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The synthesis of new analogues of cyclolinopeptide A (CLA) and their linear precursors modified with (R)- and (S)-4-methylpseudoproline in the Pro 3 -Pro 4 fragment are presented. The peptides were tested in comparison with cyclosporine A (CsA) in concanavalin A (Con A) and pokeweed mitogen (PWM)-induced mouse splenocyte proliferation and in secondary humoral immune response in vitro to sheep erythrocytes (SRBC). Their effects on expression of selected signaling molecules in the Jurkat T cell line were also determined. In addition, the structural features of the peptides, applying nuclear magnetic resonance and circular dichroism, were analyzed. The results showed that only peptides 7 and 8 modified with (R)-4-methylpseudoproline residue (c(Leu 1 -Val 2 -(R)-(αMe)Ser(ΨPro) 3 -Pro 4 -Phe 5 -Phe 6 -Leu 7 -Ile 8 -Ile 9 ) and c(Leu 1 -Val 2 -Pro 3 -(R)-(αMe)Ser(ΨPro) 4 -Phe 5 -Phe 6 -Leu 7 -Ile 8 -Ile 9 ), respectively) strongly suppressed mitogen-induced splenocyte proliferation and the humoral immune response, with peptide 8 being more potent. Likewise, peptide 8 more strongly elevated expression of Fas, a proapoptotic signaling molecule in Jurkat cells. We postulate that the increased biological activity of peptide 8, compared to the parent molecule and other studied peptides, resulted from its more flexible structure, found on the basis of both CD and NMR studies. CD and NMR spectra showed that replacement of Pro 3 by (R)-(αMe)Ser(¬Pro) caused much greater conformational changes than the same replacement of the Pro 4 residue. Such a modification could lead to increased conformational freedom of peptide 8, resulting in a greater ability to adopt a more compact structure, better suited to its putative receptor. In conclusion, peptide 8 is a potent immune suppressor which may find application in controlling immune disorders., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Phenothiazines represent a class of compounds of potential therapeutic utility. In this report we evaluated therapeutic value of an azaphenothiazine derivative, 6-acetylaminobutyl-9-chloroquino[3,2-b]benzo[1,4]thiazine (QBT), given intragastrically, in the model of dextran sodium sulfate-induced colitis in C57BL/6 mice using 5-aminosalicylic acid (5-ASA) as a reference drug. Colitis symptoms such as body weight loss, diarrhea and hematochezia (blood in stool) were observed and registered and disease activity index (DAI) was calculated. In addition, weight and cell numbers in the lymphatic organs and histological parameters of the colon wall were analyzed. The effects of QBT on viability of colon epithelial cell lines were also determined. We showed that weight and cell number of draining mesenteric lymph nodes were lower in mice treated with QBT in comparison to their control counterparts. The number of thymocytes, drastically reduced in control mice, was elevated in mice treated with the compounds with a significant effect of 5-ASA. In addition, an abnormal composition of blood cell types was partially corrected in these groups. Histological analysis of the colon revealed that the pathological changes were partially normalized by QBT and even to a higher degree by 5-ASA. In conclusion we demonstrated a therapeutic efficacy of the compound in amelioration of local and systemic pathological changes associated with chemically-induced colitis in mice. A possible mechanism of action of the compound is discussed.

Azaphenothiazines are predominantly immunosuppressive compounds. We evaluated the efficacy of an azaphenothiazine derivative, 6-chloroethylureidoethyldiquino[3,2-b;2',3'-e][1,4]thiazine (DQT) in prolongation of survival of skin allografts between BALB/c and C57Bl/6 mice. The mice were treated intraperitoneally (i.p.) with 100 μg of DQT on alternate days, on days 1-13 of the experiment (7 doses). The effect of DQT on a two-way mixed lymphocyte reaction (MLR) in the human model, as well as its effect on production of TNF α and IL-10 in a whole blood cell culture, stimulated by lipopolysaccharide (LPS), were evaluated. In addition, DQT effects were investigated regarding the proportion of T cell subsets in human peripheral blood lymphocytes (PBMC) by flow cytometry. Lastly, the effect of DQT on expression of signaling molecules involved in pro apoptotic pathways was determined by RT PCR. The results showed that DQT significantly extended skin graft survival. The compound also strongly suppressed two-way MLR in the human model at a concentration range of 2.5-5.0 μM. In addition, DQT inhibited LPS-inducible TNF α, but not IL-10 production. The compound preferentially caused a loss of the CD3-CD8+CD11b + PBMC cell subset, and transformed CD3+CD8+ high into CD3+CD8+ low cells. Lastly, we demonstrated significant increases in expression of caspases (in particular caspase 8) and of p53 in a culture of Jurkat T cells. We conclude that the immunosuppressive actions of the compound in allograft rejection may be predominantly associated with induction of cell apoptosis and inhibition of TNF α production. The apoptosis could be predominantly selective for the CD3-CD8+CD11b + cell phenotype., (Copyright © 2019 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

This work describes the synthesis of a new series of isoxazole derivatives, their immunosuppressive properties, and the mechanism of action of a representative compound. A new series of N ′-substituted derivatives of 5-amino- N ,3-dimethyl-1,2-oxazole-4-carbohydrazide ( MM1 ⁻ MM10 ) was synthesized in reaction of 5-amino- N ,3-dimethyl-1,2-oxazole-4-carbohydrazide with relevant carbonyl compounds. The isoxazole derivatives were tested in several in vitro models using human cells. The compounds inhibited phytohemagglutinin A (PHA)-induced proliferation of peripheral blood mononuclear cells (PBMCs) to various degrees. The toxicity of the compounds with regard to a reference A549 cell line was also differential. 5-amino- N ′-(2,4-dihydroxyphenyl)methylidene- N ,3-dimethyl-1,2-oxazole-4-carbohydrazide ( MM3 ) compound was selected for further investigation because of its lack of toxicity and because it had the strongest antiproliferative activity. The compound was shown to inhibit lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF α) production in human whole blood cell cultures. In the model of Jurkat cells, MM3 elicited strong increases in the expression of caspases, Fas, and NF-κB1, indicating that a proapoptotic action may account for its immunosuppressive action in the studied models.

