Immunomodulation by orally administered beta-glucan in mice

Orally administered SSG, a beta-1,3-glucan obtained from the culture filtrate of the fungus Sclerotinia sclerotiorum IFO 9395, was examined for effects on immune responses in mice. The proliferative responses of spleen cells from SSG-administered mice (40 or 80 mg/kg, daily for 5 or 10 consecutive days) to a T-cell mitogen, concanavalin A (Con A), or a B-cell mitogen, lipopolysaccharide (LPS), were higher than those from normal mice. Oral administration of SSG (80 mg/kg) to mice also enhanced the activities of both natural killer (NK) cells in spleen and the lysosomal enzyme of peritoneal macrophages. Furthermore, significant inhibition of tumor growth was observed in syngeneic tumor systems when SSG was administered directly after tumor implantation. The inhibiting effect required high doses of SSG (over 80 mg/kg). These results demonstrate that SSG can potentiate the immune response of mice following oral administration.     

Isolation of chlamydia in irradiated and non-irradiated McCoy cells.

Specimens from eye and genital tract were cultured in parallel in irradiated and non-irradiated McCoy cells and the frequency of isolation of chlamydia using these culture methods was compared. There was a significant difference between the frequencies of isolation; irradiated McCoy cells produced a greater number of positive results.     

Effect of orally administered beta-glucan on macrophage function in mice

The effect of orally administered SSG, a beta-1,3-glucan obtained from the culture filtrate of the fungus Sclerotinia sclerotiorum IFO 9395, on the function of peritoneal macrophages in CDF1 mice was examined. Oral administration of SSG (20, 40, 80 or 160 mg/kg, daily for 10 consecutive days) enhanced the acid phosphatase activity of peritoneal macrophages. The greatest enhancing effect was observed at 80 mg/kg of SSG. Relatively long periods of administration (more than 10 consecutive days) were needed to induce significant enhancing effects. Phagocytic activity, candidacidal activity, hydrogen peroxide (H2O2) production and interleukin-1 (IL-1) production of peritoneal macrophages were also enhanced after the administration of SSG by the oral route (80 or 160 mg/kg). However, the durations of the activated state after completion of administration differed depending on the activity. Enhanced activity of lysosomal enzyme (acid phosphatase) was also shown in peritoneal macrophages taken from C3H/HeJ mice, which is a nonresponder strain to bacterial lipopolysaccharide (LPS). These results demonstrate that SSG given by the oral route can activate peritoneal macrophages in mice.     

Low-dose orally administered type I interferon reduces splenic B cell numbers in mice.

The beneficial effects of low-dose orally administered type I interferon (LDOA IFN) have been demonstrated in various animal models of disease and in some human clinical trials. The mechanisms by which LDOA IFN therapy has its effects, however, remain to be established. In the present study, groups of mice were administered 10 IU murine IFN-alpha/beta (MuIFN-alpha/beta) orally for 7 days. Spleens were then collected and analyzed. No differences were detected between the spleen weights of treated mice compared with controls, although reductions in total splenic white blood cell (WBC) number ranging from 15.5% to 35% were observed. Further analysis showed this reduction to be largely restricted to the B cell population, with only minor reductions in CD4(+) or CD8(+) populations being detected. Dose-response studies showed the WBC loss from the spleen to be optimal at 1 IU MuIFN-alpha/beta, whereas both higher and lower doses showed less significant effects. Time course studies show these effects had developed after 2 days of treatment. It is hypothesized that this observed WBC movement from the spleen is part of the mechanism of action of LDOA IFN.     

Effect of orally administered monoclonal antibody on persistent Cryptosporidium parvum infection in scid mice.

Scid mice, persistently infected after exposure to 10(7) Cryptosporidium parvum oocysts, were treated daily for 14 to 17 days with 0.4 mg of monoclonal antibody (mAb) 17.41 administered by the oral route. Mice receiving mAb 17.41 shed significantly fewer (P < 0.005) C. parvum oocysts than scid mice receiving isotype control mAb. Intestinal (but not gastric) infectivity scores were also reduced for scid mice treated with mAb 17.41 (P < 0.01).     

