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.

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.

Serum antibodies against the small heat shock protein Hsp27 (HspB1) have been associated with certain diseases but have not been studied in infertility. Sera from 73 infertile patients were tested for antisperm antibodies using agglutination and immobilization tests and ELISA, and for anti-Hsp27 antibodies using ELISA. Of these sera, 31 (42.5%) were positive for anti-Hsp27 antibodies, and 46 (63%) were positive for antisperm antibodies through analysis with at least one method. A total of 17 sera (23.3%) were positive for both anti-Hsp27 and antisperm antibodies. When sera containing both anti-Hsp27 and antisperm antibodies were used for immunofluorescence, the neck and tail of spermatozoa were stained, where the related sperm-specific protein HspB10 (ODF1) is localized. These data suggest that anti-Hsp27 antibodies are elevated in a certain proportion of infertile patients, which, in some of them, could be due to cross-reactivity between Hsp27 and the related sperm cytoskeletal component ODF1.

Background: Experimental mouse and rat in vivo-models, which could suggest processes in the organism of patient with kidneys insufficiency, expressed with increased creatinine levels, were developed and investigated. Methods: Serum from blood of patient with increased creatinine levels was taken. The titers of the patient's serum IgG anti-GM1, anti-GM3 and anti-POOL antibodies were assessed by Enzyme-Linked Immuno Sorbent Assay (ELISA). The titers of the same gangliosides, but also of the IgG antibodies against them, were determined in mixture of healthy adult mouse kidneys extract with equal amounts of the patient’s serum, as well as in the total mouse kidneys extract, respectively, by ELISA modifications. Histological differences between fixed light microscopy slides from kidneys of experimental rat model of disease and healthy rat, respectively, were assessed.Results: Significantly higher was the titer of the IgG anti-POOL antibodies at dilution 1:40, and of the anti-GM1 IgG antibodies – at dilution 1:400. The highest was the titer of GM1 ganglioside. Very near was the titer of the anti-GM3 IgG antibodies. Approximately equal was the titer of gangliosides from the general POOL with the titer of the anti-POOL IgG gangliosides. Here again was suggested a possibility non-lymphoid cells, tissues and organs to produce antibodies, and the role of ganglisodies in their function regulation. The assessed biochemical data were confirmed by observed morphological differences between kidneys histological slides from experimental rat in vivo-model of kidney disorder and of healthy rat.

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 increased spontaneous fragility in the centromere regions of the chromosomes predisposes to abnormal cellular division due to abnormal interaction with the mitotic spindle, which leads to number chromosomal aberrations. The abnormal structure of the microtubule-associated proteins (MAPs) could also be among the internal factors, predisposing to these pathologies. On the other hand, the increased frequency of the spontaneous chromosomal fragility suggests abnormal structure of the respective chromosomal/chromatin components and thus an increased risk about gene mutations and structural chromosomal aberrations. The spontaneous chromosomal fragility was tested by light microscopy observation of metaphases from peripheral blood lymphocytes of 8 patients with polygene pathologies (neoplasms, neuro-degenerative diseases, and neuro-psychiatric disorders) and of 18 healthy controls. In the tested patients was established significantly higher frequency of the spontaneous chromosomal fragility, including in the centromere chromosomal regions, compared to the controls. Different inter-molecular interactions, which could prevent the pathology appearance, were investigated by appropriate experimental in vivo models. As a whole, in the most cases, the presented data showed significantly lower average titers of anti-ganglioside antibodies and gangliosides in the samples from myocardium and liver than in the samples from brain and pancreas. By taking into consideration that the myocardium and liver are known as the most enriched of GSH anatomic organs, the presented data could be explained with the literature messages of higher amounts of de novo-synthesized free GSH tri-peptide in the same two organs. Moreover, a possibility about the production of antibodies/immunoglobulins by non-lymphoid types of cells, tissues, and organs in appropriate conditions was shown. Because the so-produced antibodies are out of the germinative centers in the specialized lymphoid tissues and organs, the control of their functions by small ions and molecules as gangliosides is very important. In the current study, the attention was particularly directed to the iteractions with the participation of gangliosides, the reduced form of the tri-peptide glutathione (GSH), and the protein HACE1. Besides the tumor-suppressor activity of protein HACE1, also its neuroprotective influence was proven, namely by interactions with specific molecules.