Isoxazoles are an important class of compounds of potential therapeutic value. The aim of this study was to determine immunotropic effects of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide derivatives on spontaneous and mitogen-induced lymphocyte proliferation in young and old mice, cytokine production by peritoneal cells as well as possible mechanism of action in a model of Jurkat cells. Three-month-old and 13-month-old BALB/c mice were used as donors of the cells from a thymus, a spleen, mesenteric lymph nodes, and a peritoneal cavity. Spontaneous and concanavalin A or lipopolysaccharide (LPS)-induced cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric method. IL-1β and TNF-α production induced by LPS in macrophage-enriched peritoneal cell cultures was measured by enzyme-linked immunoassay. 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide, 01K (4-phenyl-1-(5-amino-3-methylisoxazole-4-carbonyl)-thiosemicarbazide), and 06K (4-(4-chlorophenyl)-1-(5-amino-3-methylisoxazole-4-carbonyl)-thiosemicarbazide) exhibited regulatory activity in the proliferation tests. Prevailing stimulatory activity of the hydrazide and inhibitory activity of 01K and 06K was observed. Those effects were connected with different influence of the compounds on signaling proteins expression in Jurkat cells. The regulatory effects of the compounds on IL-1β production were more profound than those on TNF-α. Differences in the compound activity in young versus old mice were mainly restricted to 01K. Immunosuppressive isoxazole leflunomide and a stimulatory RM-11 (1,7-dimethyl-8-oxo-1,2H-isoxazole [5,4-e]triazepine) were applied as reference drugs., (© 2016 John Wiley & Sons A/S.)

The nonapeptide fragment of the HLA-DR molecule, located in the exposed loop of the alpha-chain (164-172), having the VPRSGEVYT sequence, suppresses the immune response. Based on the three-dimensional structure of the HLA-DR superdimer, we designed a new cyclodimeric analog in which the two parallel peptide chains of VPRSGEVYT sequence are linked through their C-termini by spacer of (Gly5 )2 -Lys-NH2 and the N-termini are also linked by poly(ethylene glycol). The (VPRSGEVYTG5 )2 K-resin analog was synthesized using solid-phase peptide synthesis protocols. The cyclization was achieved by cross-linking the N-terminal positions of the dimeric peptide, attached to a MBHA resin, with alpha, omega-bis (acetic acid) poly(ethylene glycol), activated by esterification with pentafluorophenol. Our results demonstrate that the cyclodimerization of VPRSGEVYT results in enhanced immunosuppressive activity of the peptide. Mass spectrometry fragmentation analysis of the obtained cyclodimeric peptide is also presented. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd., (Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.)

Potential immunological activities of three compounds: RM54 and its two derivatives RM55 and RM56, were evaluated in several, selected in vitro and in vivo tests such as: mitogen-induced lymphocyte proliferation, cytokine production, the humoral immune response in vitro and carrageenan test. Leflunomide served as a reference drug. The studied compounds showed differential, generally immunosuppressive properties. RM56 exhibited stronger suppressive activities as compared to RM54 and RM55. In particular, RM56 displayed the strongest activity in suppression of the carrageenan inflammation that was correlated with strong suppression of the humoral immune response in vitro and lymphocyte proliferation. Density Functional Theory (DFT) was employed to shed a light on molecular properties of the investigated compounds. The geometrical parameters of the studied molecular structures were fully optimized at the B3LYP/6-311G(d,p) level. The atomic charges distribution derived on the base of the Mulliken population analysis was correlated with immunological activity of RM54, RM55 and RM56. The obtained relationships show that the isoxazole ring plays an important role in the observed immunological activities. We also suggest that due to strong anti-inflammatory and anti-proliferative properties of RM-56, potential therapeutic applications of this derivative can be broad.

Our previous studies revealed that ubiquitin and its decapeptide fragment with the LEDGRTLSDY sequence, located on the exposed molecule loop, strongly suppressed the immune response. This suggested that the loop may serve as a functional epitope of ubiquitin molecule and that a possible mechanism of biological action of the synthesized peptides is associated with interfering in interactions of ubiquitin with other molecules. Ubiquitin is known to exist in oligomeric forms, which can interact with various oligomeric receptors. We designed and synthesized new dimeric analogs of the ubiquitin fragment, to probe whether dimeric peptides may have higher affinity towards the ubiquitin receptors responsible for immunosuppression, which are believed to form oligomeric structures. Three dimerization strategies, N-terminus to N-terminus, C-terminus to C-terminus, and N-terminus to C-terminus (head-to-tail) via PEG derivatives were used to synthesize the dimeric peptides on solid support. In the course of our research, we developed a new and straightforward procedure of dimerization where α-amino groups of the C-terminal lysine residues of two peptide fragments were linked by PEG spacer directly on solid support. The effect of dimeric analogs on the immunological response was tested in the AFC in vitro experiment. The immunological tests showed that the head-to-tail dimerization caused a more profound increase in the biological activity than other tested dimerization methods. Our results suggest that such orientation of peptide components may correspond to orientation of the hypothetic ubiquitin receptors responsible for the immunomodulatory activity., (Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.)

