Age-Dependent Effects of Yolkin on Contact Sensitivity and Immune Phenotypes in Juvenile Mice.

Yolkin, an egg yolk immunoregulatory protein, stimulates the humoral but inhibits the cellular immune response in adult mice. The aim of this investigation was to evaluate the effects of yolkin administration on the immune response using a model of juvenile, i.e., 28-day- and 37-day-old, mice. We examined the yolkin influence on the magnitude of the cellular immune response, which was determined as contact sensitivity (CS) to oxazolone (OXA), and the humoral immune response, which was determined as the antibody response to ovalbumin (OVA). Yolkin was administered in drinking water, followed by immunization with OXA or OVA. In parallel, the phenotypic changes in the lymphoid organs were determined following yolkin treatment and prior immunization. The results showed that yolkin had a stimulatory effect on CS in the mice treated with yolkin from the 37th day of life but not from the 28th day of life. In contrast, no regulatory effect of yolkin on antibody production was found in 28-day- and 37-day-old mice. Phenotypic studies revealed significant changes in the content of B cells and T cell subpopulations, including CD4+CD25+Foxp3 regulatory T cells. The association between the effects of yolkin on the magnitude of CS and phenotypic changes in main T- and B-cell compartments, as well the importance of changes in T-regulatory and CD8+ cells in the age categories, are discussed. We conclude that the immunoregulatory effects of yolkin on the generation of CS in mice are age dependent and change from stimulation in juvenile to suppression in adult mice.

A cytotoxic effect of human lactoferrin fusion with Fc domain of IgG.

Lactoferrin (LTF) is a natural iron-binding protein with a potential for clinical utility in many human immune disorders, including cancer. A fusion of LTF with the Fc domain of IgG2 (FcLTF) was designed with inherent properties of an extended the half-life in circulation. Furthermore, the effects of LTF and FcLTF were assessed for influence on the activity of natural killer (NK) cells isolated from human peripheral blood, on the NK-92 cell line, and on human monocytes. The NK cytotoxic activity induced by LTF and FcLTF was determined against the human leukemia K562 cell line, and also for monocytes, by measuring TNFα and granzyme B production, and in an assay for Jurkat cell viability. Selected gene expression in NK-92 cells and monocytes, induced by LTF and FcLTF, was performed by Real Time PCR. No significant difference was observed in NK-92 cytotoxicity stimulated by LTF and FcLTF. The effects on NK cells isolated from the human peripheral blood were varied, possibly due to the immunoregulatory nature of LTF sensing the immune status of donors. Furthermore, only the FcLTF group strongly stimulated production of TNFα and granzyme B in isolated monocytes. In addition, only supernatants from the monocyte cultures treated with FcLTF decreased the viability of Jurkat cells. The ability of FcLTF to induce TNFα in monocytes was strongly inhibited by anti-CD32 and moderately inhibited by anti-CD14 antibody. Lastly, it was demonstrated that FcLTF, strongly induced expression of PI3K, with subsequent activation of AKT/mTOR signaling pathway. Overall, it was demonstrated that this novel fusion molecule may be a perferred choice for clinical utility than the wild type LTF.

Structural and Biofunctional Insights into the Cyclo(Pro-Pro-Phe-Phe-) Scaffold from Experimental and In Silico Studies: Melanoma and Beyond.

