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Immature B cells are susceptible to apoptosis due to ligation of surface immunoglobulin receptors. The WEHI 231 cell line represents a useful model to study the mode of action of factors preventing apoptosis. In this work we investigated the protective effects of multi-species lactoferrins in anti-mouse Ig-induced WEHI 231 cell death. Bovine milk-derived lactoferrin (bLF), recombinant human lactoferrin expressed in Chinese hamster ovary cells - rhLF(CHO) or in human endothelial kidney cells - rhLF(HEK), and recombinant mouse lactoferrin expressed in Chinese hamster ovary cells - rmLF(CHO), were used. Goat-anti-mouse Ig antibodies were used to induce cell apoptosis. Survival of WEHI 231 cells in culture was measured using the colorimetric MTT method. Expression of signalling molecules and subunits of interleukin 2 receptor was determined by the RT PCR method. The results showed that anti-mouse Ig antibodies inhibited cell growth in a dose-dependent manner. The lactoferrins alone had no effect on the cell survival. The cells exposed to LFs, prior to anti-Ig treatment, were rescued to a significant degree from cell death. Determination of the signalling molecule expression revealed almost complete suppression of caspase-3 and NF-κB1 by bLF in untreated cells, as well as deep suppression of caspase-3, block of Fas, and 4-fold increase of NF-κB1 in cells incubated with bLF prior to anti-Ig treatment. In addition, differential changes in the expression of interleukin 2 subunits upon bLF treatment were found, indicating a process of cell differentiation. In conclusion, we showed that LF-induced cell differentiation in immature B-cell line WEHI 231 was correlated with partial protection of the cells from anti-Ig-induced cell death. [ABSTRACT FROM AUTHOR]

Background: Voltage-gated sodium channels are involved in the initial depolarisation of neurones. As such, they play important roles in neurotransmission. Variants in the genes encoding these channels may lead to altered functionality and neurodevelopmental disorders. Pathogenic variants of SCN8A, which encodes the voltage-gated Na+ channel Nav1.6, have been associated with various encephalopathies characterised by developmental delay and epileptic seizures. Herein, we discuss the genotype-phenotype associations in a group of 17 novel Polish patients with SCN8A mutations, further expanding the molecular and phenotypic spectrum of SCN8A-related diseases., Methods: The participants were recruited from five clinical centres in Poland. Pathogenic and likely pathogenic SCN8A variants were identified using a next-generation sequencing (NGS) panel and exome sequencing, respectively. Magnetic resonance imaging (MRI) and electroencephalography (EEG) recordings were performed to obtain relevant clinical data on brain malformations and epileptic seizures., Results: Three phenotypes were observed in the study group: developmental and epileptic encephalopathy, early onset epileptic encephalopathy, and neurodevelopmental disorders without epilepsy. Patients in the first two phenotypic subgroups presented with epileptic seizures within the first few months of life. Their semiology evolved with age, comprising mostly tonic, clonic, and tonic-clonic seizures, with eyelid myoclonia, myoclonic seizures, and epileptic spasms. The most prevalent neurological feature was developmental delay. Alterations in muscle tone were more frequent than in previous reports., Conclusions: Seventeen patients with 11 novel mutations in SCN8A had alterations in muscular tone accompanied by typical features of SCN8A-related encephalopathies (i.e., developmental delay and a wide range of seizures)., Competing Interests: Declaration of competing interest The Authors declare no conflict of interests., (Copyright © 2024. Published by Elsevier Ltd.)

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., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