Ubiquitin is a conservative polypeptide present in every eukaryotic cell. Apart from its involvement in proteasomal degradation and other intracellular signal pathways, it was suggested to play an important role as the extracellular immunomodulator and antimicrobial agent. Moreover, ubiquitin-derived peptides were shown to express significant biological activities. Our previous studies showed a high immunosuppressive potency of the ubiquitin peptic hydrolysate in which we identified over 70 different peptides. The present work focuses on synthesizing the most abundant of these peptides and investigating their immunomodulatory potency. The peptide VKTLTGKTI possessed the highest immunosuppressory activity in AFC experiments, comparable to the previously described LEDGRTLSDY sequence (a previously discovered ubiquitin-derived peptide). Moreover, some of the investigated peptides expressed immunostimulatory effects. These findings support the idea that ubiquitin, together with products of its degradation, could represent a self-regulating immunoregulatory system. Peptide VKTLTGKTI was also tested for its activity to prolong the skin graft survival in mice. The results showed that the investigated peptide significantly extended the skin transplant rejection time, therefore it could be considered as a potential supplementary medicine in the post-transplantation therapy. Moreover, we synthesized two analogs of investigated peptides, first designed to mimic the non-linear epitope consisting of ubiquitin 16-21 and ubiquitin 52-57 fragments, and second designed to mimic the ubiquitin 5-13 hairpin. We also tested their immunosuppressory activity in in vitro experiments., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Immune response suppressors are used in the medical praxis to prevent graft rejection after organ transplantation and in the therapy of some autoimmune diseases. Cyclolinopeptide A, naturally existing immunomodulatory peptide, was modified with homophenylalanine in positions 3 (4), 4 (5) or both 3 and 4 (6). The conformational influence of the replacement of Phe by Hphe was analyzed by NMR spectroscopy. Peptides 4-6 exist as single isomers with all trans peptide bonds except cis Pro-Pro peptide bond. The peptides were tested for their ability to suppress the proliferative response of mouse splenocytes to T- and B-cell mitogens and the secondary humoral immune response to sheep erythrocytes in vitro in parallel with a reference drug--cyclosporine A. The substitution of Phe with Hphe in positions 3 and 4 of CLA led to three different activities in the studied immunological assays. Very potent inhibition of AFC number of peptide 4 was not associated with cell toxicity. This compound caused a complete block of T- and B-cell proliferation. Peptides 5 and 6, containing Hphe in position 3 or 3 and 4, respectively, gave similar effects on the proliferative response of splenocytes to mitogens. Peptide 6 was a moderate suppressor of the humoral immune response, peptide 5 was exceptionally inhibitory. The presence of Hphe in position 4 of CLA backbone markedly reduced the viability of the tested cell line, however addition of the second Hphe in position 3 improved cell survival in comparison with the solvent.

Recently, ubiquitin was suggested as a promising anti-inflammatory protein therapeutic. We found that a peptide fragment corresponding to the ubiquitin(50-59) sequence (LEDGRTLSDY) possessed the immunosuppressive activity comparable with that of ubiquitin. CD and NMR spectroscopies were used to determine the conformational preferences of LEDGRTLSDY in solution. The peptide mixture, obtained by pepsin digestion of ubiquitin, was even more potent than the intact protein. Although the peptide exhibited a well-defined conformation in methanol, its structure was distinct from the corresponding 50-59 fragment in the native ubiquitin molecule. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 423-431, 2009.

In this study, we evaluated the activities of new types of azaphenothiazines in the following immunological assays: the proliferative response of human peripheral blood mononuclear cells induced by phytohemagglutin A or anti-CD3 antibodies; lipopolysaccharide-induced cytokine production by human PBMC; the secondary, humoral immune response in mice to sheep erythrocytes (in vitro); and delayed-type hypersensitivity in mice to ovalbumin (in vivo). In some tests, chlorpromazine served as a reference drug. The compounds exhibited differential inhibitory activities in the proliferation tests, with 10H-2,7-diazaphenothiazine (compound 1) and 6-(3-dimethylaminopropyl)diquinothiazine (compound 8) being most suppressive. Compound 1 was selected for further studies, and was found to be strongly suppressive in the humoral immune response even at low concentrations (1 microg/ml). Compound 1 also inhibited the delayed-type hypersensitivity lipopolysaccharide-induced production of tumor necrosis factor and interleukin-6 in cultures of human blood cells. As there were only two subjects in this study, the effects of these compounds on human blood cells need to be confirmed. In this paper, we also discuss the structure-activity relationships of selected compounds.

Immune response suppressors are used in the medical praxis to prevent graft rejection after organ transplantation and in the therapy of some autoimmune diseases. As a continuation of our previous work searching for new, effective suppressors devoid of toxicity, we present the synthesis, conformational analysis, and biological activity of nonapeptides 1-6, analogs of naturally existing immunomodulatory peptide CLA. New CLA analogs were modified with (S)-beta(2)-iso-proline 7 or (S)-beta(3)-homo-proline 8, respectively. The conformational influence of the beta-iso-proline and beta-homo-proline building blocks was analyzed by NMR spectroscopy. Peptides 1-6 exist as a mixture of four isomers due to cis/trans isomerization of the Xxx-Pro peptide bond. The major isomers of peptides 1, 3, and 4 contain all peptide bonds of the trans geometry. The geometry of the proline-proline bond of the second populated isomer of peptides 3 and 4 is cis. The proline-proline peptide bond is cis for the major isomers of peptides 2, 5, and 6. The peptides were tested for their ability to suppress the proliferative response of mouse splenocytes to T- and B-cell mitogens and the secondary humoral immune response to sheep erythrocytes in vitro in parallel with a reference drug-cyclosporine A. The immunoregulatory actions of the peptides depended on the position and content of proline isomers and were, with some exceptions, strongly inhibitory at the highest dose tested (100 microg/ml). In addition, the peptides were practically devoid of toxicity at that dose. In conclusion, the replacement of Pro by beta-Pro may be useful for fine-tuning CLA immunosuppressive potency and undesirable toxicity., (Copyright 2008 European Peptide Society and John Wiley & Sons, Ltd.)

A series of 5-amino-3-methyl-4-isoxazolecarboxylic acid semicarbazides and thiosemicarbazides (M1-M9) were obtained by reacting 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide with isocyanate and isothiocyanate. In order to determine structure-activity relationships for the series of nine compounds (M1-M9) geometry optimization was carried out using quantum-chemical DFT calculations at B3LYP/6-31g* level. The compounds, administered to mice at 1 microg or 10 microg doses, four hours prior to immunization with sheep erythrocytes (SRBC), significantly suppressed the primary, humoral immune response in mice as measured by the number of antibody-forming cells (AFC) in the spleens. Structure/activity relationships, regarding the studied compounds, were discussed.