Antimetastatic action of orally administered lysozyme in mice bearing Lewis lung carcinoma

The pharmacological activity of orally administered lysozyme, for the control of the growth of solid tumor metastases, was examined in mice bearing Lewis lung carcinoma. Groups of at least 10 tumor-bearing mice, fed daily for three consecutive weeks from subcutaneous tumor implantation with lysozyme, prepared from hen egg-white, had a pronounced reduction of the weight of their metastatic tumor to 25–50 per cent of controls within a wide range of doses (25–200mg/kg/day). The antimetastatic effect was not related to the length of the treatment schedule employed; a short course of 7 days, given on days 1–7 after tumor implantation, proved equally active. The inhibition of the formation of lung metastases, in mice treated with lysozyme prior to tumor inoculation, lasts for at least 2 weeks after discontinuation of treatment, indicating that the antimetastatic activity observed is not associated with cytotoxic activity of the lysozyme, and is probably mediated by the elicitation of host responses. The examination of the therapeutic potential of the antimetastatic action of lysozyme supplied throught the usual diet indicates that this treatment synergizes with the antitumor effects of cisplatin, given to mice after surgical removal of the primary tumor, causing a statistically significant prolongation of the survival time of the animals as compared with chemotherapy alone.     

Divergent effects of bacterial lipopolysaccharide on immunity to orally administered protein and particulate antigens in mice.

We have investigated whether bacterial lipopolysaccharide (LPS) influences immune responses to dietary protein antigens in experimental animals. Simultaneous intravenous administration of LPS to normal mice fed ovalbumin (OVA) prevented the induction of tolerance for serum IgG antibody responses but did not alter the tolerance of systemic delayed-type hypersensitivity (DTH). In addition, exogenous LPS did not enhance the ability of spleen accessory cells to present OVA to primed T cells. LPS-unresponsive C3H/HeJ mice developed full tolerance of both humoral and cell-mediated immunity after feeding a range of doses of OVA that was equal in degree and persistence to that seen in normal, congenic C3H/HeOla mice and also had normal antigen-presenting cell (APC) activity for OVA. In contrast, C3H/HeJ mice were primed by feeding SRBC instead of developing the systemic tolerance found in normal C3H mice. Our results indicate the complexity of mechanisms that may regulate systemic immunity to orally administered antigens of different forms. Nevertheless, LPS does not modulate DTH responses to fed OVA and does not enhance APC activity, and we conclude that bacterial LPS may be unable to influence hypersensitivity to dietary proteins in man.     

Adjuvant effects of orally administered saponins on humoral and cellular immune responses in mice

We have described adjuvant effects of orally administered Quillaja saponins on the immune responses of mice fed inactivated rabies antigen (AG). The in vivo lymphocyte proliferation in mice fed antigen + saponin (AG + SAP) was significantly greater than that in mice fed antigen (AG) alone. Further, the mitogen-induced cell proliferative responses in animals primed with AG + SAP was markedly increased compared with those in the AG group. These changes in clonal expansion were associated with an enhanced helper T cell (Th) and B cell co-operation. The in vivo cell proliferation and in vitro mitogen-induced responses of mice fed AG + SAP correlated with enhanced antibody synthesis. In mice fed saponin alone, there were significant increases in clonal expansion and lymphocyte function. Our present data indicate that the immunocompetence in animals fed AG + SAP was indeed evoked by saponins. Cytotoxic T lymphocyte activity in mice fed SAP or AG + SAP was detected 7 days after booster, in contrast to 21 days in mice fed AG alone. The natural killer cell activity in mice fed SAP alone was greatly enhanced and persisted for an extended period of time.     

Systemic effects of orally administered interferons and interleukin-2.