The in vitro immunotropic actions of a calf thymus extract - thymus factor X (TFX®) preparation were investigated. The preparation did not lower the viability of the A549 epithelial cell line and mouse bone marrow cells in the investigated concentration range. TFX® exhibited a co-stimulatory action of concanavalin A (Con A)-induced mouse thymocyte proliferation and partially restored the mitogen-induced proliferation capability of mouse thymocytes exposed to hydrocortisone (HC). The preparation also inhibited Herpes virus-1 (HSV-1) replication in A549 cells when preincubated with the virus and when added to the infected cells. In addition, it weakly inhibited lipopolysaccharide (LPS)-induced TNF α, IL-1β and IL-6 by the THP-1 monocyte cell line. The determination of mitogen activated protein kinase (MAPK) expression in Jurkat T cells revealed strong increases in ERK-2 kinase and p38α subunits. In WEHI 231 immature B cells, TFX® elevated p38α, and had a particularly strong elevating effect on p38γ. In HL-60 myeloblastic cells, the expression of p38α, β and γ was not detectable, almost blocked for p38δ and JNK, but accompanied by an increase in ERK-1. In turn, the effects of TFX® in J744E macrophages resulted in a strong increase in p38γ expression, moderate elevations of ERK and a drop in p38δ. Significant increases in MAPK expression were also found in cells from the lymphoid organs. In the bone marrow cell population, p38α, β and γ, in thymocytes p38α, γ and δ, and in splenocytes p38β and γ, subunit expression was elevated. We conclude that the changes in MAPK expression may be attributed to cell maturation and differentiation, and explain the beneficial therapeutic effects of TFX®., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Congenital microcephaly causes smaller than average head circumference relative to age, sex and ethnicity and is most usually associated with a variety of neurodevelopmental disorders. The underlying etiology is highly heterogeneous and can be either environmental or genetic. Disruption of any one of multiple biological processes, such as those underlying neurogenesis, cell cycle and division, DNA repair or transcription regulation, can result in microcephaly. This etiological heterogeneity manifests in a clinical variability and presents a major diagnostic and therapeutic challenge, leaving an unacceptably large proportion of over half of microcephaly patients without molecular diagnosis. To elucidate the clinical and genetic landscapes of congenital microcephaly, we sequenced the exomes of 191 clinically diagnosed patients with microcephaly as one of the features. We established a molecular basis for microcephaly in 71 patients (37%), and detected novel variants in five high confidence candidate genes previously unassociated with this condition. We report a large number of patients with mutations in tubulin-related genes in our cohort as well as higher incidence of pathogenic mutations in MCPH genes. Our study expands the phenotypic and genetic landscape of microcephaly, facilitating differential clinical diagnoses for disorders associated with most commonly disrupted genes in our cohort.

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 1 H, 13 C 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, NH 2 deformation vibrations) in the carbonyl region of the IR (infrared) spectrum to confirm the presence of the carbonyl group.

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.)

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.

Yolkin is a product of proteolytic degradation of vitellogenin, a protein contained in eggs' yolk, with already described procognitive properties. Here, we investigated effects of yolkin on the humoral and cellular immune response in mice, phenotype of cells from lymphoid organs and function of innate immunity cells. In vitro studies included effects of yolkin on mitogen-induced thymocyte proliferation, percentage of CD19 cells in bone marrow cells culture, expression of signaling molecules in Jurkat cells, interleukin 2 receptor (IL-2R) subunits in WEHI 231 cells and susceptibility of these cells to anti-Ig-induced cell death. The results showed that repeatable i.p. injections of yolkin stimulated the humoral immune response to sheep red blood cells (SRBC) irrespective of the time of the treatment. On the other hand, yolkin inhibited contact sensitivity to oxazolone. Treatment of mice with yolkin diminished the percentage of double positive cells and increasing the content of single positive CD4+ and CD8+ cells in the thymus. At the same time an increase of percentage of CD19 + B cells in the spleen and mesenteric lymph nodes was observed. In addition, the protein, given i.p., diminished ex vivo ability to synthesize nitric oxide by resident, peritoneal macrophages, stimulated with lipopolisaccharide (LPS). In vitro studies showed that yolkin increased CD19+ cell content in bone marrow cell population. The protein also enhanced proliferation of thymocytes to concanavalin A and stimulated expression of MAP kinases in Jurkat cells. In WEHI 231 B cell line yolkin caused a loss of IL-2R gamma chain expression, correlated with an increased resistance of these cells to proapoptotic action of anti-Ig antibodies. In conclusion, this is a first demonstration of immunotropic properties of yolkin in in vitro and in vivo tests. The results provide evidence for induction of maturation and stimulatory signals in immature T and B cells by the protein, suggesting its potential role in the development of an embryo's immune system., (Copyright © 2020 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

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.)

