A low-molecular-weight dialysable leukocyte extract selectively enhances development of CD4⁺RORγt⁺ T cells and IL-17 production.

A low-molecular-weight (under 10 kDa) dialysable leukocyte extract (called transfer factor, TF) has been shown to be a prospective substance to improve or modulate immune response in autoimmunity, inflammation, infectious diseases or cancers. However, the use of TF has been limited by the absence of any data on the mechanism of its action. Here we show that TF prepared from peripheral blood leukocytes of healthy human donors displays multiple regulatory effects on individual parameters of the immune system. TF decreases proliferation of T and B lymphocytes and partially alters the production of cytokines and nitric oxide by activated macrophages. TF also inhibits production of T helper 1 (Th1) cytokines interleukin 2 (IL-2) and interferon γ, slightly stimulates production of Th2 cytokine IL-10 and considerably enhances the secretion of IL-17 by activated mouse spleen T cells. At the molecular level, TF enhances expression of genes for transcription factor RORγt and for IL-17. The enhanced expression of the RORgt gene corresponds with an increase in the number of RORγt⁺CD4⁺ Th17 cells and with enhanced IL-17 production. In contrast, the expression of the Foxp3 gene and the proportion of CD4⁺CD25⁺Foxp3⁺ regulatory T cells are not significantly changed in the presence of TF. These results suggest that the activation of pro-inflammatory Th17 cells, which have multiple immunoregulatory properties, could be the main mechanism of the immunomodulatory action of a low-molecular-weight leukocyte extract.

The effect of bacterial contamination of semen on sperm chromatin integrity and standard semen parameters in men from infertile couples.

A considerable proportion of male factor infertility cases are associated with inflammatory processes. The most common sexually transmissible agents causing sexually transmitted diseases in industrial countries are Chlamydia trachomatis, genital Ureaplasma and Mycoplasma spp. This study was undertaken to investigate whether these bacterial contaminants in semen affect sperm quality parameters and particularly DNA integrity (detected by sperm chromatin structure assay) in males from infertile couples (n = 293). The results showed that semen contaminations with the investigated bacterial species were not associated with sperm DNA fragmentation. However, contaminations with Mycoplasma spp. and C. trachomatis were associated with decreased sperm concentrations. Total sperm numbers in contaminated semen samples tended to be decreased, but not significantly. Mycoplasma had the highest adverse effect on sperm quality (concentration, motility, morphology and DNA condensation). Antibiotic therapy of the selected 47 men was successful in 55%, but semen quality parameters did not improve at least up to 3 months after the therapy. The presence of pathogenic bacteria in semen is primarily associated with low sperm production. Our data showed that Mycoplasma spp. contamination of semen had the highest adverse effect on sperm quality. Sperm chromatin integrity assessed by the presence of DNA breaks was not disturbed.

Sperm chromatin integrity in young men with no experiences of infertility and men from idiopathic infertility couples.

Damage to the genetic component of spermatozoa seems to play the main role in a majority of cases where current approaches fail to reveal the specific cause of male infertility. In this study, we compared semen quality in men assigned to two defined groups: men from couples with unexplained infertility - idiopathic infertility (A) and young men with no experiences of infertility (B). All samples were examined by standard ejaculate analysis and sperm chromatin structure assay (SCSA). Sperm chromatin damage was significantly higher in men from group A than in those from group B. Similar results were obtained by comparison of men from group A (all men were normozoospermic) with normozoospermic men from group B. According to these results, we can suppose that chromatin disorders may be the causal factor of subfertility or infertility in some of these men. No evidence for a strong association between chromatin disorders and standard parameters of ejaculates was found. We failed to confirm a relationship between smoking and sperm quality in men from any of the investigated groups. SCSA is a method that facilitates the identification of infertile men who otherwise show normal semen variables.

Immunosuppressive effects of vermiculine in vitro and in allotransplantation system in vivo.