Short peptides have great potential as safe and effective anticancer drug leads. Herein, the influence of short cyclic peptides containing the Pro-Pro-Phe-Phe sequence on patient-derived melanoma cells was investigated. Cyclic peptides such as cyclo(Leu-Ile-Ile-Leu-Val-Pro-Pro-Phe-Phe-), called CLA, and cyclo(Pro-homoPro-ß3homoPhe-Phe-), called P11, exert the cytotoxic and the cytostatic effects in melanoma cells, respectively. CLA was the most active peptide as it reduced the viability of melanoma cells to 50% of control at about 10 µM, whereas P11 at about 40 µM after 48 h incubation. Interestingly, a linear derivative of P11 did not induce any effect in melanoma cells confirming previous studies showing that cyclic peptides exert better biological activity compared to their linear counterparts. According to in silico predictions, cyclic tetrapeptides show a better pharmacokinetic and toxic profile to humans than CLA. Notably, the spatial structure of those peptides containing synthetic amino acids has not been explored yet. In the Cambridge Structural Database, there is only one such cyclic tetrapeptide, cyclo((R)-ß2homoPhe-D-Pro-Lys-Phe-), while in the Protein Data Bank-none. Therefore, we report the first crystal structure of cyclo(Pro-Pro-ß3homoPhe-Phe-), denoted as 4B8M, a close analog of P11, which is crucial for drug discovery. Comparative molecular and supramolecular analysis of both structures was performed. The DFT findings revealed that 4B8M is well interpreted in the water solution. The results of complex Hirshfeld surface investigations on the cooperativity of interatomic contacts in terms of electrostatic and energetic features are provided. In short, the enrichment ratio revealed O…H/H…O and C…H/H…C as privileged intercontacts in the crystals in relation to basic and large supramolecular H-bonding synthon patterns. Furthermore, the ability of self-assemble 4B8M leading to a nanotubular structure is also discussed.

Antiviral Activity of a Cyclic Pro-Pro-ß3-HoPhe-Phe Tetrapeptide against HSV-1 and HAdV-5.

The core of Cyclolinopeptide A (CLA, cyclo(LIILVPPFF)), responsible for its high immunosuppressive activity, contains a Pro-Pro-Phe-Phe sequence. A newly synthesized cyclic tetrapeptide, cyclo(Pro-Pro-ß3-HoPhe-Phe) (denoted as 4B8M) bearing the active sequence of CLA, was recently shown to exhibit a wide array of anti-inflammatory properties in mouse models. In this investigation, we demonstrate that the peptide significantly inhibits the replication of human adenovirus C serotype 5 (HAdV-5) and Herpes simplex virus type-1 (HSV-1) in epithelial lung cell line A-549, applying Cidofovir and Acyclovir as reference drugs. Based on a previously established mechanism of its action, we propose that the peptide may inhibit virus replication by the induction of PGE2 acting via EP2/EP4 receptors in epithelial cells. In summary, we reveal a new, antiviral property of this anti-inflammatory peptide.

Effects of Modifications on the Immunosuppressive Properties of Cyclolinopeptide A and Its Analogs in Animal Experimental Models.

The consequences of manipulations in structure and amino acid composition of native cyclolinopeptide A (CLA) from linen seeds, and its linear precursor on their biological activities and mechanisms of action, are reviewed. The modifications included truncation of the peptide chain, replacement of amino acid residues with proteinogenic or non-proteinogenic ones, modifications of peptide bond, and others. The studies revealed changes in the immunosuppressive potency of these analogs investigated in a number of in vitro and in vivo experimental models, predominantly in rodents, as well as differences in their postulated mechanism of action. The modified peptides were compared with cyclosporine A and parent CLA. Some of the synthesized and investigated peptides show potential therapeutic usefulness.

Synthesis and Biological Activity of New 7-Amino-oxazolo[5,4-d]Pyrimidine Derivatives.