Malformations of cortical development (MCDs) manifest with structural brain anomalies that lead to neurologic sequelae, including epilepsy, cerebral palsy, developmental delay, and intellectual disability. To investigate the underlying genetic architecture of patients with disorders of cerebral cortical development, a cohort of 54 patients demonstrating neuroradiologic signs of MCDs was investigated. Individual genomes were interrogated for single-nucleotide variants (SNV) and copy number variants (CNV) with whole-exome sequencing and chromosomal microarray studies. Variation affecting known MCDs-associated genes was found in 16/54 cases, including 11 patients with SNV, 2 patients with CNV, and 3 patients with both CNV and SNV, at distinct loci. Diagnostic pathogenic SNV and potentially damaging variants of unknown significance (VUS) were identified in two groups of seven individuals each. We demonstrated that de novo variants are important among patients with MCDs as they were identified in 10/16 individuals with a molecular diagnosis. Three patients showed changes in known MCDs genes  and a clinical phenotype beyond the usual characteristics observed, i.e., phenotypic expansion, for a particular known disease gene clinical entity. We also discovered 2 likely candidate genes, CDH4, and ASTN1, with human and animal studies supporting their roles in brain development, and 5 potential candidate genes. Our findings emphasize genetic heterogeneity of MCDs disorders and postulate potential novel candidate genes involved in cerebral cortical development.

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.

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cyclolinopeptide A (CLA), an immunosuppressive nonapeptide derived from linen seeds, was modified with S or R-γ 4 -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(Pro 1 -Pro 2 -Phe 3 -S-γ 4 -hhPhe 4 -Leu 5 -Ile 6 -Ile 7 -Leu 8 -Val 9 ), 13 c(Pro 1 -Pro 2 -S-γ 4 -hhPhe 3 -R-γ 4 -hhPhe 4 -Leu 5 -Ile 6 -Ile 7 -Leu 8 -Val 9 ) and 15 c(Pro 1 -Pro 2 -R-γ 4 -hhPhe 3 -Phe 4 -Leu 5 -Ile 6 -Ile 7 -Leu 8 -Val 9 ) 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 -CH 2 - 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(Pro 1 -Pro 2 -R-γ 4 -hhPhe 3 -Phe 4 -Leu 5 -Ile 6 -Ile 7 -Leu 8 -Val 9 ) 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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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.)

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., (Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

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.

These studies were performed to confirm the diagnostic value of objective factors routinely assessed during clinical examination in an aim to distinguish between patients with chronic and aggressive periodontitis. Moreover, these factors were verified with subjective description of symptoms reported by patients. To obtain the appointed targets multinominal logistic regression analysis was used. Sixty three patients, aged from 21 to 56 years, treated in the Department of Periodontology of Wroclaw Medical University, were examined as to their oral hygiene and periodontal status. We determined Approximal Plaque Index (API), Papilla Bleeding Index (PBI), probing pocket depths of less than 4 millimetres, furcation involvement, patient's age and the time of appearance of the first symptoms of the disease. The measured data of each parameter was divided into two groups with the use of standard deviation and the multinominal logistic regression analysis. The obtained statistical results proved high clinical value of the studied parameters and gave evidence that the established protocol consisting of five objective factors and one subjective factor used in our clinical practice allowed for good diversification of patients into the two mentioned types of disease.

Background: Differences in duration of bone healing in various parts of the human skeleton are common experience for orthopaedic surgeons. The reason for these differences is not obvious and not clear., Methods: In this paper we decided to measure by the use of real-time RT-PCR technique the level of expression of genes for some isoforms of bone morphogenetic proteins (BMPs), whose role is proven in bone formation, bone induction and bone turnover. Seven bone samples recovered from various parts of skeletons from six cadavers of young healthy men who died in traffic accidents were collected. Activity of genes for BMP-2, -4 and -6 was measured by the use of fluorescent SYBR Green I., Results: It was found that expression of m-RNA for BMP-2 and BMP-4 is higher in trabecular bone in epiphyses of long bones, cranial flat bones and corpus mandibulae then in the compact bone of diaphyses of long bones. In all samples examined the expression of m-RNA for BMP-4 was higher than for BMP-2., Conclusion: It was shown that m-RNA for BMP-6 is not expressed in the collected samples at all. It is postulated that differences in the level of activation of genes for BMPs is one of the important factors which determine the differences in duration of bone healing of various parts of the human skeleton.