Orally administered interferons (IFN-alpha, IFN-beta, and IFN-gamma) have been shown to exert a number of systemic effects. Orally administered IFNs exert dose-dependent suppressive effects on the peripheral white blood cell (WBC) count. The suppression of the peripheral WBC count is mediated by a suppression of the function of the bone marrow, as measured in an in vitro bone marrow colony-forming assay. The peripheral WBC and bone marrow suppressive effects of orally administered IFNs are at least as potent as those occurring with parenterally administered IFNs. However, the mechanism by which orally administered IFNs exert these peripheral WBC suppressive and bone marrow suppressive effects differs significantly from that of parenterally administered IFNs: orally administered IFN is not detectable in the serum, the effect of orally administered IFN is not blocked by circulating antibody, the effect of orally administered IFN can be adoptively transferred by injection with peripheral white blood cells from donor mice, and the effect of orally administered IFN develops more slowly than that of parenterally administered interferon. Orally administered IFN-alpha employed alone and in synergistic combination with intraperitoneally administered IFN-gamma can exert an antitumor effect. Finally, orally administered interleukin-2 can exert a suppressive effect on both the peripheral white blood cell count and on the bone marrow. These observations suggest that the oral route may be an effective and novel mechanism for the efficacious administration of IFNs and other lymphokines/cytokines.     

Antigenicity of purified glutaraldehyde-treated cholera toxoid administered orally.

The antigenicity of orally administered glutaraldehyde-treated cholera toxoid was investigated in healthy volunteers. Fourteen volunteers ingested two or three 2-mg doses of toxoid with saline, with the doses spaced at 28-day intervals. Thirteen other volunteers received comparable toxoid doses with NaHCO3 and milk to neutralize gastric acid. Increments in circulating antitoxin levels were used to assay the antigenicity of oral toxoid. Antitoxin was measured by adrenal cell, rabbit skin permeability factor, and passive hemagglutination assays in sera collected on days 0, 28, 35, 56, 63, and 84 after primary immunization. Adrenal cell and rabbit skin assays exhibited identical sensitivity in detecting antitoxin rises in the 27 vaccinees (19/27) and were significantly more sensitive than passive hemagglutination (11/27) (P less than 0.03). Volunteers who ingested toxoid with NaHCO3 and milk had a higher rate of seroconversion (77%) than those who received toxoid with saline (64%); they also had earlier rises in antitoxin titer and consistently higher geometric mean titers on all days tested. These studies demonstrate that purified cholera toxoid is antigenic in humans after oral administration. The possible role of oral toxoid in enhancing the protective effect of killed whole-cell vaccines can now be investigated.     

Evaluation of orally administered PEGylated phenylalanine ammonia lyase in mice for the treatment of Phenylketonuria

Phenylketonuria (PKU), a Mendelian autosomal recessive phenotype (OMIM 261600), is an inborn error of metabolism causing impaired postnatal cognitive development in the absence of treatment. We used the Pah^enu2/enu2 PKU mouse model to study oral enzyme substitution therapy with various chemically modified formulations of phenylalanine ammonia lyase (Av-p.C503S/p.C565S/p.F18A PAL). In vivo studies with the most therapeutically effective formulation (5 kDa PEG-Av-p.C503S/p.C565S/p.F18A PAL) revealed that this conjugate, given orally, yielded statistically significant (p = 0.0029) and therapeutically relevant reduction (~ 40%) in plasma phenylalanine (Phe) levels. Phe reduction occurred in a dose- and loading-dependent manner; sustained clinically and statistically significant reduction of plasma Phe levels was observed with treatment ranging between 0.3 IU and 9 IU and with more frequent and smaller dosings. Oral PAL therapy could potentially serve as an adjunct therapy, perhaps with dietary treatment, and will work independently of phenylalanine hydroxylase (PAH), correcting such forms of hyperphenylalaninemias regardless of the PAH mutations carried by the patient.     

