Colostrum and Lactoferrin Protect against Side Effects of Therapy with Antibiotics, Anti-inflammatory Drugs and Steroids, and Psychophysical Stress: A Comprehensive Review.

In this article, we review the benefits of applying bovine colostrum (BC) and lactoferrin (LF) in animal models and clinical trials that include corticosteroid application and psychic stress, treatment with non-steroid anti-inflammatory drugs (NSAIDs) and antibiotics. A majority of the reported investigations were performed with native bovine or recombinant human LF, applied alone or in combination with probiotics, as nutraceutics and diet supplements. Apart from reducing adverse side effects of the applied therapeutics, BC and LF augmented their efficacy and improved the wellness of patients. In conclusion, LF and complete native colostrum, preferably administered with probiotic bacteria, are highly recommended for inclusion in therapeutic protocols in NSAIDs and corticosteroid anti-inflammatory, as well as antibiotic, therapies. These colostrum-based products can also be of value for individuals subjected to prolonged psychophysical stress (mediated by endogenous corticosteroids), especially at high ambient temperatures (soldiers and emergency services), as well as physically active people and training athletes. They are also recommended for patients during recovery from trauma and surgery, which are always associated with severe psychophysical stress.

Colostrum Proteins in Protection against Therapy-Induced Injuries in Cancer Chemo- and Radiotherapy: A Comprehensive Review.

In this article, we review the benefits of application of colostrum and colostrum-derived proteins in animal models and clinical trials that include chemotherapy with antimetabolic drugs, radiotherapy and surgical interventions. A majority of the reported investigations was performed with bovine colostrum (BC) and native bovine or recombinant human lactoferrin (LF), applied alone, in nutraceutics or in combination with probiotics. Apart from reducing side effects of the applied therapeutics, radiation and surgical procedures, BC and LF augmented their efficacy and improved the wellness of patients. In conclusion, colostrum and colostrum proteins, preferably administered with probiotic bacteria, are highly recommended for inclusion to therapeutic protocols in cancer chemo- and radiotherapy as well as during the surgical treatment of cancer patients.

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.

Antimicrobial and Prebiotic Activity of Lactoferrin in the Female Reproductive Tract: A Comprehensive Review.

Women's intimate health depends on several factors, such as age, diet, coexisting metabolic disorders, hormonal equilibrium, sexual activity, drug intake, contraception, surgery, and personal hygiene. These factors may affect the homeostasis of the internal environment of the genital tract: the vulva, vagina and cervix. This equilibrium is dependent on strict and complex mutual interactions between epithelial cells, immunocompetent cells and microorganisms residing in this environment. The microbiota of the genital tract in healthy women is dominated by several species of symbiotic bacteria of the Lactobacillus genus. The bacteria inhibit the growth of pathogenic microorganisms and inflammatory processes by virtue of direct and multidirectional antimicrobial action and, indirectly, by the modulation of immune system activity. For the homeostasis of the genital tract ecosystem, antimicrobial and anti-inflammatory peptides, as well as proteins secreted by mucus cells into the cervicovaginal fluid, have a fundamental significance. Of these, a multifunctional protein known as lactoferrin (LF) is one of the most important since it bridges innate and acquired immunity. Among its numerous properties, particular attention should be paid to prebiotic activity, i.e., exerting a beneficial action on symbiotic microbiota of the gastrointestinal and genital tract. Such activity of LF is associated with the inhibition of bacterial and fungal infections in the genital tract and their consequences, such as endometritis, pelvic inflammation, urinary tract infections, miscarriage, premature delivery, and infection of the fetus and newborns. The aim of this article is to review the results of laboratory as well as clinical trials, confirming the prebiotic action of LF on the microbiota of the lower genital tract.

Lactoferrin as an Adjuvant for the Generation of Delayed Type Hypersensitivity to Orally Administered Antigen.

OBJECTIVE: The aim of this investigation was to evaluate the property of bovine lactoferrin (LF) in the generation of delayed type hypersensitivity (DTH) as an oral adjuvant during immunization with ovalbumin (OVA) and BCG. METHODS: LF admixed with OVA or BCG was used for immunization of CBA or C57BL/6 mice when given via oral or subcutaneous routes. Elicited DTH response was measured post immunization. Inhibition studies using mannose or galactose were accomplished by gavage prior to oral administration of antigens. LF was also examined for effects on BCG uptake by bone marrow derived macrophages (BMM). RESULTS: LF at doses of 1.0 mg and 10.0 mg, admixed with OVA (10.0 mg), significantly enhanced the antigen-specific DTH reaction. The stimulatory effects of LF were inhibited by the oral pretreatment of mice with 50.0 mg of mannose but not galactose. LF also enhanced the DTH reaction to orally administered BCG. LF enhanced uptake of BCG by BMM in a dose-dependent manner. CONCLUSION: LF was able to augment development of DTH when orally administered with OVA or BCG antigens. Inhibition studies suggest the involvement of the receptor with an affinity to mannose in mediation of the adjuvant effect. LF augmentation of the DTH response was partially effective when given in advance of oral delivery of the antigen; this effect could also be saturated by mannose. BCG studies provide preliminary evidence for LF in the potential augmentation of oral vaccination to prevent mycobacterial infection. In vitro experiments provide evidence that LF plays a role in modulation of antigen presenting cell activation.