Case Report: A 24-year-old woman suffering from post-influenza otitis media infection was initially treated with several series of a steroid (Elocon) and a combination of steroids and antibiotics (Atecortin, Dicortineff) without significant medical benefit. The isolated bacterial strains were identified as Staphylococcus homis and Staphylococcus epidermidis. Specific phage therapy applied sequentially over a period of three weeks resulted only in a partial reduction in inflammation and limited improvement in overall health condition. Oral application of lactoferrin (LF; 50-mg daily oral doses for seven days with two-week intervals) led to a complete clearance of both bacterial strains and full recovery of the patient. The recovery was associated with increased myelopoiesis and a sustained elevation of serum endogenous LF. In conclusion, specific bacteriophage therapy combined with the administration of lactoferrin proved to be effective in the treatment of antibiotic-resistant external ear infection.

CD59 is one of the key molecules involved in cell protection against autologus complement. The fact that complement regulatory proteins are able to prevent hyperacute rejection of organs in pig to primate model, raises the question of possible complement regulatory protein (CRP) involvement in the maturation of immunological system. We report here that in foetal and postnatal human thymus, CD59 and CD55 are primarily located on Hassall's corpuscles and medullary epithelial cells. This localization highly correlates with the expression of CD30L, which is the member of the tumour necrosis factor superfamily. Additionally, TUNEL technique was used to visualize distribution of apoptotic cells in the thymus, which revealed the presence of apoptotic cells closely associated with the Hassall's corpuscles. The observed co-localization of CD59, CD55 and CD30L might suggest an involvement of the complement system in thymic selection in humans.

In this paper the effect of osteoprotegerin (OPG) on slowing down the resorption process of heterotopically induced bone tissue is described. The induced ossicle is resorbed ex inactivitate. This system is analogous to osteoporosis in immobilised skeletal bones. Bone induction was achieved in BALB/c mice after injection of a suspension of 3 x l0(6) HeLa cells into thigh muscle of animals immuno-suppressed by a single dose ofhydrocortisone. To slow down the process of induced bone resorption, OPG was administered and the effect was measured quantitatively by weighing the mass of the induced ossicle after hydrolysis of soft tissues surrounding the induced ossicles. As an effect of the application of OPG, dry bone mass of the induced ossicles exceeded 340-540% of the values of the control specimens following 9 applications of0.05 mg OPG per mouse every second day or 14 doses every day.

In this paper, the effect of osteoprotegerin (OPG) on slowing down the resorption process of heterotopically induced bone tissue is described. The induced ossicle is resorbed ex inactivitate. This system mimics osteoporosis in immobilised skeletal bones. Bone induction was achieved in BALB/c mice after the injection of the suspension of 3 x 10(6) HeLa cells into thigh muscle of animals immuno-suppressed by a single dose of hydrocortisone. To slow down the process of resorption we applied OPG and measured quantitatively the effect by weighing the mass of mineral deposited in the induced ossicle after hydrolysis of soft tissues surrounding the induced ossicles. As the effect of application of OPG more than 340-540% of bone mineral is found in the induced ossicles following nine applications of 0.05 mg OPG per mouse, every second day--in comparison to the control animals.

The mechanism of formation of the maxillary sinuses is not elucidated as yet, although their morphology during embryogenesis is well described. In the prenatal period, the pneumatization hypothesis is not valid. As the molecular approach to this problem is difficult to apply to human samples, we decided to apply immunohistochemical reactions to analyse the synthesis of selected molecules involved in the rebuilding of tissues. Hematoxylin-eosin staining and immunohistochemical reactions for the detection of MMPs (matrix metalloproteinases), one of their inhibitor TIMP 1 (tissue inhibitor of MMPs), BMP 6 (bone morphogenetic protein 6) and TGF-beta (transforming growth factor beta) were performed in the epithelium the mucosa of the maxillary sinuses of several human foetuses from the collection of the Anatomical Institute. The age of the foetuses was 8, 11, 15, 16, 17, 18 and 22 weeks. An intense positive reaction for MMPs 1, 2 and 3 was found in the mucosal epithehum of developing sinuses in the whole series of foetuses was found. The reaction was more intense in advanced stages of foetal development. Tissue derived inhibitor TIMP was hardly detectable, regardless of the age of samples. However, the intensity of the reaction for TGFbeta was strong in both young and more mature sinus epithelium. The presence of BMP 6, a member of the superfamily of TGFbeta, was detected although the intensity of this reaction in the epithelium was rather weak. Both TGFbeta and BMP 6 are well known as regulators of differentiation in the course of organogenesis. Results of the histochemical analysis suggest the possible involvement of the epithelium in the growth and formation of the maxillary sinuses. The main argument for this is intense reaction for MMP proteases which, as in bone, regulate the turnover and rebuilding processes of the extracellular matrix (ECM).