Vermiculine, a macrocyclic aglycosidic dilactone isolated from Penicillium vermiculatum, has been shown to have immunomodulatory properties. Here, we tested the effects of vermiculine on selected parameters of cell-mediated immunity in vitro and on skin allograft survival in vivo. Vermiculine inhibited in a dose-dependent manner the proliferation of mouse spleen cells stimulated with Concanavalin A ((Con A), i.e. T-cell mitogen), bacterial lipopolysaccharide ((LPS), B-cell mitogen) or with irradiated allogeneic cells. In addition, vermiculine dose-dependently inhibited the production of Thl (IL-2, IFN-gamma) and Th2 (IL-4, IL-10) cytokines and suppressed the production of nitric oxide (NO) by activated macrophages. When compared with cyclosporine (CsA), vermiculine was less inhibitory for IL-2 gene expression and IL-2 synthesis, comparably suppressive on IL-10 production and even more inhibitory for NO synthesis. These observations suggest that vermiculine and CsA inhibit immune reactions by different mechanisms. Treatment of graft recipients with vermiculine or CsA prolonged survival of skin allografts in a mouse model. The combination of both drugs enhanced the survival of allografts significantly more than either drug alone. The results thus suggest that vermiculine is a potential immunosuppressive drug acting by a mechanism distinct from that of CsA, and thus it may be used alone or in combination with other drugs for immunoregulatory purposes.

Prevention of corneal allograft rejection in a mouse model of high risk recipients.

AIM: To determine the effectiveness of treatment with immunosuppressive drugs and monoclonal antibodies (mAb) after penetrating keratoplasty in two different models of high risk mouse recipients. METHODS: Corneas were grafted orthotopically in mouse models of high risk recipients with either neovascularisation of the graft bed or presensitisation to graft donor antigens. Recipients were treated with mAb against CD4(+) or CD8(+) cells or against T cells, or were treated with cyclosporin A (CsA) or mycophenolate mofetil (MMF), or a combination of both drugs. RESULTS: Control untreated recipients with neovascularised graft bed or presensitised to the graft donor antigens rejected corneal allografts in 12.5 (SD 2.3) and 9.9 (1.6) days, respectively. Treatment of graft recipients with a neovascularised graft bed with mAb anti-CD4 or anti-T cells, but not with mAb anti-CD8 or with immunosuppressive drugs, resulted in a significant prolongation of graft survival; 75% and 28.5%, respectively, of grafts survived for more than 45 days after grafting. However, none of the treatments were successful in presensitised recipients. CONCLUSIONS: Treatment of high risk recipients with mAb anti-CD4 is more effective in preventing corneal allograft rejection than the treatment with mAb anti-CD8 or the immunosuppressive drugs MMF and CsA. However, the effectiveness of the treatment depends on the recipients' pretransplantation risk type.

Reverse effect of indomethacin on the immunosuppressive activity of boar seminal immunosuppressive fraction.

The inhibitory activity of seminal immunosuppressive fraction (ISF) on mitogen-stimulated lymphocyte proliferation and on production of antibody to a soluble antigen was modified by indomethacin or monoclonal antibody to ISF. The ability of indomethacin or monoclonal antibody to ISF to reverse the ISF-induced inhibition of mitogen-stimulated lymphocyte proliferation was estimated by measuring bromodeoxyuridine incorporation into replicated DNA. Splenocytes from mice treated with indomethacin or monoclonal antibody to ISF prior to the application of ISF were tested. The ability of indomethacin or monoclonal antibody to ISF to reverse ISF-induced suppression of antibody production was estimated by measuring antibody titers by ELISA in the blood sera from mice immunized with keyhole limpet hemocyanin (KLH). These animals were treated with indomethacin or monoclonal antibody to ISF prior to the application of ISF. The results showed that both indomethacin and monoclonal antibody to ISF reversed the inhibitory effect of ISF on mitogen-stimulated lymphocyte proliferation as well as on antibody production.Recently, we have identified ISF as a complex of the major seminal glycoproteins PSP I and PSP II. PSP II is the part that is responsible for immunosuppressive properties of the complex. To learn whether the ISF immunosuppressive effect is associated with its protein or saccharide part, we examined the deglycosylated PSP II for its antiproliferative effect on mitogen-stimulated mouse lymphocytes. The results suggest that deglycosylation of PSP II did not affect its antiproliferative activity. This suggest that PSP II immunosuppressive properties are associated with the protein and not the saccharide part of the molecule.

Active suppression of the proliferative response to interleukin-2 in tumour-bearing mice.

Spleen cells from mice bearing progressively growing syngeneic methylcholanthrene-induced sarcomas are immunologically hyporeactive and also their proliferative responses to interleukin-1 (IL-1) or interleukin-2 (IL-2) stimulation are considerably decreased. The hypo-reactivity to IL-2, but not to IL-1, is due to an active suppression: spleen cells from tumour-bearing mice inhibit the IL-2-induced proliferation of cells from normal donors. Supernatants obtained after cultivation of spleen cells from mice bearing tumours show similar suppressive effects. The hyporeactivity to IL-2 and IL-1 in tumour-bearerers is not improved by indomethacin, an inhibitor of prostaglandin synthesis. The results show that the low reactivity to IL-2 in tumour-bearing mice is due to an active suppression mediated by spleen cells and their factor(s) and that more different mechanisms regulate responsiveness to interleukins in tumour-bearing hosts.