The aim of this study was to evaluate efficacy of an isoxazole derivative RM11 to accelerate reconstitution of selected immune activities in cyclophosphamide (CP)-immunocompromised mice. We demonstrated that administration of fifteen 10 mug intraperitoneal doses of RM11, following a sublethal (200 mug/kg) dose of CP, significantly stimulated the number of antibody-forming cells (AFC) to sheep erythrocytes (SRBC) as determined 35 days after the CP treatment. Similarly, treatment of the CP-injected mice with 7 doses of RM11 significantly enhanced generation of delayed type hypersensitivity (DTH) to ovalbumin (OVA). Moreover, in that model, the treatment of mice with RM11 accelerated the process of myelopoiesis. RM11 also counteracted the suppressive action of methotrexate (MTX) in the in vitro model of the humoral immune response to SRBC. The phenotypic studies with fluorocytometer revealed that intraperitoneal 10 mug dose of RM11 significantly elevated the percentage of mature (CD3(+), CD4(+) and CD8(+)) T cells in the spleen and down-regulated the content of CD19(+) cells. We conclude that RM11 may be of potential therapeutic value in restoration of the immune status in patients undergoing chemotherapy.

The aim of this investigation was to evaluate effectiveness of RM-33, a new isoxazolotriazepine, in the model of carrageenan-induced inflammation in rats. Wistar rats were pretreated with intraperitoneal (ip) or oral (po) doses of RM-33, at daily doses ranging from 250 to 1000 microg, administered 1-3 days before elicitation of the carrageenan reaction. We showed that both routes of RM-33 administration were effective in significantly diminishing the footpad edema. The effects were dose-dependent and better pronounced at the ip administration of the compound. We found a lower production of tumor necrosis factor alpha (TNF-alpha) by mitogen-stimulated splenocytes isolated from rats pretreated with RM-33 and injected with carrageenan, as well as lower serum TNF-alpha levels in these rats, as compared to the respective control. Histological analysis of the skin reaction site revealed that in the rats pretreated with RM-33, the carrageenan-induced inflammation was reduced, as reflected by a lesser damage of mast cells, smaller infiltration by macrophages and a diminished edema of the connective tissue. Together with our previous data, indicating the antagonistic action of RM-33 in the adjuvant-induced footpad inflammation in mice, the present results confirm the anti-inflammatory activity of RM-33 compound.

Experimental evidence from previous studies supports the conclusion that orally administered lactoferrin (LF) restores the immune response in mice treated with a sublethal dose of cyclophosphamide (CP). The aim of this study was to elucidate potential benefit of LF in mice undergoing chemotherapy with busulfan (BU) and CP, followed by intravenous (i.v.) injection of bone marrow cells. CBA mice were treated orally with busulfan (4 mg/kg) for 4 consecutive days, followed by two daily doses of CP delivered intraperitoneally (i.p.) at a dose of 100 mg/kg and reconstituted next day with i.v. injection of 10(7) syngeneic bone marrow cells. One group of these mice was given LF in drinking water (0.5% solution). After treatment, mice were immunized with ovalbumin (OVA) to subsequently measure delayed type hypersensitivity responsiveness and with sheep red blood cells to determine humoral immunity by evaluation of splenic antibody-forming cells. As expected, both humoral and cellular immune responses of mice that were treated with these chemotherapeutic agents was markedly impaired. Here we report that this impairment was remarkably attenuated by oral administration of LF. Humoral immunity fell to levels that were 66-88% lower than that of untreated animals. Humoral immunity of LF-treated animals was equivalent to that of untreated mice within 1 month. Cellular immune responses were inhibited by chemotherapy treatment to a lesser degree, reaching levels that were approximately 50% lower than those of untreated animals. Again, LF mitigated this decrease, resulting in responses that were only slightly lower than those observed in untreated animals. Furthermore, when mice were given a lethal dose of BU (4 x 25 mg daily doses, i.p.) followed by a bone marrow transplant, LF caused enhanced lympho-, erythro-, and myelopoiesis in the bone marrow and appearance of transforming splenic lymphoblasts, similar to effects caused by administration of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). In summary, our study suggests that LF may be a useful agent to accelerate restoration of immune responsiveness induced by chemotherapy in bone marrow transplant recipients.

The aim of this investigation was to evaluate immunotropic properties of a new isoxazolotriazepine (compound RM33) in mice. We found that RM33 significantly inhibited induction of the humoral immune response to sheep red blood cells (SRBC), given 2 h prior to immunization. The development of the cellular immune response (delayed type hypersensitivity--DTH) to SRBC and to ovalbumin (OVA) was also suppressed, as well as the effector phase of the DTH to OVA. The compound was also effective when administered per os. The suppressive effects of RM33 on the immune response were comparable to those of cyclosporine A(CsA). We also showed that RM33 inhibited DTH to OVA if admixed with the sensitizing dose of an antigen and complete Freund's adjuvant (cFa) suggesting that the compound may affect initial events of antigen presentation. Such a hypothesis was supported by finding that RM33 significantly inhibited foot pad edema elicited by administration of cFa. The effects of RM33 on the activities of cytokines relevant to development and control of the immune response and inflammation such as: tumor necrosis factor alpha (TNF-alpha), interleukin 6 and 10 (IL-6 and IL-10) were also studied. The compound markedly (by 63%) inhibited lipopolysaccharide (LPS)-induced TNF-alpha serum level whereas IL-6 activity was lowered to a lesser extent (by 17%). The inducible IL-10 level in the splenocyte cultures was not affected at all. In summary, the presented results revealed immunosuppressive properties of RM33, which could be associated with its selective interference with co-stimulatory signals provided by adjuvant at initiation of the immune response.