Heterotopic ossicles were induced in thigh muscles of immunosuppressed mice by implantation of suspension of several HeLa cell lines. These ossicles are the object of our research on osteoporosis. It was found that various HeLa cell lines differ in their potential to induce osteogenesis. In the previous paper we demonstrated the involvement of bone morphogenetic proteins (BMP-4 and BMP-6) secreted by HeLa cells in this phenomenon. In the present paper we try to find out the reason of the heterogeneity of various cell lines regarding the differences in their osteoinductive potencies. By the use of semiquantitative RT-PCR the differences in mRNA expression for several isoforms of BMP proteins in examined HeLa cell lines were found. The presence or absence of some of the BMP isoforms seems to be correlated with the quantity of heterotopically induced mineralised tissues. This was measured by weighing the deposited mineral after digestion of soft tissues surrounding the induced ossicles. This finding is supporting the thesis on high and uncontrolled heterogeneity of various HeLa cell lines used all over the world.

We have previously shown that bovine lactoferrin (BLF) given intravenously (i.v.) protected mice against a lethal dose of Escherichia coli and strongly stimulated both the clearing and killing activities in liver, lungs, spleen and kidney. Since some studies indicated a reduction of the manifestation of experimental pancreatitis with lactoferrin (LF), we decided to examine the protective activity of BLF against lethal E. coli infection in animals with alloxan (Alx)-induced diabetes. It appeared that 48 h diabetes substantially lowered the killing activity in all four organs as well as the clearing rate of E. coli from the circulation. BLF given i.v. reduced this undesirable effect of diabetes. However, in 10- and 20-day diabetic animals, the diabetes alone stimulated the killing activity in the organs investigated, and upregulated the clearing rate of E. coli from the circulation. Lactoferrin significantly increased both the killing and the clearing activity in these long-term diabetic animals. In some cases the stimulating effect of BLF was very high, suggesting a concerted action of BLF and diabetes in that category of mice. Despite these beneficial effects of BLF and diabetes on the killing process in the investigated organs, the survival time of animals from all the diabetic groups (48 h, 10 and 20 days) was not prolonged by BLF. The protective properties of BLF did not depend on the blood glucose levels in the diabetic animals. BLF partly delayed the development of experimental Alx-induced diabetes, measured by the glucose level, but only if administered shortly after Alx injection. In conclusion, we demonstrated that the state of diabetes alone could increase killing of bacteria in the investigated organs and LF enhanced this process. However, LF had no protective effect against the mortality of diabetic mice infected with a lethal dose of E. coli.

Six ELISA variants exploiting two monoclonal antibodies, one rabbit antibody and their peroxidase conjugates were applied in assays of purified human myoglobin, apomyoglobin and the protein in human muscle extracts. The myoglobin was accurately determined with monoclonal antibody no. 82 used for coating of ELISA plates while assays performed with monoclonal antibody no. 49 or rabbit antibody used for coating were weak or none. Determinations of human apomyoglobin with ELISA variants were somewhat more sensitive than those of myoglobin. Obtained in this work results were compared with those done using commercial Seratec kit for immunoassay of human myoglobin. Addition to the muscle extracts not only concentrated salts but also acetone, ethanol, sodium dodecyl sulfate or some other denaturing agents markedly increased assays of myoglobin by ELISA with monoclonal antibody no. 49 and antibody no. 82 conjugated with peroxidase. Removal of acetone or ammonium sulfate from extracts resulted in dramatic decrease of the estimated myoglobin. Filtration of the extract through Bio-Gel A5m column did not affect low assays of myoglobin in fractions without pretreatment with acetone. Myoglobin was isolated from human heart extract by immunoaffinity chromatography on Sepharose-antibody no. 82 column and the isolated protein was identified by gel electrophoresis and Western blot.