The synthesis of a series of novel 7-aminooxazolo[5,4-d]pyrimidines 5, transformations during their synthesis and their physicochemical characteristics have been described. Complete detailed spectral analysis of the intermediates 2-4, the N'-cyanooxazolylacetamidine by-products 7 and final compounds 5 has been carried out using MS, IR, 1D and 2D NMR spectroscopy. Theoretical research was carried out to explain the privileged formation of 7-aminooxazolo[5,4-d]pyrimidines in relation to the possibility of their isomer formation and the related thermodynamic aspects. Additionally, the single-crystal X-ray diffraction analysis for 5h was reported. Ten 7-aminooxazolo[5,4-d]pyrimidines 5 (SCM1-10) were biologically tested in vitro to preliminarily evaluate their immunological, antiviral and anticancer activity. Compounds SCM5 and SCM9 showed the best immunoregulatory profile. The compounds displayed low-toxicity and strongly inhibited phytohemagglutinin A-induced proliferation of human peripheral blood lymphocytes and lipopolysaccharide-induced proliferation of mouse splenocytes. Compound SCM9 caused also a moderate suppression of tumor necrosis factor α (TNF-α) production in a human whole blood culture. Of note, the compounds also inhibited the growth of selected tumor cell lines and inhibited replication of human herpes virus type-1 (HHV-1) virus in A-549 cell line. Molecular investigations showed that the compounds exerted differential changes in expression of signaling proteins in Jurkat and WEHI-231 cell lines. The activity of SCM5 is likely associated with elicitation of cell signaling pathways leading to cell apoptosis. The compounds may be of interest in terms of therapeutic utility as inhibitors of autoimmune disorders, virus replication and antitumor agents.

Isoxazole Derivatives as Regulators of Immune Functions.

In this review, we present reports on the immunoregulatory properties of isoxazole derivatives classified into several categories, such as immunosuppressive, anti-inflammatory, immunoregulatory, and immunostimulatory compounds. The compounds were tested in various models using resident cells from rodents and humans, cell lines, and experimental animal disease models corresponding to human clinical situations. Beneficial features of the described isoxazole derivatives include low toxicity and good bioactivity at low doses. In a majority of studies, the activities of investigated compounds were comparable or even higher than registered reference drugs. Whenever possible, a plausible mechanism of action of the investigated compounds and their potential therapeutic utility were proposed. Among the described compounds, particular attention was paid to the class of immune stimulators with a potential application in chemotherapy patients.

Synthesis, Immunosuppressive Properties, and Mechanism of Action of a New Isoxazole Derivative.

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.

An Anti-Inflammatory Azaphenothiazine Inhibits Interferon ß Expression and CXCL10 Production in KERTr Cells.

An azaphenothiazine derivative, 6-chloroethylureidoethyldiquino[3,2-b;2',3'-e][1,4]thiazine (DQT), has recently been shown to exhibit immunosuppressive activities in mouse models. It also inhibited the expression of CXCL10 at the protein level, at non-toxic concentrations, in the culture of KERTr cells treated with double-stranded RNA, poly(I:C). In this report, we demonstrated that DQT inhibits the transcription of the CXCL10 gene. Although CXCL10 is an IFNγ-inducible protein, we found that the CXCL10 protein was induced without the detectable release of IFNγ or IκB degradation. Hence, we concluded that IFNγ or NFκB was not involved in the regulation of the CXCL10 gene in KERTr cells transfected with poly(I:C), nor in the inhibitory activity of DQT. On the other hand, we found that IFNß was induced under the same conditions and that its expression was inhibited by DQT. Kinetic analysis showed that an increase in IFNß concentrations occurred 4⁻8 h after poly(I:C) treatment, while the concentration of CXCL10 was undetectable at that time and started to increase later, when IFNß reached high levels. Therefore, DQT may be regarded as a new promising inhibitor of IFNß expression and IFNß-dependent downstream genes and proteins, e.g., CXCL10 chemokine, which is implicated in the pathogenesis of autoimmune diseases.

Synthesis and biological activity of cyclolinopeptide A analogues modified with γ4-bis(homo-phenylalanine).