The alterations of immunological reactivity in heroin addicts and their normalization in patients maintained on methadone.

Drug addiction influences many physiological functions including reactions of the immune system. The higher occurence of infectious and other diseases in drug addicts has been explained by the depression of immunity due to the harmful effects of the drug. To test this assumption, we tested the proliferative responsiveness and cytokine production of PBL from a group of heroin addicts (N = 19), patients maintained on methadone (N = 15) and healthy controls (N=15). The results show that Con A-induced proliferation of PBL from heroin addicts was even enhanced in comparison with PBL from the control group. Similarly, production of IL-2, IL-10 and IFNgamma was higher in the group of heroin addicts than in healthy controls. The enhanced proliferation of PBL or the increased production of cytokines observed in heroin addicts was partially or completely normalized in the group of patients maintained on methadone. A significantly higher production of IL-6 was found in both unstimulated and stimulated PBL from heroin addicts and patients maintained on methadone, when compared with PBL from healthy controls. The results thus showed enhanced proliferative activity and increased production of various cytokines in heroin addicts and partial or complete adjustment of these alterations in patients maintained on methadone.

Phenotype, immunological reactivity and cytokine production profile of Peyer's patch cells from mice immunized orally with allogeneic cells.

Mice of inbred strain BALB/c were immunized orally for 10 consecutive days with fresh spleen cells from allogeneic C57BL/10 (B10) donors. The immunized mice displayed significant allotransplantation immunity in vivo as demonstrated by resistance to the growth of allogeneic tumours induced by high doses of tumour cells. No significant changes in the proportion of the major T cell subsets in PP of immunized mice were found 1 or 7 days after the last immunization dose. However, PP cells from orally immunized mice displayed stronger proliferative response after stimulation with cells used for oral immunization than the cells from control animals. Similarly, after stimulation in vitro with specific alloantigens, PP cells from orally immunized mice produced more IFN-gamma than the cells from control recipients. On the contrary, the production of IL-4 was significantly decreased in the immunized mice. The production of IL-2 by PP cells after oral immunization was not significantly changed and IL-10 was only slightly increased. The results thus show that oral immunization with allogeneic cells induces systemic transplantation immunity which can be demonstrated already in Peyer's patches by increased cell proliferation after immunization with specific alloantigens and by changes in cytokine production.

Ultrastructure of the tracheal epithelium in rabbits after acetylcholine administration.

The ultrastructure of the tracheal epithelium was studied 5 and 20 min after intravenous (i.v.) administration of 0.5 mg of acetylcholine. As a result of this treatment, the goblet cells were overstimulated and damaged, and the mechanism of their secretion was accelerated. Only a mild pathological alteration was encountered in the ciliated cells. According to our classification, the degree of secretory element damage was severe, the injury to the ciliary border was moderate. The morphological signs of the severe impairment of the self-cleaning ability of the epithelium were inspissated mucus and very numerous bacteria in the area among free cilia.

The role of macrophages and nitric oxide in the effector phase of allotransplantation reaction.

OBJECTIVES: In spite of the extensive research, the exact mechanism of graft rejection remains not well understood. Since the recipients which have inactivated all known mechanisms of T cell-mediated cytotoxicity can reject allografts, the graft infiltrating macrophages have been considered as a potential effector cell population capable to reject incompatible allografts. METHODS: Skin grafts were transplanted in mice in fully allogeneic strain combination or in xenogeneic system and the production of nitric oxide (NO) by graft infiltrating macrophages was determined in graft explants. RESULTS: A significant production of NO was found in rejected allografts and xenografts. The production of NO in graft explants was dependent on the presence of alloimmune T cells and was inhibited by the specific inhibitors of inducible NO synthase. Treatment of allograft recipients with inhibitors of NO synthase enhanced grafts survival. CONCLUSIONS: T cell-dependent production of NO by graft infiltrating macrophages may represent at least one of the effector mechanisms of allograft and xenograft rejection.

An abnormally high occurrence of malformed kinocilia in the tracheal epithelium of a clinically healthy rabbit.

