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.

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.

Corneal stromal cells selectively inhibit production of anti-inflammatory cytokines by activated T cells.

The eye has been described as an immunologically privileged site where immunity is purely expressed. It has been demonstrated that administration of antigen into the eye induces only a weak immune response. However, the anterior part of the eye represents an important protective barrier against pathogens and other harmful invaders from the outer environment. Therefore, effective immune mechanisms, which operate locally, need to be present there. Because the cornea has been shown to be a potent producer of various cytokines and other molecules with immunomodulatory properties, we investigated a possible regulatory role for the individual corneal cell types on cytokine production by activated T cells. Mouse spleen cells were stimulated with the T cell mitogen concanavalin A in the presence of either corneal explants or cells of corneal epithelial or endothelial cell lines and the production of T helper 1 (Th1) or Th2 cytokines was determined by enzyme-linked immunosorbent assay (ELISA) or reverse transcription-polymerase chain reaction (RT-PCR). We found that the cornea possesses the ability to inhibit, in a dose-dependent manner, production of the inhibitory and anti-inflammatory cytokines interleukin (IL)-4 and IL-10 by activated T cells. The production of cytokines associated with protective immunity [IL-2, IL-1beta, interferon (IFN)-gamma ] was not inhibited under the same conditions. Corneal explants deprived of epithelial and endothelial cells retained the ability to suppress production of anti-inflammatory cytokines. This suppression was mediated by a factor produced by corneal stromal cells and occurred at the level of cytokine gene expression. We suggest that by this mechanism the cornea can potentiate a local expression of protective immune reactions in the anterior segment of the eye.

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.

Effects of boar seminal immunosuppressive fraction on production of cytokines by Concanavalin A-stimulated spleen cells and on proliferation of B lymphoma cell lines.

PROBLEM: The immunosuppressive fraction (ISF) of boar seminal vesicle fluid has recently been demonstrated to inhibit mitogen-stimulated proliferation of lymphocytes and antibody response to corpuscular and soluble antigens. The effects of ISF on in vitro and in vivo production of cytokines as well as its possible inhibitory effect on proliferation of B lymphoma cells remain to be elucidated. METHODS: The effect of ISF on proliferation of normal mouse spleen cells stimulated by Concanavalin A (Con A) and on mouse B lymphoma cells was measured by 3H-thymidine incorporation. Cytokines were determined in the supernatants of mouse spleen cells stimulated with Con A in the presence or absence of ISF by enzyme-linked immunosorbent assay (ELISA). In vivo cytokine production in the sera samples of mice treated with ISF and immunized with keyhole limpet hemocyanin (KLH) was followed by ELISA, too. RESULTS: We confirmed the inhibitory effect of ISF on Con A-stimulated lymphocyte proliferation. ISF affected cytokine production in the Con A-stimulated spleen cells: production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) was lowered, but production of IL-4, IL-6, and IL-10 was enhanced. Similarly, in the sera samples of mice immunized with keyhole limpet hemocyanin (KLH), IL-2 and IFN-gamma levels were decreased by ISF. ISF inhibited proliferation of Ag 8 and X 63-IL-2 B lymphoma cells as well. CONCLUSIONS: ISF inhibited production of T helper1 (Th1) cytokines (IL-2 and IFN-gamma) and enhanced production of Th2 cytokines (IL-4, IL-6, and IL-10). ISF seems to shift the Th1/Th2 pattern in favor of Th2. ISF exhibited an antiproliferative activity on mouse B lymphoma cells.

[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.

Augmented production of proinflammatory cytokines and accelerated allotransplantation reactions in heroin-treated mice.

Heroin treatment or abusive drug addiction influences many physiological functions, including the reactions of the immune system. Although suppression of various manifestations of the immune system after heroin (or morphine) administration has been reported, we show here that production of proinflammatory cytokines and nitric oxide (NO) was enhanced and allotransplantation reactions were accelerated significantly in heroin-treated recipients. Mice were treated by a subcutaneous administration of heroin (diacetylmorphine) given in one or repeated daily doses. The ability of spleen cells from treated mice to respond in vitro to alloantigens and to produce IL-2, IL-4, IL-10 and IFN-gamma, and the production of IL-1beta, IL-12 and NO by peritoneal macrophages, were tested. Within 2 h after heroin administration, proliferative responses to alloantigens and the production of IL-1beta, IFN-gamma, IL-12 and NO were enhanced significantly. In contrast, the production of anti-inflammatory cytokines IL-4 and IL-10 was at the same time rather decreased. As a consequence, skin allografts in heroin-treated mice were rejected more promptly than in untreated or vehicle-treated recipients. Similarly, the growth of allogeneic tumours induced by high doses of tumour cells was suppressed significantly in heroin-treated mice. The enhancing effects of heroin on the production of proinflammatory cytokines were antagonized by naltrexone, a specific inhibitor of classic opioid receptors. These results show that heroin treatment augments production of proinflammatory cytokines and accelerates allotransplantation reactions. The observations thus illustrate the complexity of the effects of heroin on the immune system and should be taken into account during medical treatment of opiate addicts and in the use of morphine to decrease pain in various clinical situations.