Cyclolinopeptide A (CLA), an immunosuppressive nonapeptide derived from linen seeds, was modified with S or R-γ4-bis(homo-phenylalanine) in positions 3 or 4, or both 3 and 4. These modifications changed the flexibility of new analogues and distribution of intramolecular hydrogen bonds. Analogues 11 c(Pro1-Pro2-Phe3-S-γ4-hhPhe4-Leu5-Ile6-Ile7-Leu8-Val9), 13 c(Pro1-Pro2-S-γ4-hhPhe3-R-γ4-hhPhe4-Leu5-Ile6-Ile7-Leu8-Val9) and 15 c(Pro1-Pro2-R-γ4-hhPhe3-Phe4-Leu5-Ile6-Ile7-Leu8-Val9) existed as a mixture of stable cis/trans isomers of Pro-Pro peptide bond. The comparison of the relative spatial orientations in crystal state of the two carbonyl groups, neighboring γ-amino acids, revealed conformational similarities to α-peptides. The addition of two -CH2- groups in γ-amino acids led to a more rigid conformation, although a more flexible one was expected. A significant difference in the relative orientation of the carbonyl groups was found for cyclic γ-peptides with a dominance of an antiparallel arrangement. As carbonyl groups may be engaged in the interactions with plausible receptors through hydrogen bonds, a similar biological activity of the modified peptides was expected. Our biological studies showed that certain cyclic, but not the corresponding linear peptides, lowered the viability of peripheral blood mononuclear cells (PBMC) at 100µg/mL concentration. The proliferation of PBMC induced by phytohemagglutinin A (PHA) was strongly inhibited by cyclic peptides only, in a dose-dependant manner. On the other hand, lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-α) production in whole blood cell cultures was inhibited by both linear and cyclic peptides. Peptide 15 c(Pro1-Pro2-R-γ4-hhPhe3-Phe4-Leu5-Ile6-Ile7-Leu8-Val9) blocked the expression of caspase-3, inhibited the expression of caspases-8 and -9 in 24h culture of Jurkat cells, and caused DNA fragmentation in these cells, as an indicator of apoptosis. Thus, we revealed a new mechanism of immunosuppressive action of a nonapeptide.

Antiviral Resistance of Splenocytes in Aged Mice.

We compared the susceptibility to viral infection of splenocytes, isolated from young versus old CBA mice, and evaluated the antiviral actions of lactoferrin in splenocytes infected with Encephalomyocarditis virus (EMCV). Recombinant mouse lactoferrin (rmLF) and bovine lactoferrin (bLF) were used. There were no differences in the susceptibility to EMCV infection in the studied age categories. Both types of lactoferrins were protective in young and old mice. The study confirmed the undisturbed viral resistance in old mice and the protective actions of lactoferrin in viral infection. The antiviral action of the homologous mouse lactoferrin was demonstrated for the first time.

Cathepsin G-mediated proteolytic degradation of MHC class I molecules to facilitate immune detection of human glioblastoma cells.

To mount an adaptive immune response, MHC I molecules present antigenic peptides to CTLs. Transcriptional reduction of MHC I molecules is a strategy of immune evasion, which impairs the detection of infected or tumorous cells by CTLs. Natural killer (NK) cells, on the other hand, eliminate target cells specifically in the absence of MHC I. Consequently, infected or tumorous cells partly retain their MHC I at the cell surface to avoid NK recognition. However, it remains unclear which protease degrades MHC I molecules and how these cells maintain a limited set of MHC I at the cell surface. Here, we demonstrate that cathepsin G (CatG), a serine protease, found in the endocytic compartment of APCs and, to a lesser extent, CatD and CatS proteolytically degrade MHC I molecules. Inhibition of CatG boosted MHC I expression at the cell surface of primary human immune cells. In contrast, human glioblastoma cells do not harbor active CatG and might have lost the ability to proteolytically degrade MHC I during tumorigenesis to avoid NK-mediated killing. Overexpression of CatG in glioblastoma cells resulted in a rapid and efficient MHC I degradation. In conclusion, CatG is an essential protease for regulating MHC I molecules and thus modulation of CatG activity might present a new avenue for therapeutic intervention.

Cyclodimerization of immunosuppressive fragment of HLA-DR molecule. Design, synthesis and ESI-MS/MS analysis.

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.

Synthesis and biological evaluation of novel propargylquinobenzothiazines and their derivatives as potential antiproliferative, anti-inflammatory, and anticancer agents.

