Gangliosides are acidic glycosphingolipids localized mainly on the outer membrane layer of the membranous cell structures. These molecules participate in important mechanisms at molecular, cellular, tissue, organ, and organism levels. It has been proven that gangliosides play a role as regulators of various biological processes but also as markers in a number of multifactor pathologies. In this regard, the present study determined the titers of the GM3, GM1, and GD1a gangliosides, as well as the titers of IgG-class antibodies against each of them by enzyme-linked immunosorbent assay (ELISA), in several different anatomic organs: brain, pancreas, myocardium, liver, and small intestine from rodents. A total extract (control sample) containing the complete set of molecules is prepared from each isolated anatomic organ. An equivalent amount of the extract is passed through a GSH-agarose column in order to select the molecules from each organ possessing affinity to the reduced form of glutathione tripeptide (GSH). GSH is known as an antioxidant, immunomodulator, cardioprotector, neuroprotector, hepatoprotector, anticancer, and antiaging agent. As a whole, significantly lower titers of the three gangliosides and the antibodies to them are reported in the myocardium and liver samples compared to the brain and pancreas samples. Taking into account that the myocardium and liver are the organs known with the highest content of GSH, the obtained results can be explained by the possibly high content of free and/or newly synthesized GSH in them, which does not participate in intermolecular interactions compared to the other investigated organs. Complete absence of each of the three tested gangliosides or of antibodies against them at certain dilutions of the small intestine samples, as well as the highest titers of the same parameters compared to the corresponding samples from the other organs of each of the gangliosides or of the specific antibodies at other dilutions, is observed. One of the explanations for those peculiarities is associated with the presence of the intestinal microflora, including the influence of intestinal bacteria neuraminidases (sialidases). The presented data also show a possibility of antibodies/immunoglobulins production by non-lymphoid cells, tissues, and organs in suitable conditions. Since the immunoglobulins thus produced reside outside the germinal centers of the specialized lymphoid tissues and organs, regulation of their production and functions by interactions with small ions and/or molecules is also important. Gangliosides are namely such small molecules. Special attention is paid to intermolecular interactions involving the listed gangliosides and GSH. The main objective is related to understanding the mechanisms underlying the interaction between the individual organs and systems in the body.
Abstract A major factor affecting male fertility is excessive death of germ cells, both immature germ cells and mature spermatozoa. It can be due to various factors causing testicular and/or post-testicular damage, such as infections, obstructive conditions, toxins, oxidative stress, hormonal imbalance, hyperthermia, and anti-sperm antibodies. Massive death of spermatozoa leads to a high proportion of dead sperm cells in the ejaculate (necrozoospermia or necrospermia) while death of immature germ cells can lead to low sperm count (oligozoospermia or oligospermia). Cell death can occur both by necrosis and by apoptosis; in recent decades, it has been found that apoptosis of mature spermatozoa is not only possible but quite common, and can contribute to infertility. Treatment approaches are primarily directed to the underlying condition, i.e. removing the cause(s) of sperm cell death whenever possible, but include also attempts to bypass the cell death event by intracytoplasmic sperm injection with testicular spermatozoa.
The spontaneous chromosomal fragility was tested by light microscopy observation of metaphases from peripheral blood lymphocytes of 8 patients with malignancies and of 8 healthy controls. In the tested patients, a significantly higher frequency of the spontaneous chromosomal fragility was observed compared to the controls, especially in the centromere chromosomal regions. Of particular interest were interactions involving gangliosides, the reduced form of tri-peptide glutathione (GSH) and/or of tumor-suppressor protein HACE1. The average titers of gangliosides and of anti-ganglioside antibodies in extracts from experimental in vitro models of laboratory-incubated cultures of mouse embryonic 3T3 fibroblasts, of mouse malignant myeloma cells, as well as of mixed cultures of both cellular types, were determined after previous passing of each one through GSH-agarose columns about the “selection” of the molecules in each one of the described samples, possessing affinity to GSH. Additionally, the presence and expression of the tumor-suppressor gene HACE-1 in the genome of mouse embryonic stem cells (mESCs) and malignant human cervical carcinoma HeLa cells, both containing an additional copy of this gene, inserted by transfection with appropriate recombinant DNA vectors containing a copy of the tumor-suppressor gene, were tested. The developed experimental in vitro models show specific intermolecular interactions, which could prevent the disease development. Furthermore, a possibility about the production of antibodies/immunoglobulins by non-lymphoid cellular types was shown. Because the antibodies produced in this manner are outside the germinal centers in the specialized lymphoid tissues and organs, the control of their functions by small ions and molecules, such as gangliosides, is important.