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.

Alloantigen-induced, T-cell-dependent production of nitric oxide by macrophages infiltrating skin allografts in mice.

The immunological rejection reaction occurring after organ or tissue transplantation is characterized by a strong infiltration of the graft by T cells and macrophages. Since the rejection reaction is highly specific, we tested the role of T cells in the activation of macrophages and in the induction of nitric oxide (NO) production during graft rejection. The rejection of both MHC and non-MHC antigen-disparate skin allografts was associated with a significantly increased production of NO in the graft. The kinetics of NO production after transplantation correlated with the rejection reaction and with the fate of the allograft. A significant reduction in NO production was found in immunologically hyporeactive mice treated with cyclosporine, and no specific production of NO was found in tolerated skin allografts from neonatally tolerant mice. The production of NO was completely suppressed in graft explants from mice with depleted CD4(+) cells, but remained at a normal level in skin allografts from mice treated with anti-CD8 monoclonal antibody. The treatment of recipients of fully allogeneic skin grafts with 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), a specific inhibitor of the inducible NO synthase, resulted in a significant prolongation of graft survival. The results thus show CD4(+) T-cell-dependent, alloantigen-induced production of NO by graft-infiltrating macrophages and the role of NO in the rejection reaction. We suggest that this pathway may represent one of the local effector mechanisms of graft rejection.

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.

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.

Immunoreactivity in kainate model of epilepsy.

Seizure-related changes in function of the peripheral immune system, especially in its cell component are poorly recognized. In the present study, we examined the effect of seizures induced by intraperitoneal injection of kainate to mice and rats on weight of central and secondary immunological organs and metabolic activity of splenocytes (MTT test). In kainate-injected mice the production of cytokines: interleukin 2 (IL-2) and IL-10 was also estimated. Seventy two hours after kainate administration, the mice and rats showed a marked decrease in the thymus weight by 36% and 50%, respectively, whereas the spleen weight tended to decrease in rats only. Splenocytes of kainate-injected mice and rats showed significant increase in metabolic activity. The ability of splenocytes of kainate-injected mice to produce IL-2 and IL-10 was reduced but only the former effect reached statistical significance. The results suggest a decrease in T helper-cell dependent immunoreactivity and enhanced phagocytic activity of macrophages in kainate-treated rodents.

Induction of specific transplantation immunity by oral immunization with allogeneic cells.

Oral administration of antigen has been shown to be effective for both positive and negative modulation of immune responses. In the present study we characterized changes in the reactivity of the immune system after oral immunization with allogeneic spleen cells. Mice were orally immunized for 10 consecutive days with fresh allogeneic spleen cells, and the phenotype, proliferative response, cytotoxic activity and cytokine production profile of recipient spleen cells were assessed 1 or 7 days after the last immunization dose. Although no significant changes in the proportion of CD4+, CD8+ or CD25+ cells were observed in the spleen of orally immunized mice, significant activation of alloreactivity in spleen cells was found. Cells from orally immunized mice exhibited enhanced proliferation and cytotoxic activity after stimulation with specific allogeneic cells in vitro, and produced considerably higher concentrations of interferon-gamma (IFN-gamma) and significantly less interleukin (IL)-4 than did cells from control mice. The production of IL-2 was essentially unchanged and that of IL-10 was only slightly increased. The systemic allosensitization induced by oral immunization was demonstrated in vivo by increased resistance to the growth of allogeneic tumours induced by subcutaneous inoculation of high doses of tumour cells. In addition, orthotopic corneal allografts in orally immunized recipients were rejected more rapidly (in a second-set manner) than in control, untreated recipients. These data show that oral immunization with allogeneic cells modulates individual components of the immune response and that specific transplantation immunity, rather than tolerance, is induced in the treated recipients.

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.

Urocanic acid enhances IL-10 production in activated CD4+ T cells.

The immunosuppressive effects of UV radiation have been well documented. This suppression has been attributed to the action of the cis form of urocanic acid (UCA), a photoproduct of trans-UCA, a natural constituent of the skin. Here, we show that mouse spleen cells preincubated with cis-UCA have a diminished proliferative response to allogeneic cells in MLC and to stimulation with anti-CD3 mAb. Cells preincubated with cis-UCA also had a decreased ability to serve as APC and to stimulate the proliferation of allogeneic lymphocytes in MLC. Simultaneously, the production of IL-2 and IFN-gamma by cells preincubated with cis-UCA was decreased. However, IL-10 gene expression and IL-10 protein secretion by spleen cells stimulated in the presence of cis-UCA were significantly enhanced. The principal cell population displaying the cis-UCA-induced elevated production of IL-10 was CD4+ T cells, which were shown to be a direct target of cis-UCA action. This was also supported by the observation that production of IL-10 by stimulated splenic non-T cells or by macrophages was not altered by cis-UCA. The enhanced production of IL-10 by activated CD4+ T cells may represent a novel pathway of UVB radiation-induced, cis-UCA-mediated immunosuppression. We suggest that the elevated production of IL-10 by activated CD4+ T cells may account for the suppressor T cell phenomena described in UV-irradiated recipients.

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.

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.