to be effective in elimination of less susceptible strain of C. albicans (C. albicans JMCR) in Balb/c mice, but may not be recommended due to toxicity constraints. On the other hand, liposomal nystatin was shown to possess higher efficacy as compared to that of Amp B, and was pertinent in treatment of C. albicans JMCR strain. The data of present work reveals that the incorporation of nystatin in tuftsin-bearing-liposomes results in a significant increase in its efficacy against experimental murine candidiasis. Interestingly, the pre-treatment of animals with liposomised-tuftsin prior to challenge with C. albicans infection was more effective in elimination of the pathogen from host and shows an advantage in prophylactic perspectives.

Tuftsin-bearing liposomes in treatment of macrophage-based infections.

The use of liposomes as drug carriers in treatment of various diseases has been explored extensively for more than 20 years. 'Conventional' liposomes, when administered in vivo by a variety of routes, rapidly accumulate in the mononuclear phagocyte system (MPS). The inherent tendency of the liposomes to concentrate in MPS can be exploited in enhancing the non-specific host defence against infections by entrapping in them the macrophage modulators, and as carriers of antibiotics in treatment of intracellular infections that reside in MPS. This must further be enhanced by grafting on the liposome surface the ligands, e.g. tuftsin, that not only binds specifically to the MPS cells but also enhances their natural killer activity. Keeping this in view, we designed and developed tuftsin-bearing liposomes as drug carriers for the treatment of macrophage-based infections and outline these studies in this overview.