The potential of viruses as appropriate vectors for the development of new therapeutic strategies, as well as for the design of molecular (DNA, RNA, and/or protein) vaccines via substitution of nucleotide sequences, has been proven. Among the most appropriate DNA and/or RNA fragments, members belonging to families Parvoviridae (particularly adeno-associated virus, AAV) and Poxviridae have frequently been suggested for this purpose. In previous studies, the vaccine avipoxvirus strains FK (fowl) and Dessau (pigeon) have been proven able to infect mammalian cells (as well as avian cells), and to replicate productively in a small number of them; thus, we may be able to adapt them using incubation, and in these conditions. Additionally, we have previously proved, based on AAV recombinant DNA vectors, that it is possible to transfer appropriate genes of interest via mouse embryonic stem cells (mESCs). In the current study, we develop methods for the application of the same vaccine avipoxviral strains, based on the AAV DNA genome recombinant constructs, to be used for gene transfer in cells, for the transfer of DNA and/or RNA fragments (for the suppression of unwanted viral and/or cellular genes), and for the production of molecular (DNA, RNA, and/or protein) anti-cancer and anti-viral vaccines. To this end, sub-populations of embryonic mammalian cells infected with the two forms of both vaccine avipoxviral strains were frozen in the presence of cryo-protector dimethylsulfoxide (DMSO), subsequently thawed, and re-incubated. In most cases, the titers of the intra-cellular forms of the two strains were higher than those of their extra-cellular forms. These data were explained by the probable existence of the intra-cellular forms as different sub-forms, including those integrated in the cellular genome proviruses at a given stage of the cellular infection, and suggest the possibility of transferring nucleotide (DNA and/or RNA) fragments between cellular and viral genomes; this is due to the influence of activated fusion processes on DMSO, as well as drastic temperature variations.
The aim of the present investigation was to establish the sperm antibody incidence among patients included in a program for assisted reproduction by the means of the classical and new methods for sperm antibody detection; to analyze the correlation between the results demonstrated by the different techniques; to evaluate the obtained data in the context of the application of new assisted reproduction technologies (ART). In the Laboratory of Reproductive Immunology, Department of Biology, Medical University of Sofia, 73 sera from patients (35 couples and 3 men) with primary and secondary infertility, aged 23-46 years, grouped according to the diagnosis and included in the program for assisted reproduction "Technobioassistance" were tested. Our results demonstrated the highest incidence of sperm antibodies amongst patients with primary unexplained infertility. The percentage of positive reacting couples for at least one of the applied tests was 31%. The highest relative share of those reacting positively, was observed with ELISA and the tray agglutination test (TAT) of Friberg. For ELISA 31.48% of the sera reacted positively, while clinically relevant liters of sperm antibodies were found with TAT in 21.92% of the tested sera. We also found that 17.46% and 13.7% of the tested sera were positive, respectively in the sperm immobilization test (SIT) of Isojima and the gelatin agglutination test (GAT) of Kibrick. The high degree of correlation (P < 0.0001) between the tests of Kibrick and Friberg showed the appropriateness of their application for patients in an ART program. At the other hand, the lack of correlation between the other applied tests (the Isojima test and ELISA) confirmed the assumption that immunity against spermatozoa should be sought with at least two diagnostic tools. Five of the tested families with clinically relevant TAT titers were included in our ART program for "in vitro" fertilization and embryo transfer, after preliminary absorption of the seminal fluid with autologous semen, or underwent the ICSI technique. For these patients, one ICSI pregnancy finished with the successful birth of a healthy child and one "in vitro" pregnancy is developing at the moment.