Our previous studies revealed that lactoferrin (LF) reconstitutes the cellular and humoral immune response in cyclophosphamide-treated mice. The aim of this investigation was to establish whether the suppressory effects of methotrexate (MTX) on the cellular and humoral immune response can be modulated by LF. We found that MTX, given intraperitoneally (i.p.) at a dose of 200 mg/kg b.w., 48 h following sensitization of CBA mice with ovalbumin (OVA), reduced by 80% the delayed type hypersensitivity (DTH) response. Co-administration of LF in drinking water (0.5% solution) for the duration of the experiment (4 days) restored the DTH response almost to the control level. However, LF was not able to restore the primary humoral immune response, measured by the number of antibody-forming cells (AFC) to sheep erythrocytes (SRBC) in the spleens when MTX (1 mg/kg b.w.) was administered to mice i.p. 48h post immunization. On the other hand, mice treated with LF after second challenge with SRBC showed significant restoration of the MTX-suppressed humoral immune response following the booster immunization. In addition, LF (1 microg/ml) restored the secondary humoral immune response to SRBC in vitro when MTX (0.05-1 mM) was added to cell cultures on day 2 following cell culture initiation. These data demonstrate that LF preferentially restores the cellular immune response impaired by MTX treatment. It seems that LF also prevents the block of the activity of T memory cells in the secondary, humoral immune response. Taken together, we demonstrated that LF given orally can reduce the toxic effects of MTX.

Previous studies on cyclophosphamide (CP)-immunocompromised mice showed accelerated reconstitution of immune system function following oral treatment with lactoferrin (LF). The aim of this investigation was to evaluate the ability of mice, treated with a sublethal dose of CP and given LF, to combat bacterial infections. Mice were injected with a single, intraperitoneal dose of CP (350 mg/kg body weight). One group of CP-treated mice was also given LF in drinking water (0.5% solution) for 14 days. Untreated and LF-treated mice served as controls. On day 15 following CP administration, mice were infected intravenously with 10(8) Escherichia coli or 5 x 10(7) Staphylococcus aureus. Twenty-four hours later, the number of colony-forming units (CFU) in spleens and livers were determined. Phenotypic analysis of blood leukocytes was determined, as well as the ability of splenic and peritoneal cells to produce IL-6 spontaneously and in the presence of lipopolysaccharide (LPS). Treatment with CP, or with CP and LF, led to profound reduction of E. coli CFU in the liver and the spleen; treatment with LF alone had significant inhibitory effects on organ enumerated CFU. S. aureus CFUs were also significantly reduced in spleens of mice treated with CP or CP/LF and, to a lesser degree, after LF alone. These effects were also significantly reduced in the livers. Analysis of blood cellular phenotype revealed total number of peripheral leukocytes was lower in the CP-treated group (52.6%) but not significantly different from control values in CP/LF and LF-treated groups (90.7% and 104.6%, respectively). Conversely, percentage of blood neutrophils was markedly elevated in CP and CP/LF groups--62% and 42.5% vs. 18.4% in controls. These findings were accompanied by production of IL-6 by splenic and peritoneal cells which was significantly increased in CP- and CP/LF-treated groups. It was concluded that the increased clearance of bacteria in the organs of mice treated with CP and CP/LF may result from a rise in the number of neutrophils infiltrating the organs and contributing to accelerated clearance of bacteria. The study also suggests that the ability of cells from CP- and CP/LF-treated mice to produce significantly more IL-6 may also contribute to increased resistance to infections. Lastly, together with our previous data, this study indicates that LF used to reconstitute the antigen-specific immune response in CP-treated mice does not impair their resistance to infection.

A peptide fragment corresponding to the ubiquitin(50-59) sequence (LEDGRTLSDY) (U50-59) possesses a very high immunosuppressory activity, comparable to that of cyclosporine, both in the cellular and humoral immune responses. We found that the pentapeptide DGRTL (U52-56) is the shortest, effective immunosuppressory fragment of ubiquitin, although its potency is weaker than that of U50-59. Replacement of each consecutive residue with alanine in U52-56 allowed identification of essential amino acids involved in the immunosuppression. We also evaluated the roles of its N- and C-terminal groups by their acetylation and/or amidation, respectively. The active sequence is located in the external loop of the molecule and therefore it may serve as an important functional epitope for intermolecular binding. Based on the crystal structure of ubiquitin molecule, we designed and synthesized the cyclic analogue with a restricted conformation, cyclo(Glt-Gln-Leu-Glu-Asp-Gly-Arg-Thr-Leu-Ser-Asp-Lys)-NH2 (Glt = glutaryl) by reacting the C-terminal Lys side chain with the glutarylated N-terminus. The peptide was designed to mimic the ubiquitin(48-59) loop, in order to obtain the ligand that may interact with hypothetical receptors of the loop. The cyclization product selectively but strongly suppresses the cellular immune response. The results indicate that the 48-59 loop may serve as an important functional epitope in the ubiquitin molecule for intermolecular binding., (Copyright 2004 Wiley Periodicals, Inc.)

Methotrexate (MTX) is one of the immunosuppressory compounds applied in the prophylaxis and treatment of several diseases, including: rheumatoid arthritis, systemic lupus erythematosus, psoriasis, graft-versus-host disease and, in combination with other drugs, neoplastic diseases. Studies in humans and in animal model, of clinical disease, have demonstrated that MTX diminishes the clinical symptoms of various immunological disorders. MTX, an antagonist of folic acid synthesis, causes apoptosis in activated cells, primarily in the G1 and S phases of the cell cycle. One of its actions, is inhibition of the synthesis or activity of several proinflammatory cytokines. At high doses, MTX is cytotoxic to hemopoietic cells; low doses, however, promote hemopoiesis. MTX also induces the differentiation of monocytic tumor cells, which may explain, in part, its therapeutic effects in the treatment of some disorders. The compound suppresses both cellular and humoral immune response and mitogen-induced lymphocyte proliferation. These effects are dose- and time-dependent. The strongest suppressory activity is exerted when MTX is applied 24 or 48 hours after immunization or mitogen stimulation. Administration of MTX in unfavorable conditions such as stress or other diseases, diminishes its tolerance and increases its toxicity. Side-effects of MTX may be ameliorated by application of pharmacological synthetic agents and plant extracts. In summary, MTX has been an effective agent in suppressing immunological disorders (for 50 years) and is still finding many applications.