Azaphenothiazines containing the quinoline ring, 8-10-substituted 6H-quinobenzothiazines and 6H-diquinothiazine were transformed into new 6-propargyl and 6-dialkylaminobutynyl derivatives containing the triple bond. Most of them displayed strong antiproliferative actions against human peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglutinin A (PHA), strongly suppressed lipopolysaccharide (LPS)-induced TNF-α production by whole blood human cell cultures, and exhibited low cytotoxicity. Three propargylquinobenzothiazines with the bromine, trifluoromethyl, and methylthio groups at position 9 and propargyldiquinothiazine exhibited comparable actions to cisplatin against the L-1210 and SW-948 tumor lines. 6-Propargyl-9-trifluoromethylquinobenzothiazine was shown to block caspase 3 expression and inhibit expression of caspase 8 and 9 in Jurkat cells indicating its possible mechanism of action. These derivatives could be promising, potential therapeutics for treatment of neoplastic diseases and autoimmune disorders.

SYNTHESIS AND IMMUNOREGULATORY PROPERTIES OF SELECTED 5-AMINO-3-METHYL-4-ISOXAZOLECARBOXYLIC ACID BENZYLAMIDES.

The aim of the study was to characterize a series of isoxazole derivatives in several immunological tests in vitro and in vivo, in mouse and human models. The human model included measurement of: viability of peripheral blood mononuclear cells (PBMC), phytohemagglutinin A (PHA)-induced proliferation of PBMC, production of tumor necrosis factor a (TNF a) in whole blood cultures stimulated with lipopolysaccharide (LPS) and growth of SW-948 and L1210 tumor cell lines. Experiments in mice encompassed the following tests: secondary, humoral immune response splenocytes to sheep erythrocytes (SRBC) in vitiv, delayed type hypersensitivity (DTH) to ovalbumin (OVA) and carrageenan-induced foot edema. All compounds were non-toxic against PMBC and displayed differential, dose-dependent suppressive properties in the model of PHA- induced PMBC proliferation. They also exhibited differential, mostly inhibitory effects on TNF a production. The inhibitory actions on growth of tumor cell lines were moderate. M05 (5-amino-3-methyl-N-(4-methyl-benzyl)-4-isoxazolecarboxamide) was most suppressive in the proliferation and TNF a production tests, it was, therefore, selected for in vitro and in vivo studies in the mouse models. The compound inhibited the humoral immune response in vitro, stimulated the inductive phase of DTH in vivo, although it inhibited the eliciting phase of that response. The compound also inhibited the carrageenan skin reaction. M05 combines strong anti-proliferative and anti-inflammatory activities, it is therefore attractive for further studies in more advanced animal models as a potential therapeutic.

Synthesis and selected immunological properties of 10-substituted 1,8-diazaphenothiazines.

A new type of tricyclic azaphenothiazines-1,8-diazaphenothiazines-was obtained in the reaction of 2,3- and 3,4-disubstituted pyridines. The reaction ran as the Smiles rearrangement. The 1,8-diazaphenothiazine system was determined using NOE experiment and 2D NMR spectra (COSY, HSQC, HMBC). 10H-1,8-diazaphenothiazine was transformed into 10-derivatives with alkyl, aminoalkyl, amidoalkyl, sulfonamidoalkyl, and nitrogen half-mustard groups. The compounds were tested for their effects on phytohemagglutinin A-induced proliferative response of human peripheral blood mononuclear cells (PBMC) and lipopolysaccharide-induced tumor necrosis factor alpha production by human whole blood cultures. The compounds exhibited differential, dose-dependent inhibitory activities in these tests. All the compounds were low toxic against PBMC. The compounds showing the highest antiproliferative activity strongly inhibited the growth of leukemia L-1210 and colon cancer SW-948 cell lines, similarly as cisplatin, a reference drug.

The effect of carbohydrate moiety structure on the immunoregulatory activity of lactoferrin in vitro.