Synthesis, Physicochemical Characteristics and Plausible Mechanism of Action of an Immunosuppressive Isoxazolo[5,4-e]-1,2,4-Triazepine Derivative (RM33).

Previous studies demonstrated strong anti-inflammatory properties of isoxazolo[5,4-e]-1,2,4-triazepine (RM33) in vivo. The aim of this investigation was to describe synthesis, determine physicochemical characteristics, evaluate biological activities in murine and human in vitro models, as well as to propose mechanism of action of the compound. The compound was devoid of cell toxicity up to 100 µg/mL against a reference A549 cell line. Likewise, RM33 did not induce apoptosis in these cells. The compound stimulated concanavalin A (ConA)-induced splenocyte proliferation but did not change the secondary humoral immune response in vitro to sheep erythrocytes. Nevertheless, a low suppressive effect was registered on lipopolysaccharide (LPS)-induced splenocyte proliferation and a stronger one on tumor necrosis factor alpha (TNFα) production by rat peritoneal cells. The analysis of signaling pathways elicited by RM33 in nonstimulated resident cells and cell lines revealed changes associated with cell activation. Most importantly, we demonstrated that RM33 enhanced production of cyclooxygenase 2 in LPS-stimulated splenocytes. Based on the previous and herein presented results, we conclude that RM33 is an efficient, nontoxic immune suppressor with prevailing anti-inflammatory action. Additionally, structural studies were carried out with the use of appropriate spectral techniques in order to unequivocally confirm the structure of the RM33 molecule. Unambiguous assignment of NMR chemical shifts of carbon atoms of RM33 was conducted thanks to full detailed analysis of 1H, 13C NMR spectra and their two-dimensional (2D) variants. Comparison between theoretically predicted chemical shifts and experimental ones was also carried out. Additionally, N-deuterated isotopologue of RM33 was synthesized to eliminate potentially disturbing frequencies (such as NH, NH2 deformation vibrations) in the carbonyl region of the IR (infrared) spectrum to confirm the presence of the carbonyl group.

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.

Prolongation of skin graft survival in mice by an azaphenothiazine derivative.

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.

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.

Topically applied azaphenothiazines inhibit experimental psoriasis in mice.

The therapeutic efficacy of topically applied azaphenothiazine derivatives: 9-chloro-6-acetylaminobutylquinobenzo[3,2-b][1,4]thiazine (compound 4) and 6-chloroethylureidoethyldiquino[3,2-b;2';3'-e][1,4]thiazine (compound 5) in the amelioration of inflammatory symptoms of imiquimod-induced psoriasis in mice was investigated. Clobederm®, containing clobetasol propioniate, served as a reference drug. The application of the compounds led to thinning of the epidermis and reduction of the cell layers. The suppressive actions of the compounds were even stronger with regard to pathological changes of the dermis. The compounds also exerted generalized, anti-inflammatory effects by decreasing the number of circulating leukocytes, lowering subiliac lymph node weight and partially normalizing an altered blood cell composition. The changes in the composition of main cell types in the epidermis and dermis were less affected by the compounds. In addition, both compounds inhibited to a similar degree production of tumor necrosis factor α (TNF α) in human whole blood cell culture. Whereas compound 5 strongly inhibited IL-8 and CXCL10 chemokines in human keratinocytes - KERTr cell line, transfected with poly(I:C), the suppressive action of compound 4 in this model was weak. In addition, compound 5, but not compound 4, exhibited at low doses proapoptotic properties with regard to colonic cell lines. In summary, we demonstrated the therapeutic potential of two selected azaphenotiazines in the amelioration of the skin pathology elicited in a mouse experimental model of psoriasis.

Immunomodulatory properties of human recombinant lactoferrin in mice: Implications for therapeutic use in humans.

BACKGROUND: Trauma and major surgery cause extensive immune hyporeactivity in patients. Thus, the preventive, preoperative application of immunoregulatory therapeutics may normalize this immune reactivity and decrease morbidity and mortality in these subjects. OBJECTIVES: The aim of this study was to investigate the immunomodulatory actions of recombinant human lactoferrin (rhLF) in mice, and to relate these effects to in vitro actions of rhLF on tumor necrosis factor alpha (TNF-α) production in lipopolysaccharide-stimulated whole blood cell cultures (LPS-stimulated WBCC) from patients admitted to intensive care units. MATERIAL AND METHODS: BALB/c and CBA mice were used. rhLF was tested for allergic response to ovalbumin (OVA), delayed-type hypersensitivity (DTH) to OVA, and carrageenan-induced inflammation in an air pouch. Blood samples from 30 patients diagnosed with severe sepsis/septic shock (Apache II 21 ±1, mortality rate 40%) were collected on days 1, 3 and 5 of observation. The effects of rhLF on LPS-induced TNF-α production were measured in WBCCs. RESULTS: Recombinant human lactoferrin reduced the parameters of OVA-induced inflammation and inhibited the elicitation phase of DTH and carrageenan-induced inflammation in mice. The majority of patients from whom whole blood cell cultures (WBCC) were established showed a strong hyporeactivity to LPS upon admission. rhLF exerted differential effects on the production of LPS-induced TNF-α in those cultures on days 1, 3 and 5 of observation. Cytokine production was upregulated only in patients with sustained anergy to LPS, and inhibited or unchanged in moderately reactive patients. CONCLUSIONS: Evidence for the potential preventive or therapeutic utility of rhLF in patients with impaired immune reactivity has been demonstrated.