Two newly isolated monoclonal anti-beta hLH antibodies did not recognize both human chorionic gonadotropin (hCG) and three synthetic peptide fragments corresponding to beta hLH sequence. These antibodies were applied for ELISA of human luteinizing hormone (hLH), however, sensitivities of these assays were much lower than those with two other monoclonal antibodies obtained earlier in our laboratory. No significant differences between assays of hLH from pituitary and urine with 6 different pairs of monoclonal antibodies (4 anti-beta and 1 anti-alpha-subunit) were noticed. The highest hLH concentration in urine samples collected during 53 menstrual cycles of 6 women was demonstrated at different times of the day. In 14 out of 52 "preovulatory urine" portions, preserved for several weeks, over 80% increase of assayed hLH was shown. The highest assays of hLH from pituitary and urine were noted at pH 7.5-9.0. Using affinity chromatography the whole pituitary hLH was bound to ConA-Sepharose column and was eluted with alpha-methyl-D-mannoside as a single peak. However, a small part of hLH from urine was eluted from the column as non retarded peak. When HPLC analysis with DEAE column was performed, pituitary hLH was separated in two fractions while lutropin from urine was eluted as a single peak.

It was noted that human and horse sera as well as human heart and skeletal muscle homogenates or extracts distinctly decrease immunoassays of purified myoglobins. The assays of homogenate and extract myoglobins could be many times increased by precipitation certain proteins with concentrated ammonium sulfate or sodium chloride. Also in homogenates and extracts incubated for several days increased assays of myoglobins were noted. The obtained results indicate that both myoglobins occur in complex with other tissue component(s).

Rabbit antisera against human myoglobin and horse myoglobin cross-reacted with both myoglobins but only one of them recognized human hemoglobin. Two mouse monoclonal antibodies anti-human myoglobin were obtained, but only one of them (No. 49) cross-reacted with horse myoglobin. Antibody No. 49 and rabbit antibodies reacted also with apo-, FITC- and treated with hydrochloric acid or TPCK-trypsin horse myoglobin, but their binding to myoglobin pretreated with NaOH was reduced. Thirteen peptides overlapping sequence of human myoglobin were synthesized on polyethylene pins. Rabbit and mouse polyclonal antibodies reacted with some of these peptides but no reaction was noted with mouse monoclonal antibodies. Two monoclonal antibodies were applied for specific immunoassay of human myoglobin.

Luteinizing hormone (LH) is a heterodimeric glycoprotein containing varied amount of sialic acid. This is a reason of numerous LH isoforms called also isohormones. The hormone isoforms were separated usually by gel electrophoresis, isoelectrofocusing or chromatofocusing. They differ in biological and immunological activity. Human and some animals LH isoforms were reviewed. Also some genetic mutants of LH are described. Problems of the human isoforms for pathology and diagnostics are presented.

Results of studies on birth control vaccines accomplished by many research groups within the WHO Special Programe of Research, Development and Research Training in Human Reproduction has been reviewed. The mostly investigated contraceptive vaccines contain human chorionic gonadotropin, and therefore structure, biological role and immunogenicity of the hormone were presented. Also, vaccines obtained on the basis of subunit beta of the human chorionic gonadotropin, its heterodimer with subunit beta of ovine lutropin or C-terminal peptide of beta subunit were described. Results of two phases of clinical trials with immuno-contraceptive vaccines were presented. The use of contraceptive vaccines for treatment of some trophoblastic tumors and the idea of using contraceptive recombinant vaccines were also mentioned.