Previous studies have shown that oral treatment with lactoferrin (LF) restores the immune response in cyclophosphamide (CP) immunocompromised mice. The aim of the present investigation was to determine the regulatory ability of LF on the production of interleukin 6 (IL-6) in peritoneal and alveolar cells, derived from CP-treated mice. CBA mice were injected with a single, intraperitoneal (i.p.) dose of CP (350 mg/kg body weight) followed by LF administered in drinking water (0.5% solution) for 21 days. The control counterparts were given water. Peritoneal and alveolar cells were isolated from mice and the production of IL-6, both spontaneous and lipopolysaccharide (LPS) induced, was determined in 24h cell cultures using a bioassay. The results showed increased production of IL-6 in both CP-treated mice and in mice given, in addition, LF. The administration of LF alone led also to an increase in IL-6 production by the cell cultures. Intravenous (i.v.) administration of LPS resulted in a significant increase in IL-6 serum levels in CP and CP/LF but not in LF-treated mice. Analysis of cell type composition in the peritoneal cavity revealed a strong increase in mastocyte and neutrophil content in CP and CP/LF-treated groups. Our findings suggest that enhanced IL-6 production in CP and CP/LF-treated mice may contribute to reconstitution of immune system function in immunocompromised mice.

Looking for active immunosuppressant, a series of substituted phenylamides of 5-aminomethinimino-3-methylisoxazole-4-carboxylic acid was obtained, which showed immunosuppressory activities in the in vitro and in vivo tests, comparable with that of cyclosporine A. Rentgenostructural studies of three most representative derivatives were performed and the molecular modelling of compounds, demonstrating most characteristic biological activities, was performed. In the next stage, quantum-chemical investigations were conducted in order to determine structure-activity relationships.

In this communication we reveal differential inhibitory effects of cyclosporine A (CsA) on generation of the cellular and humoral immune responses to sheep erythrocytes (SRBC) in mice. For the analysis of the regulatory effects of CsA, we analyzed the results of 45 separate experiments performed in recent years where CsA served as a reference drug for our research on various immunoregulatory compounds. The humoral immune response was determined as the number of plaque-forming cells (PFC), and the delayed type hypersensitivity (DTH) was measured by foot pad swelling. We demonstrated that treatment of mice intraperitoneally (ip) with a dose of 100 microg of CsA/mouse, 2 h after immunization resulted in a differential pattern of inhibition of these two types of the immune response depending on the magnitude of the response in a given experiment. In the case of the antibody response (mean number of PFC 2312 median 2200), high PFC numbers were inhibited stronger than low ones; mean values in respective quarters and inhibitory actions were the following: 1,552 (42.3%), 2,049 (52.4%), 2,441 (61.2%) and 3042 (62.5%). In consequence, high, medium and low responses were down-regulated to approximately the same level. Another inhibitory pattern was observed in the DTH model (mean 10.35 units, median 10.8 units), i.e. low DTH responses were suppressed more strongly than high ones. The mean DTH responses and suppression effects in respective quarters were: 7.4 (58.2%), 10.1 (52.7%),11.8 (51.8%) and 13.1 (38.8%). As the result of such CsA action, the DTH response profile was parallel to that of the control response. In summary, the humoral immune response was down-regulated by CsA proportionally to the immune response observed in control mice, while DTH response was inversely proportional. The possible mechanisms of the observed regulatory CsA actions are discussed.

The aim of this study was to evaluate immunotropic properties of vratizolin, a known antiviral drug, in several in vitro and in vivo assays in mouse and human models. We demonstrated that vratizolin exerted strong immunosuppressive actions both in the humoral and cellular immune response to SRBC in mice. The compound affected not only the inductive phase of delayed type hypersensitivity (DTH) but also the effector phase of that response. Vratizolin was effective when given intraperitoneally and orally. The inhibitory action of vratizolin was comparable to that of cyclosporin A (CsA), the reference drug. Vratizolin exhibited also suppressory properties with regard to PHA-induced proliferation of human peripheral blood lymphocytes and that effect exceeded the inhibitory action of CsA. We also showed that vratizolin inhibited to some degree LPS-induced cytokine production in human peripheral blood cultures. The activities of TNF-alpha, IL-1 and IL-6 were inhibited on average by 37, 26 and 35%, respectively. This was in contrast to the effects of CsA which strongly inhibited only IL-1 production. Lastly, we demonstrated that vratizolin markedly inhibited growth of several tumor cell lines. In particular, the compound significantly inhibited growth of mouse leukemia L-1210 and human acute lymphoblastoid leukemia CCRF-CEM cell lines. The presented data suggest that the immunosuppressory action of vratizolin, although similar to that of CsA, is mediated by a different mechanism. The properties of vratizolin, described in this report, indicate that the drug should be further investigated for possible immunosuppressory and antitumor application.

A series of 4-imino derivatives of the 5-amino-3-methylisoxazole-4-carboxylic acid hydrazide and 5-amino-3-methylisoxazole[5,4-d]-6,7-dihydropyrimidine has been prepared by condensation of 5-amino-3-methylisoxazole-4-carboxylic acid hydrazide with carbonyl compounds. The resulting products were evaluated for their immunological activities in the models of the humoral and cellular immune responses of mice in vivo and concanavalin A (Con A) and lipopolysaccharide (LPS)-induced splenocyte proliferation. In addition, effects on polyclonal antibody production by human peripheral blood cells in culture were investigated. For all studied compounds we carried out quantum chemical calculations at ab initio B3LYP 6-31G(d, p) level. The stimulatory or inhibitory effects depended strongly on the origin and location of substitunets, which is described in the conclusions and was supported by QSAR studies.