Mucosal and systemic immune responses in BALB/c mice to Bacteroides gingivalis fimbriae administered orally

A 41,000-molecular-weight fimbrial protein was isolated from freshly cultivated whole cells of Bacteroides gingivalis 381 and purified chromatographically. Salivary and serum antibody responses to the fimbriae, which had been orally administered in the presence of an acyl derivative of muramylpeptides, i.e., either N2-[(N-acetylmuramyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-L-lysine [MDP-Lys(L18)] or sodium beta-N-acetyl-glucosaminyl-(1----4)-N-acetylmuramyl-L-alanyl-D-isoglu tam inyl- (L)-stearoyl-(D)-meso-2,6-diaminopimelic acid-(D)-amine-D-alanine (GM-53), or in the absence of adjuvant, were examined in BALB/c mice when administered by gastric intubation on days 0 and 1 as primary immunizations and on days 27 and 28 as booster immunizations. Gastric intubation of the fimbriae with an adjuvant significantly enhanced the production of anti-fimbria immunoglobulin A (IgA) in saliva. Subcutaneous injection of fimbriae along with an adjuvant also raised anti-fimbria IgA levels, as well as IgG levels, in saliva. Both immunization procedures enhanced the levels of anti-fimbria IgG, IgA, and IgM in serum, and the major class of fimbria-specific antibody was IgG, followed by IgA and IgM. However, subcutaneous injection was more effective than gastric intubation to enhance the production of serum antibody in mice. The subclasses of IgG antibody specific for fimbriae in serum were mainly IgG1, followed by IgG2a, IgG2b, and IgG3. These results demonstrated that the combined use of B. gingivalis fimbrial antigen and either GM-53 or MDP-Lys(L18) resulted in a sharply increased IgA antibody response in saliva and a predominantly stimulated IgG antibody response in serum, respectively. Both antibodies were found to be specific for the fimbriae used for immunization.     

Effects of immune colostrum and orally administered antisporozoite monoclonal antibodies on the outcome of Cryptosporidium parvum infections in neonatal mice.

A neonatal BALB/c mouse model of cryptosporidiosis was used to examine the potential passive transfer of immunity via immune colostrum and oral treatment with anticryptosporidial monoclonal antibodies. Neonates suckled by dams that recovered from Cryptosporidium parvum infections were equally susceptible to infection as their control counterparts suckled by naive dams. Parasite loads among the control and immune colostrum-fed mice were indistinguishable. Neonates receiving orally administered antisporozoite monoclonal antibodies were equally susceptible to infections compared with the control and immune colostrum-fed mice. Parasite loads among the mice receiving daily oral treatment with monoclonal antibody mixtures exhibited significantly lower parasite loads compared with the control mice (P less than 0.05).     

Pharmacokinetics and tissue penetration of orally administered pefloxacin

The pharmacokinetics of the quinolone pefloxacin were determined following a 400mg oral dose given to each of six male volunteers. Concentrations were determined in serum and urine by high-performance liquid chromatography, and in cantharidin-induced inflammatory fluid by a microbiological assay. The mean peak serum level of 6.6 g/ml was attained rapidly 0.8 h after administration. The mean serum elimination half-life was 11.6 h. Inflammatory fluid was penetrated quickly with a mean peak level of 3.9 g/ml occurring at 2.4 h. Pefloxacin was excreted in the urine as the parent compound and its two metabolites, norfloxacin and pefloxacin N-oxide (24h urinary recovery being 8.0%, 12.0% and 13.1% respectively of the dose). This study suggests that a twice or possibly once daily dosage may be sufficient to treat systemic infections caused by susceptible pathogens. Once daily dosing should be sufficient for urinary tract infections.     

Curative activity of spiramycin adipate by parenteral route in experimental septicemia in mice. Comparison with orally administered basic spiramycin

Experimental septicemia was induced in mice by intraperitoneal injection of 10 to 100 lethal doses of Staphylococcus aureus and Streptococcus pneumoniae. Animals were treated by a mixture of adipic acid and spiramycin (subcutaneous route) or by spiramycin base (oral route), 1 and 6 hours after infection. To determine the effective dose 50% that achieves survival of half the mice after 7 days, each drug was used in 6 dosages (mg/kg) and each dosage was given to 12 mice. In 21 independent experiments, ED50S of spiramycin adipate by the subcutaneous route were found to be 5 to 50 times lower than those of spiramycin base per os. These results are consistent with the high serum peak concentrations of spiramycin adipate observed following subcutaneous administration.     