Human sera obtained from healthy individuals, patients with cancer diseases, infertile and pregnant women were investigated for the presence of immunoglobulins (Ig) binding to synthetic fragments corresponding to sequence of human chorionic gonadotropin (hCG). Four synthetic peptides corresponding to some regions of alpha hCG and beta hCG subunits, coupled to polystyrene beads and 16 peptides overlapping sequences of alpha- and beta hCG subunits, synthesized on polyethylene pins, were used. IgG and IgM bound to hCG-synthetic peptides were assayed using goat or mouse monoclonal antibodies anti-human IgG labeled with horseradish peroxidase. Most of the investigated sera contained IgG which bound to examined synthetic peptides, but obtained results were dependent on serum donors. Some relationships between results obtained with peptides synthesized on modified pins according to Geysen et al. method and corresponding peptides synthesized by Merrifield method and coupled to beads were noted.

Characteristics of two monoclonal antibodies against human lutropin and their use in enzyme immunoassays of the hormone are presented. Diluted solution of pituitary lutropin was unstable in buffered saline and could be stabilized with some proteins. Concentration of the lutropin in woman urine, collected during "lutropin-peak", was markedly increased during long time storage at 5 degrees C or at room temperature. Using HPLC it was demonstrated that pituitary lutropin, normal urine lutropin and "increased" urinary lutropin were eluted from ion exchange column almost with the same retention time.

Composition, properties and occurrence of human myoglobin (hMb) were described. Methods for immunological assay of hMb were presented. Localization of antigenic determinants in myoglobin molecule was shown. Value of assay of hMb in serum and urine for early diagnosis of myocardial infarct and some other muscle diseases was discussed.

Coating of Immulon polystyrene plates with 0.2% casein in phosphate buffered saline (PBS) completely abolishes adsorption of human serum proteins to these plates. However, pretreatment of such plates with PBS or 0.15 M NaCl in deionized water (10 microS) before coating makes possible adsorption of human immunoglobulins G (HIgG) and M but not serum albumin (HSA) and transferrin (HTrf). Effect of pretreatment with PBS on adsorption of HIgG is long-term and rather stable. Results of experiments with radioiodinated HIgG, mouse IgG, HSA and human chorionic gonadotropin confirm the peculiar effect of pretreatment with PBS on casein coating. Pretreatment with deionized water, instead of PBS, markedly diminish adsorption but tap or commercial spring waters (> 1000 microS), even without 0.15 M NaCl, affect adsorption similarly to PBS in deionized water. Super pure water (0.05 microS) even with NaCl used for pretreatment does not influence adsorption of HIgG to plates coated with casein. Distinct differences in adsorption of HIgG and HTrf was demonstrated using solutions for ELISA prepared from super pure, deionized and tap waters.

Heterodimeric composition and amino acid sequence of hLH is reviewed. The hormone isoforms and methods of their assay are summarized. Releasing of the hormone, its control by synthetic antagonists and agonists and their significance for therapy are described. Structure and function of hLH receptor are presented.

Five peptides corresponding to human lutropin (hLH) subunit fragments were synthesized by a solid phase method and their physicochemical characteristics are presented. Antibodies induced in rabbits with 3 peptides of beta hLH fragments coupled to thyrotropin did not bind hLH and human chorionic gonadotropin (hCG). Also 2 synthetic peptides of alpha hLH fragments did not react with rabbit anti-hLH, anti-hCG and anti-alpha hCG antibodies. Using 2 monoclonal anti-alpha hCG and anti-beta hLH antibodies a sensitive sandwich ELISA technique was elaborated. Using this technique stability of hLH was investigated. The ELISA was also applied for assays of urine hLH in normal and ovulation days.

Human chorionic gonadotropin, its two subunits and its conjugate with thyroglobulin were used for immunization. The strongest immunogens were demonstrated to be the intact hormone and beta subunit, the weakest was alpha subunit. Using three peptides corresponding to hormone subunits coupled to Sepharose 4B various antibodies were isolated from goat antiserum by immunoaffinity chromatography. Specificity of the purified antibody preparations was studied. It was demonstrated that enzyme linked immunosorbent assay with two goat antibodies--one for coating of microtiter plates and the other one conjugated with peroxidase--is suitable for sensitive assays of both human chorionic gonadotropin and luteinizing hormone. Specific and sensitive assays were performed using combination of goat antibody anti-122-145-beta hCG and monoclonal antibody anti-alpha hCG.