2-Aminobenzimidazole reacted with selected esters of alpha, beta-unsaturated acids and alpha, beta-unsaturated ketones to form derivatives of 1,2,3,4-tetrahydropyrimido[1,2-a]benzimidazol-2-ones, 1,4-dihydropyrimido[1,2-a]benzimidazoles, and 2-acetylaminobenzimidazole. 2-Cinnamoylaminobenzimidazole, 4-phenyl-1,2,3,4-tetrahydropyrimido[1,2,-a]benzimidazol-2-on e, 4-(benzimidazol-2-ylamino)-4-phenylbutan-2-one, and 4-methyl-2-phenyl-1,2,3,4-tetrahydropyrimido[1,2-a]benzimidazole were tested for their potential activity in immunological assays in the mouse model. Compound 1, at a dose of 100 micrograms/mouse, significantly inhibited the humoral immune response and, at the same time, stimulated the cellular type of that response. The proliferative response of mouse splenocytes to concanavalin A was inhibited only at a higher dose (2 micrograms/ml). The immunotropic activity of 4, on the other hand, was uniformly strongly inhibitory in all applied tests. The suppressive activity of 4 was lower in the cellular immune response and proliferation tests than that of cyclosporin A.

The synthesis of two series of derivatives containing the quinazolinone-4 moiety is described. 3-Amino-2(1H)-thioxo-4(3H)-quinazolinone (1) was subjected to reactions with halogenoketones and halogenoaldehydes, leading to the production of the corresponding ketones, aldehydes, Schiff bases, and 6-oxo-1,4,5-thiadiazin[2,3-b]quinazoline derivatives. Subsequently, 1 was condensed with selected alpha, beta-unsaturated carbonyl compounds, aldehydes, ketones, acid chlorides, and esters. The compounds were tested for their potential activity in a model of humoral and cellular immune response. The tests showed that the compounds exhibited differential immunotropic activities. Of particular interest is compound 19, exhibiting a strong stimulatory activity with regard to cellular immune response and compound 16 exerting a strong inhibitory action in both types of the immune response.

Linear and cyclic analogues of cyclolinopeptide A (CLA) in which one or both phenylalanine residues in fragment Pro6-Pro7-Phe8-Phe9 were substituted by their sulfonated derivatives have been synthesized by SPPS method and cyclization with the BOP reagent. The peptides were examined for their immunosuppressive activity in the humoral and cellular immune response by PFC and DTH tests. All of the analogues retain some immunosuppressive activity of native CLA. Their CD spectra confirm that the optical activity of aromatic residues in CLA depends on their position in the peptide chain. Only the residue in position 8 seems to be optically active. CD spectrum of the cyclic analogue modified in position 9 is very similar to that of native CLA which correlates with its high biological activity. The chiroptical properties of the p-sulfonated Phe-residue are established.

The effects of Lys residue deletion from peptides, related to immunosuppressive regions of lactoferrin (LF), were studied in the humoral and cellular immune responses to sheep erythrocytes (SRBC) in mice. We found that the deletion of Lys residue, vicinal to Pro, influences mostly the immunomodulatory activity of short fragments (tetra-, and pentapeptides) of immunosuppressive 231-245 mini-domain of LF. The activity of longer peptides, related to this mini-domain, is influenced to much lower extent by Lys removal. In particular, the tetrapeptide derived from Arg-Lys-Pro-Val-Asp sequence by deletion of Lys, exhibits strongly reduced immunosuppressive activity. On the contrary, the similar change within Arg-Lys-Pro-Val-Thr sequence, derived of 575-589 loop of LF, produces a tetrapeptide Arg-Pro-Val-Thr, that is active as immunosuppressor both in humoral and cellular immune response. A quite strong immunosuppressive activity of tripeptide Lys-Arg-Pro is also worth consideration, especially when compared with the loss of activity observed for Thr-Arg-Pro tripeptide. The data presented herein indicate that exclusion of Lys residue from the LF-derived immunomodulatory peptides differentially alters their effects on the immune response to SRBC in mice.

It was found that tuftsin (Thr-Lys-Pro-Arg) and its tetrapeptide analogues with the same amino acid composition, but different sequences, demonstrate immunosuppressive activity for the humoral, but not (excepting the Thr-Lys-Arg-Pro tetrapeptide) for the cellular, immune response. The splitting of N-terminal residues from these tetrapeptides leads, however, to formation of tripeptides that are active in both humoral and cellular immune response tests. Thus, the biological degradation of peptides of the type investigated can seriously modulate their immunobiological activities. We also found that tetrapeptides Thr-Arg-Lys-Pro and Thr-Lys-Arg-Pro, as well as tripeptides Arg-Lys-Pro and Lys-Arg-Pro, distinctly differ in their immunomodulatory activities, although the structural differences consist in this case only in the displacement of two basic amino acid residues.

The immunosuppressive activity of three pentapeptides related to the C-terminal fragments of tachykinins was investigated using the PFC (plaque forming cell) test. The peptides were of the general structure Phe-X-Gly-Leu-Met-NH2, with Tyr, Val and Ile in X position, respectively. The results were compared with those obtained previously for the substance P (SP) C-terminal pentapeptide. It was found that peptides containing aromatic residues in X position were more potent immunosuppressors than peptides containing aliphatic residues in the same position. The results also suggest that in mammalian organisms substance SP may be involved in immunomodulation which is rather not the case with other mammalian tachykinins (neurokinin A and neurokinin B).

Synthesis of four analogs, containing D-amino acid residues, of the tripeptide immunosuppressor Lys-Arg-Pro is described. In addition, acetyl and amide derivatives of this peptide were synthesized. Biological effects of all peptides were studied using a delayed type hypersensitivity (DTH) test. It was found that incorporation of D-amino acid residues in definite positions of the peptide chain gives the substances with immunosuppressive activity. A strong immunosuppressive activity was observed also for all-D-analogs (D-Lys-D-Arg-D-Pro).