Azaphenothiazines - promising phenothiazine derivatives. An insight into nomenclature, synthesis, structure elucidation and biological properties.

For the last two decades, classical phenothiazines have attracted attention of researchers, as the hitherto investigations have revealed many significant biological activities within this class of compounds, other than originally discovered neuroleptic ones. Important, new pharmaceutical results on phenothiazines, as 10-substituted dibenzothiazines, were recently highlighted in several reviews. Azaphenothiazines are structurally modified phenothiazines by substitution of one or both benzene rings in the phenothiazine ring system with the azine rings, such as: pyridine, pyridazine, pyrimidine, pyrazine, 1,2,4-triazine, quinoline, quinoxaline, benzoxazine and benzothiazine. They form over 50 different heterocyclic systems, of tri-, tetra-, penta- and hexacyclic structures, and contain from one to even four azine nitrogen atoms. This review summarizes the methodical knowledge on azaphenothiazines, referring to their nomenclature, synthesis, structure analysis and above all significant varied biological activities, examined in vitro and in vivo. It describes, in addition, current trends in the synthesis of azaphenothiazines. The influence of the azaphenothiazine ring system, the nature of the substituents, predominantly at the thiazine nitrogen atom, as well as at the azine nitrogen atom and carbon atom, on the biological activities, were also discussed.

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.

5-Amino-3-methyl-4-isoxazolecarboxylic acid hydrazide derivatives with in vitro immunomodulatory activities.

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-1ß 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-1ß 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.

Exogenous cathepsin G upregulates cell surface MHC class I molecules on immune and glioblastoma cells.

Major histocompatibility complex (MHC) class I molecules present antigenic peptides to cytotoxic T cells. During an adaptive immune response, MHC molecules are regulated by several mechanisms including lipopolysaccharide (LPS) and interferon gamma (IFN-g). However, it is unclear whether the serine protease cathepsin G (CatG), which is generally secreted by neutrophils at the site of inflammation, might regulate MHC I molecules. We identified CatG, and to a higher extend CatG and lactoferrin (LF), as an exogenous regulator of cell surface MHC I expression of immune cells and glioblastoma stem cells. In addition, levels of MHC I molecules are reduced on dendritic cells from CatG deficient mice compared to their wild type counterparts. Furthermore, cell surface CatG on immune cells, including T cells, B cells, and NK cells triggers MHC I on THP-1 monocytes suggesting a novel mechanism for CatG to facilitate intercellular communication between infiltrating cells and the respective target cell. Subsequently, our findings highlight the pivotal role of CatG as a checkpoint protease which might force target cells to display their intracellular MHC I:antigen repertoire.

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.

Anti-inflammatory properties of an isoxazole derivative - MZO-2.

BACKGROUND: A series of new isoxazole derivatives of expected immunosuppressive activities was synthesized. Following in vitro screening in the human cell models, the activity of MZO-2 compound (ethyl N-{4-[(2,4-dimethoxybenzyl)carbamoyl]-3-methylisoxazol-5-yl}acetimidate) in mouse in vivo models was evaluated. METHODS: In vitro tests included evaluation of: peripheral blood mononuclear cells (PBMC) viability, phytohemagglutinin (PHA)-induced PBMC proliferation and lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNF α) production in whole blood cell cultures. MZO-2 was studied in mice for its effects on: humoral immune response to sheep erythrocytes (SRBC), delayed type hypersensitivity (DTH) to ovalbumin (OVA), contact sensitivity to oxazolone and carrageenan-induced foot pad edema. In addition, the effect of MZO-2 on expression of caspases in Jurkat cells was determined. RESULTS: The studied compounds exhibited differential, dose-dependent effects to suppress PHA-induced PBMC proliferation and a weak property to suppress LPS-induced production of TNF α. MZO-2 had no effect on the induction phase of the humoral immune response to SRBC in vitro and in vivo, but moderately suppressed the induction phase of DTH to OVA. Its inhibitory effect on carrageenan-induced paw inflammation was potent. Likewise, MZO-2, applied in ointment, was very effective in reducing ear edema and number of lymphocytes in draining lymph nodes of mice sensitized to oxazolone, comparably to tacrolimus, the reference drug. The expression of caspases 3, 8 and 9 in Jurkat cells was inhibited by the compound. CONCLUSION: MZO-2, applied systemically or locally, may serve as a potential drug for amelioration of inflammatory processes.