An abnormally high occurrence of malformed kinocilia containing axonemes with different number or arrangement of microtubules compared with the typical 9+2 pattern of motile cilia was encountered in the tracheal epithelium of one clinically healthy rabbit. The malformed cilia amounted to 6.87% of all kinocilia. Individual types of ciliary malformations were further classified. The frequency of ciliary malformations was compared with that in all other rabbits the ciliary border of which has been quantitatively evaluated in our laboratory. Using Grubbs test the extremely low probability of occurrence of such a high number of malformed kinocilia in the rabbits' population was verified. The studied rabbit suffered from a mild form of the immotile cilia syndrome, but the loss of less than 10% of moving cilia did not lead to the expression of the clinical signs of the impaired function of cilia in the organism.

Effects of oral intake of nitrates on reproductive functions of bulls.

Effects of oral intake of nitrates on selected biochemical and endocrinological indices and its impact on reproductive functions were investigated in five feeder bulls aged 16-18 months. The bulls were tested prior to (30 days), during (30 days) and after (35 days) the period of the nitrate administration. The initial dose of 100 g potassium nitrate per day was increased at weekly intervals by 50 g up to 250 g per day. The administration of nitrates resulted in a highly significant (P < 0.01) increase in methaemoglobin concentration and a non-significant decrease in the concentration of beta-carotene and a highly significant (P < 0.01) decrease in the concentration of E vitamin in blood serum. A significant (P < 0.01) increase in blood serum concentration of bile acids and prolonged biological half-life of progesterone were suggestive of an impairment of liver metabolism. Prolonged intake of excessive doses of nitrates resulted in a significant (P < 0.05) increase in cortisol concentration during and after the administration period, while depressed thyroid gland activity was evident from a significant (P < 0.05) decrease in thyroxin concentration during the administration period. A suppression of hypothalamic functions after the administration period was documented by non-detectable levels (< 0.001 microgram/ml) of thyrotropin in TRH test. Depressive effects of nitrates on the function of Leydig cells during and particularly after the administration period were apparent from weakening testicular responses to a treatment with GnRH. Biochemical analyses of seminal plasma revealed a highly significant (P < 0.01) increase in total acid phosphatase activity and a significant (P < 0.05) decrease in the concentration of fructose. No other significant changes in seminal plasma components were observed. Adverse effects of excessive intake of nitrates were also evident from reduced sperm motility in the 120-min thermal test. While no difference was found in the frequency of primary morphological abnormalities, the number of secondary abnormalities rose by 115% in the post-administration period and was suggestive of damaged membrane integrity. Histological examinations revealed degenerative lesions in cells of the spermiocyte and spermatid layers.

[Opioids and their immunomodulatory properties]. / Opiáty a jejich imunomodulacní vlastnosti.

Opiates have been recently used for suppression of the neuropathic pain or to relieve pain in patients with cancer diseases. However, opiates are also used by drug abusers to achieve feeling of euphoria. These drugs influence not only the nervous system but they can also modulate many other physiological functions including those of the immune system. Since opioid receptors have been found on the surface of cells of the immune system, two possible mechanisms of opiate actions have to be considered. The first one represents a direct action of the opiates through the opioid receptors on immune cells; the second mechanism is mediated by the nervous system. The immunomodulatory properties of the opiates have been demonstrated in numerous models. Especially the enhanced sensitivity to viral and bacterial infections, observed in drug abusers, is accounted to the side effects of opiates. Experimental animal models have shown even more complex actions of opiates, which can lead to suppression as well as to stimulation of individual immunological parameters. Although proliferation of lymphocytes tested in vitro after application of opiates in vivo is generally reduced, production of the pro-inflammatory cytokines and some functions of macrophages can be enhanced. Effects of opiate action depend on the experimental model used, the drug dose, way of drug application, time of testing and on the tested immunological parameter. This article summarizes recent knowledge of effects of opiates on the functions of cells of the immune system. It also refers global problems of exploitation of illegal drugs and the importance of methadone in the substitution treatment.

Relationship between the saccus endolymphaticus and the fourth ventricle of the brain of the domestic fowl (Gallus gallus f. domestica).