A series of linear and cyclic hexapeptides, consisting of: Phe-Phe-Val-Pro-Gly-Ala, Tyr-Phe-Val-Pro-Gly-Ala, Phe-Tyr-Val-Pro-Gly-Ala, D-Phe-Tyr-Val-Pro-Gly-Ala, D-Tyr-Phe-Val-Pro-Gly-Ala, D-Tyr-Tyr-Val-Pro-Gly-Ala, c-(Phe-Tyr-Val-Pro-Gly-Ala-), c-(Tyr-Tyr-Val-Pro-Gly-Ala-) was synthesized and examined for the influence on humoral immune response by the plaque forming cell test. The peptides are related to Tyr-Val-Pro-Gly-Phe-Pro hexapeptide (an active fragment of immunomodulatory polypeptide found in sheep colostrum) and to the retro-sequence of cycloamanide A, a peptide immunosuppressor present in the mushroom Amanita phalloides. We found that substitution of one or both phenylalanine residues of the linear retro-sequence of cycloamanide A by tyrosine, enhances the immunosuppressive activity of the peptide. The inversion of configuration of N-terminal aromatic residues preserves the immunosuppressive activity. However, in this case, application of higher doses of the peptides decreases their activity. The highest immunosuppressive potency was found for Phe-Tyr-Val-Pro-Gly-Ala hexapeptide, which is most closely related to the active fragment of a peptide immunomodulator from the ovine colostrum, investigated by us previously.

The immunosuppressive activity of a linear peptide related to cyclolinopeptide A, Leu-Ile-Ile-Leu-Val-Pro-Pro-Phe-Phe, and its nine analogs, where each successive amino acid residue was substituted by alanine, was investigated using the plaque forming cell and delayed type hypersensitivity tests for the humoral and cellular immune response, respectively. The linear peptide was less active than its cyclic counterpart in the humoral, and equally active in the cellular immune response. All alanine analogs showed quite strong immunosuppressive potencies in the humoral immune response tested in vitro. In the in vivo humoral immune response their activities were much less differentiated than it was observed in the in vitro experiments. All alanine analogs retain the immunosuppressive activity in the cellular immune response. However, their activities are practically not differentiated, and it is impossible to decide which amino acid side chains of the parent peptide are the most important for generation of the immunosuppressive effects. CD spectra of alanine analogs show that, in comparison with the parent peptide, conformational equilibria of these peptides are shifted towards unordered conformations. This finding correspond well to the results of the biological tests which show that all alanine analogs are less biologically active than the parent peptide. The results of CD measurements suggest also that interaction of the hydrophobic residues located at the ends of the peptide chain may be of importance for stabilization of the folded structure of the investigated compounds.

The immunosuppressor activity of the cycloamanides A, B, C, and D, and two of their D-amino acid residue-containing analogues, was examined using PFC (plaque forming cell) and DTH (delayed type hypersensitivity) tests. It was found that cycloamanide A (CyA A, II) [c-(Phe-Phe-Ala-Gly-Pro-Val-)] and its D-Phe-containing analogue III [c-(Phe-D-Phe-Ala-Gly-Pro-Val-)] are the most potent immunosuppressors of the whole series. The retroanalogue of III [c-(D-Phe-Val-Pro-Gly-Ala-)] was found to be less active than III. The immunosuppressor activity of O-carboxymethyl-Tyr6-antamanide (I) was also tested. It was found that the substitution of one of the Phe residues of ANT by O-carboxymethyl-Tyr does not substantially affect the immunosuppressor activity.

We demonstrated that a linear peptide Gly-Ile-Ile-Leu-Val-Pro-Pro-Phe, the analog of a cyclic nonapeptide c-(Leu-Ile-Ile-Leu-Val-Pro-Pro-Phe-Phe) possesses a distinct immunosuppressive activity. We synthesized a series of eight other analogs of the linear peptide. The series consists of the nonapeptides protected on N- and/or C-terminus by acetyl-, succinyl-, and amido- functions and of Gly-Ile-Ile-Leu-Val-Pro-Pro-Phe-Phe elongated on N- and/or C-terminus by additional glycine residues. It was found that the protection of the terminal functional groups of the peptide slightly increases its immunosuppressor potency. At the same time the elongation of the peptide chain on both termini by additional glycine residues evokes a distinct increase of the immunosuppressive activity.

In connection with our discovery of a strong immunosuppressive activity of cyclolinopeptide A (CLA), we investigated immunosuppressive properties of antamanide and a number of its analogues, including symmetrical antamanide, and compared them with the activities of cyclosporin A and CLA. The peptides were investigated by using plaque forming cell (PFC), graft-versus-host (GvH), delayed type hypersensitivity (DTH), and autologous rosette formation cell (ARFC) tests. Antamanide and symmetrical antamanide exhibit an immunosuppressive activity lower than CLA. Linear antamanide fragments are also active. At higher concentrations of the latter peptides, toxic effects occur.

Immunosuppressive properties of six tyrosine analogues of cyclolinopeptide A were studied and compared with the activity of cyclosporin A. The peptides were investigated using PFC (in vitro) and DTH tests.

It was found that substitution of Val2 and/or Leu4 residue in a hexapeptide Tyr-Val-Pro-Leu-Phe-Pro (I) transforms this peptide immunostimulant into analogues possessing the immunosuppressor activity--Tyr-Gly-Pro-Leu-Phe-Pro (II), Tyr-Val-Pro-Gly-Phe-Pro (III), and Tyr-Gly-Pro-Gly-Phe-Pro (IV). Biological effects of peptides I-IV were studied using PFC (plaque forming cell) test, GvH (graft vs host) reaction in mice, and ARFC (autologous rosette forming cell) test. The strongest immunosuppressor activity was observed for III--in this case the immunosuppressor effect was observed even in PFC test in vitro in which II and IV showed no such activity. These results suggest that simultaneous presence of both Val2 and Leu4 residues is necessary for the generation of immunostimulation. The CD study of I-IV in methanol solution suggests that the conformational preferences of II-IV change towards stabilization of the beta-turn structure, whereas in the case of I the gamma-turn on Leu4 and cis' orientation of the Pro3 carbonyl (distorted beta-turn of type I) was found as the preferred conformation. Competition in biological effects observed for I and III in PFC in vitro test suggests that these analogues may interact with the same cellular receptor. Drastic changes in the activity which accompany changes in the sequence are discussed in the terms of our stereochemical hypothesis.