The aim of this study was to evaluate the immunoregulatory effects of recombinant human lactoferrin (rhLF) in two in vitro models: (1) the secondary humoral immune response to sheep erythrocytes (SRBC); and (2) the mixed lymphocyte reaction (MLR). We compared the non-sialylated glycoform of rhLF as expressed by glycoengineered Pichia pastoris with one that was further chemically sialylated. In an earlier study, we showed that sialylated rhLF could reverse methotrexate-induced suppression of the secondary immune response of mouse splenocytes to SRBC, and that the phenomenon is dependent on the interaction of lactoferrin (LF) with sialoadhesin (CD169). We found that the immunorestorative activity of sialylated rhLF is also dependent on its interaction with the CD22 antigen, a member of the immunoglobulin superfamily that is expressed by B lymphocytes. We also demonstrated that only sialylated rhLF was able to inhibit the MLR reaction. MLR was inhibited by bovine lactoferrin (bLF), a glycoform that has a more complex glycan structure. Desialylated bLF and lactoferricin, a bLF-derived peptide devoid of carbohydrates, did not express such inhibitory activity. We showed that the interaction of LF with sialic acid receptors is essential for at least some of the immunoregulatory activity of this glycoprotein.

Antimicrobial properties of substituted quino[3,2-b]benzo[1,4]thiazines.

Our previous studies demonstrated that among phenothiazines several derivatives could be found showing strong antiproliferative actions and the property of inhibiting inducible tumor necrosis factor alpha (TNF a) production in human blood cultures. The aim of this investigation was to determine potential antimicrobial actions of forty four new phenothiazine derivatives with the quinobenzothiazine structure. The compounds showed differential antibacterial and antifungal activities against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans depending on the compound structures, concentrations and bacterial strains. More specifically, 6-(1-methyl- 2-piperidylethyl) quinobenzothiazine displayed strongest actions against S. aureus and E. coli whereas 6-methanesulfonylaminobutyl-9-methylthioquinobenzothiazine exhibited the most universal antimicrobial properties. The correlation between antimicrobial activity and the chemical structure of quinobenzothiazines was discussed.

New lead structures in the isoxazole system: relationship between quantum chemical parameters and immunological activity.

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.

Milk-derived proteins and peptides in clinical trials.

Clinical trials are reviewed, involving proteins and peptides derived from milk (predominantly bovine), with the exception of lactoferrin, which will be the subject of another article. The most explored milk fraction is α-lactalbumin (LA), which is often applied with glycomacropeptide (GMP) - a casein degradation product. These milk constituents are used in health-promoting infant and adult formulae as well as in a modified form (HAMLET) to treat cancer. Lactoperoxidase (LCP) is used as an additive to mouth hygiene products and as a salivary substitute. Casein derivatives are applied, in addition, in the dry mouth syndrome. On the other hand, casein hydrolysates, containing active tripeptides, found application in hypertension and in type 2 diabetes. Lysozyme is routinely used for food conservation and in pharmaceutical products. It was successfully used in premature infants with concomitant diseases to improve health parameters. When used as prophylaxis in patients with scheduled surgery, it significantly reduced the incidence of hepatitis resulting from blood transfusion. Lysozyme was also used in infected children as an antimicrobial agent showing synergistic effects in combination with different antibiotics. Proline-rich polypeptide (PRP) was introduced to therapy of Alzheimer's disease patients. The therapeutic value of PRP was proved in several clinical trials and supported by studies on its mechanism of action. Concentrated immunoglobulin preparations from colostrum and milk of hyperimmunized cows showed efficacy in prevention of infections by bacteria, viruses and protozoa. A nutrition formula with milk-derived TGF-ß2 (Modulen IBD®) found application in treatment of pediatric Crohn's disease. In conclusion, the preparations containing milk-derived products are safe and effective measures in prevention and treatment of infections as well as autoimmune and neoplastic diseases.