The fine structure of the wall of the SE was determined exactly and its relationship to the cisternae (the evaginations of the roof of the fourth ventricle extending to the SE) was defined. The way in which the cisterna is formed was defined and the development of its fine structure was described by comparing serial sections from 19-day embryos and adult fowls. Like the SE, the cisternae are lodged in the angle between the cerebellum and the medulla oblongata, in the subarachnoid space. The terminal segment of the cisterna lies in the immediate vicinity of the mesenchymal epithelium bordering the basal labyrinth of the SE cells. Collagen trabeculae keep the SE and the cisternae suspended in the subarachnoid space. The cisternae and trabeculae are wrapped in mesenchymal epithelium. The cisterna is avascular and does not communicate with the SE. The cisterna is lined internally with simple squamous epithelium (modified neural epithelium of the roof of the fourth ventricle). The bodies of the cells bulge into the lumen of the cisterna in the region of localization of their nucleus. The epithelium is seated on a pronounced basal lamina. The surface turned towards the subarachnoid space is lined continuously with mesenchymal epithelium without a basal lamina. The cells of the cisternal epithelium are connected by tight junctions of the type of zonulae occludentes and desmosomes. The basal lamina is continuous and distinct. The mesenchymal epithelium of the subarachnoid space has no basal lamina, as on the subarachnoid surface of the SE, the cisternae, the trabeculae, the pia mater and the arachnoidea.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the gene for tumor necrosis factor-beta but not for tumor necrosis factor-alpha is impaired in tumor-bearing mice.

Spleen cells from mice bearing progressively growing syngeneic sarcomas are immunologically hyporeactive and respond by significantly decreased proliferative response to stimulation with mitogens and cytokines. Here we show that these hyporeactive cells synthesize, after mitogen stimulation, comparable amount of mRNA for tumor necrosis factor (TNF)-alpha as do cells from control mice. However, stimulated spleen cells from the same tumor-bearing mice produce considerably less mRNA for TNF-beta than cells from control mice. These observations were further confirmed using purified peritoneal macrophages and enriched splenic T cells. The results thus demonstrate a distinct regulation of expression of genes for TNF-alpha and TNF-beta, two functionally very similar cytokines, and simultaneously a selective impairment of T-cell function in the course of growth of syngeneic tumors in mice.

[Participation of the myocardium in the formation of the pulmonary vein wall of small mammals (a quantitative study)]. / Uchastie miokarda v formmirovanii stenki legochykh ven melkikh mlekopitaiushchikh (kolichestvennoe issledovanie).

The thickness of the layer of cardiac muscle cells in the wall of the pulmonary veins was studied and quantitatively evaluated. The thickness of the intrapulmonary myocardial layer in the tunica adventitia of the venous wall depends on the body mass of the animal. This relation can be expressed by a hyperbola. In animals with the body mass of approximately 50-400 g, the venous pulmonary myocardium can be observed only as a continuous coat in the intrapulmonary veins up to the diameter of 170 mcm. In animals with the body mass of approximately 3--50 g, the myocardial layer reaches all branches of the pulmonary veins up to the diameter of 50--100 mcm.

Inability of mitogen-stimulated spleen cells from newborn mice to synthesize interleukin-2 receptors.

Spleen cells from newborn mice do not respond by proliferation to concanavalin A (Con A) or bacterial lipopolysaccharide (LPS) stimulation. This non-reactivity cannot be reversed to a positive response by exogenous interleukin-2 (IL-2). The stimulation with Con A of spleen cells from newborn mice, in contrast to cells from adult animals, does not result in synthesis of mRNA for inducible 55,000 molecular weight (MW) IL-2 receptors (IL-2R). The failure of neonatal spleen cells to synthesize IL-2R mRNA is an intrinsic property of the cells themselves, and it is not due to activity of natural suppressor cells present in newborn animals. Since the expression of functional IL-2R represents one of the early and pivotal events in immune cell activation, we propose that the inability to synthesize IL-2R may be one of the primary reasons for the immunological immaturity of newborns.

Immunological nonreactivity of newborn mice: immaturity of T cells and selective action of neonatal suppressor cells.

Mitogen-stimulated spleen cells from newborn mice do not synthesize mRNA for the 55-kDa interleukin-2 receptor (IL-2R). The kinetic of development after birth of ability to synthesize IL-2R correlated well with the functional immaturity of T cells, as was tested by responsiveness to T-cell mitogen concanavalin A (Con A). This functional immaturity of T cells was not due to the activity of neonatal suppressor cells (NSC) which inhibited immune responses induced by mitogens or antigens. The suppressor cells did not inhibit proliferation of spleen cells stimulated with IL-1 or IL-2, nor did they inhibited expression of genes for tumor necrosis factor (TNF)-beta, TNF-alpha, and IL-2R in stimulated cells from adult mice. The results thus show functional immaturity of T cells in newborn mice and selectivity of the immunosuppressive action of NSC, which allow for production and for functional activity of cytokines at a time when the specific immune system is not functional because of both immaturity and a selective activity of inhibitory cells.