Enhancement of murine alveolar macrophage functions by orally administered beta-glucan.

The effect of orally administered SSG, a beta-1,3-glucan obtained from the culture filtrate of the fungus Sclerotinia sclerotiorum IFO 9395, on alveolar macrophage (AM) functions of CDF1 mice was examined. SSG administered orally (20, 40, 80 or 160 mg/kg) for 10 consecutive days enhanced the lysosomal enzyme activity of AM. The greatest enhancing effect was observed at 80 mg/kg of SSG. Multiple oral administrations of SSG (10 consecutive days) were needed to induce significant enhancing effects. Phagocytic activity and interleukin-1 (IL-1) production of AM were also augmented by oral administration of SSG, and the kinetics of the activated state differed depending on the kind of activity. However, H2O2 production of AM was not affected by SSG. Orally administered SSG also (40 or 80 mg/kg, 10 consecutive days) increased the number of AM and the greatest increment was observed 14 days after the first administration. On the other hand, the supernatant of Peyer's patch (PP) cells from mice administered SSG (80 mg/kg) orally stimulated the lysosomal enzyme activity of AM in vitro, and enhanced colony stimulating activity (CSA) was detected from this supernatant. These results demonstrate that SSG given by the oral route can activate murine AM both qualitatively and quantitatively, and it would mediated, at least in part, by the activation of PP cells in the intestine.     

Biologic effects of orally administered deuterium oxide on rat liver.

Rats were made to drink D2O mixed water (30: 70) for 6 weeks in order to study the biological effects of orally administered D2O on the liver. Heavy water administration results in gradual decrease in the body weight whereas the liver showed marginal increase in weight throughout the experimental period. Phosphatases and dehydrogenases were analyzed biochemically. Acid phosphatase, glucose-6-phosphatase and adenosine triphosphatase registered fall in contrast to alkaline phosphatase, SDH and LDH, all of which showed a definite increase. Lipids, nucleic acids and proteins, estimated biochemically, gradually decreased throughout the experimental period in response to D2O feeding.     

T-cell lymphokine response to orally administered proteins during priming and unresponsiveness.

Feeding antigens induces an immunological unresponsiveness termed oral tolerance but under some conditions, for example following the administration of cyclophosphamide (CY), immunity can be induced. These observations have usually been made by studying antibody production and delayed hypersensitivity with little attention given to other measurements of cellular activation. We have therefore examined the lymphokines produced by T cells obtained after the induction of oral tolerance or intragastric priming. Cells isolated from the spleen and Peyer's patches (PP) of tolerized mice could secrete high levels of interferon-gamma (IFN-gamma) and moderate levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) in response to antigen while interleukin-2 (IL-2), IL-3 and IL-4 could not be detected. Mesenteric lymph node (MLN) cells of tolerized mice did not respond to antigen unless spleen adherent cells were added to the cultures where IFN-gamma and GM-CSF were produced. Intragastric priming was achieved by feeding antigen to CY-treated mice. T cells from the spleen, MLN and PP of these mice could produce GM-CSF, IFN-gamma, some IL-3 but little or no IL-2 and IL-4. The ability of MLN cells to proliferate with antigen in vitro was low and corresponded to low IL-2 production. Thus T cells from fed mice secrete a defined pattern of lymphokines which differs in tolerizing and priming regimes.     