Natural killer (NK) cells can discriminate between normal and cancer cells and are known to directly recognize and kill malignant cells or induce apoptosis. Thus, activation of NK cells is considered as a promising strategy for cancer treatment. However, clinical application has been somewhat limited because of difficulties in the preparation of sufficient number of highly cytotoxic/activated NK cells in vitro. We used cytokine stimulation to provide a suitable environment (activating receptor-ligand interactions) for the expansion of NK cells. This method potently expanded NK cells, and the final product was composed of highly proliferating NK cells. The expanded NK cells showed significant upregulation of various activation receptors such as CD69 and NKG2D. The latter is a particularly important receptor for triggering NK cell responses toward tumor cells.
Summary. The object of the present study was to study if there are differences in the presence of CD4-like molecules on human ejaculated spermatozoa in fertile donors and infertile patients (with globozoospermia). Indirect and absorption enzyme-linked immunosorbent assay and indirect immunofluorescence were applied. The enzyme-linked immunosorbent assay data showed that monoclonal anti-human CD4-antibody recognizes an epitope common to the human spermatozoa with normal morphology and round-headed spermatozoa. Localization of the antigenic determinants, identified by anti-human CD4-monoclonal antibody, in the acrosomal region, including equatorial segment, postnuclear cap and tail was determined in normozoospermic samples. A positive reaction was found on the sperm head both in the acrosomal and postacrosomal region of some round-headed spermatozoa in the samples with globozoospermia. The tails of the normozoospermic spermatozoa and of some round-headed spermatozoa were weakly immunopositive. The results of the experiments carried out are evidence of heterogeneity in the presence of CD4-like antigen determinants on human spermatozoa. These data increase the information about the CD4-antigen characteristic of the spermatozoa from fertile donors and infertile patients.
Mammalian cells from lines derived from bovine embryonic trachea (EBTr) were used in the present study. After the formation of semi-confluent monolayers, one subpopulation of cells was inoculated with the vaccine avian poxvirus strain FK (fowl) and the other with the vaccine avian poxvirus strain Dessau (pigeon) ( Poxviridae family). Twenty-four hours after viral inoculation, individual subsets of infected cells were treated with the purine derivative aminophylline and the remaining subsets with the indole derivative ergotamine tartrate, respectively. The effects of the analogues thus administered on the cells were recorded at the 24 th hour and 48 th hour after treatment with each substance, respectively. In cells inoculated with the FK viral strain and treated with both aminophylline and ergotamine tartrate, decreased cell viability was observed at all dilutions at the 48 th hour post-treatment compared to the 24 th hour. In aminophylline-treated cells, these differences were not statistically significant, unlike in ergotamine tartrate-treated cells, where they were statistically significant. In the same cells infected with the Dessau strain and treated with aminophylline, although decreased cell viability was found at the 48 th hour of treatment compared to the 24 th hour, in most cases no statistically significant differences were found. In cells infected with the same strain but treated with ergotamine tartrate, despite the lack of statistically significant differences, increased cell viability was seen at the 48 th hour post-treatment compared to the 24 th hour, specifically at the higher concentrations of 10 -3 and 10 -4 M/mL. These results suggest a lower cytotoxicity of aminophylline compared to ergotamine tartrate, but on the other hand, a higher anti-viral activity of ergotamine tartrate against the Dessau virus strain compared to aminophylline in in vitro conditions. Further studies need to be conducted in this regard.