Stability of orally administered immunoglobulin in the gastrointestinal tract

Oral administration of immunoglobulin in the colostrum or egg yolk has been considered an effective tool for preventing enterobacterial infection via passive immunization. During this process, the transmission and residence of the active immunoglobulin are the most important conditions for successful protection. We investigated the stability of encapsulated colostrum and egg yolk immunoglobulin for the effective transmission of immunoglobulin in the gastrointestinal (GI) tract. First, we measured GI transit time. Contrast media passed through and reached the stomach within 10min, the small intestine within 3.5h, and the cecum within 5h. Both the encapsulated colostrum containing anti-hepatitis A virus (HAV) antibody (IgG) and egg yolk with anti-rotavirus antibody (IgY) showed lower antibody activity than the non-encapsulated colostrum did in the stomach after administration; however, significantly higher antibody activities were observed in the encapsulated groups than in the non-encapsulated groups in the small intestine 3.5h after the administration. In the large intestine, the antibody activities of the encapsulated groups were maintained or slightly increased in a time-dependent manner; however, the titers of each non-capsulated control were as low as the negative controls. Therefore, this encapsulation is considered a useful tool for the delivery of active antibody through the GI tract.     

Differential effects of orally versus parenterally administered qinghaosu derivative artemether in dogs.

Artemether (AM) is an antimalarial drug derived from artemisinin (Qinghaosu), an extract of the herb Artemisia annua L., sweet wormwood. Its antiparasitic effect is that of a schizontocide and is explained by rapid uptake by parasitized erythrocytes and interaction with a component of hemoglobin degradation resulting in formation of free radicals. It has been shown to exhibit a high clinical cure rate. Previous animal safety studies with Qinghaosu derivatives revealed dose-dependent neurotoxicity with movement disturbances and neuropathic changes in the hindbrain of intramuscularly treated dogs, rats and monkeys. Such effects have not been seen in man. The objective of our present studies was to compare the effects of high levels of AM administered to dogs p.o. versus i.m. In a pilot study 20 mg/kg/day of AM was given i.m. to groups of 3 male Beagle dogs for 5 and 30 days, respectively. Clinical signs of neurotoxicity were noted in some individual dogs from test day 23 on. One dog had to be sacrificed pre-term. Hematologic findings indicated a hypochromic, microcytic anemia. Microscopic examination demonstrated neuropathic changes only at 30 days, but not at 5 days. The animals had neuronal and secondary axonal damage, most prominent in the cerebellar roof, pontine and vestibular nuclei, and in the raphe/paralemniscal region. The affected neurons showed loss of Nissl substance, cytoplasmic eosinophilia, shrinkage of the nucleus and in advanced stages scavenging by microglia. In a subsequent experiment, AM was administered to groups of 4 male and 4 female dogs, respectively, at 8 daily doses of 0, 20, 40 and 80 mg/kg i.m., or 0, 50, 150 and 600 mg/kg p.o. Neurologic signs were seen at high i.m. doses only. In most animals they were inconspicuous and consisted of reduced activity with convulsions seen in single dogs shortly before death. Neuronal damage occurred in all animals at 40 and 80 mg/kg following i.m. treatment. At 20 mg/kg minimal effects occurred in 5/8 dogs only, indicating that this level was close to tolerated exposure. No comparable lesions were observed after oral administration. Both i.m. and p.o. exposure at high dose levels was associated with a prolongation of mean QT interval of ECG, suggesting slowing of repolarization of the myocardium. Individual data indicated that in 1 of 4 females at 80 mg/kg i.m. this prolongation was above the 25% level considered as threshold for concern. After intramuscular administration pharmacokinetics indicated peak plasma levels of AM at 2 to 4 hours post-dose, slow elimination and a tendency to accumulate after repeated administration. Only low levels of the major metabolite, dihydroartemisinin (DHA), were found. AM levels in the cerebrospinal fluid (CSF) were < 10% of plasma levels. After oral administration AM concentrations were considerably lower than after i.m. administration. The concentration of DHA was high on day 1 but almost nil on day 7 indicating its fast inactivation in dogs. Two hours after the 8th oral administration neither AM nor DHA was detected in CSF which may explain the absence of neurotoxicity in dogs after oral administration of AM.