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.     

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.     

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.     

Immunomodulation by 8-week voluntary exercise in mice

This study was designed to evaluate the effects of voluntary exercise on macrophage and lymphocyte functions in mice. Male A/He inbred mice aged 19 weeks were divided into two groups: a group given voluntary exercise and a control group (n = 10 in each group). Exercise consisted of spontaneous running in wheels for 8 weeks (3 days week-1). Glucose consumption of peritoneal macrophages in the exercise group during incubation up to 72 h was significantly higher than that in the control group (70 and 13%, respectively). Also, activities of acid phosphatase (APH) (10.75 +/- 0.37 IU), beta-glucuronidase (GLU) (1.55 +/- 0.07 IU) and lactate dehydrogenase (LDH) (43.3 +/- 0.7 IU) in the peritoneal macrophages in the exercise group was significantly increased (P < 0.01). Compared with the control group, the exercise group had a significant increase of about twofold in macrophage production of nitric oxide (NO2-) stimulated by lipopolysaccharide (LPS) (11.1 +/- 0.1 vs. 5.9 +/- 0.1 microM mL-1 in exercise and control groups, respectively; P < 0.01). Stimulation indices both by concanavalin A (Con A) and phytohaemagglutinin were also significantly higher in the exercise group (P < 0.01). A significant increase in the splenocyte production of interleukin-2 (IL-2) stimulated by Con A was noticed in the exercise group (354.1 +/- 28.8 vs. 218.9 +/- 23.5 pg mL-1 in exercise and control groups, respectively; P < 0.01). These findings suggest that voluntary exercise enhances not only macrophage function but also lymphocyte responsiveness in mice. In the studies of voluntary exercise, evaluation of NO2- production, as an indicator of macrophage function, is recommended.     

Mucosal immunity induced by orally administered transgenic plants

Oral immunization is an efficient means to induce protection at the portal entrance for many pathogens. Therefore, the design of efficient edible vaccines through transgenic plants represents a challenging alternative to the traditional injectable ones. We have previously reported the construction of transgenic potato plants expressing the genes coding for the immunogenic proteins of Newcastle Disease Virus (NDV) and their immunogenicity in mice. All mice receiving transgenic plant extracts in incomplete Freund's adjuvant produced specific antibodies. Animals fed with transgenic leaves also showed a specific response against NDV. The aim of the present study was to continue the evaluation of the mucosal immune response. Adult Balb/c mice were fed with potato leaves for a month and on day 36 mucosal samples were collected. ELISAs performed on intestinal washes showed that transformed plants elicited the synthesis of NDV-specific IgG and IgA antibodies. In addition, anti-NDV IgA antibodies were detected in supernatants of cultured small intestine fragments of mice fed with the recombinant immunogens, suggesting the presence of NDV-specific IgA secreting plasma cells in the intestinal tissue. Moreover, we detected specific anti-NDV antibodies in intestinal fluids after oral immunization with F and HN transgenic plants. Also, indirect immunofluorescence on intestinal tissue was performed. The present results suggest that these immunogens, F and HN glycoproteins of NDV, when orally administered, would enhance the number of IgA(+) B cells, and the cytotoxic cellular immune response via CD8(+) T cells, found in the gut lamina propria that is in accordance with our first findings.     

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.     

Pathological evaluation of WR-151327 administered orally in irradiated and non-irradiated male mice.

Studies were made on the radioprotective and toxic effects of orally administered WR-151327 in male CD2F1 mice. The lowest dose of orally administered drug permitting probit analysis of data was 450 mg per kg. The calculated radioprotective dose reduction factors (DRF) at 450 mg per kg and 900 mg per kg of body weight (BW) WR-151327 were 1.2 and 1.3, respectfully. Pathological examination at 8, 30 or 90 days post administration of 100, 450, or 900 mg per kg of the drug demonstrated that the major target organ for orally dosed mice was the testes. There was a decrease in the number of cells in the germinal cell layers of testes from animals administered 450 mg per kg WR-151327 or 10 Gy whole body irradiation after eight days. Moreover, there was a dramatic reduction in the germinal cells in mice seminiferous tubules treated with a combination of 450 mg per kg WR-151327 plus 10 Gy radiation after eight days.     

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.     

Immunomodulation of GvH reaction in mice by Listeria monocytogenes

In the first part of experiments the GvHR activity of the spleen cell suspensions from normal parental donors (Balb/c) or infected with Listeria monocytogenes was compared. GvHR was examined in (Balb/c X AKR) F1 mice. Full suspensions from these donors or depleted of adherent cells or with addition of macrophages were studied. It has been proved that adherent cells population plays a significant role in the GvHR development. The addition of syngeneic macrophages from F1 hybrids results in a greater augmentation of GvH reactivity of parental lymphocytes. However, the addition of macrophages harvested from F1 hybrids immunized with L. monocytogenes 7 days before, brings about a weaker GvH reaction. Spleen cells of parental donors injected with L. monocytogenes 5-6 days earlier induced stronger GvHR as compared with the splenocytes of normal donors. Similarly, F1 hosts treated with L. monocytogenes 3-5 days before injection of normal donor's spleen cells showed increased GvHR, but those infected 7-9 days before injection of spleen cells, developed only very weak GvHR.     

Quantitative elemental analysis on aluminum accumulation by HVTEM-EDX in liver tissues of mice orally administered with aluminum chloride

Quantitative elemental analysis on Al was carried out by high-accelerating voltage transmission electron microscopy (HVTEM) equipped with energy-dispersive X-ray microanalysis (EDX) using an accelerating voltage at 300 kV with high permeability in 1-μm-thick samples obtained from mice administered with aluminum chloride solution for 3, 9, and 17 weeks. By light microscopic observation, no morphological changes were observed in the hepatocytes and macrophages in the liver tissues of mice that were administered with excess Al as compared with the normal control mice. In contrast, by electron microscopic observation, ultrastructural changes were observed in the lysosomes in the hepatocytes as well as the pinocytotic vesicles in the macrophages in the experimental animals. Therefore, the concentrations of Al detected in lysosomes in hepatocytes and pinocytotic vesicles in macrophages of livers of mice administered with Al were measured in relationship to those administration periods. Moreover, transitional changes of hepatocyte lysosome ratios by image analysis and the macrophage counts in the unit area increased in liver tissues of mice administered with Al as compared with normal control mice. From the results, it was demonstrated that hepatocyte lysosome ratio and macrophage count increased in liver tissues of treated mice during those short-term excessive Al administration periods. It was also clarified that the concentrations of Al in both hepatocytes and macrophages increased as observed by HVTEM-EDX. In conclusion, Al accumulated in hepatocytes and macrophages at 3 and 9 weeks administration, while the ultrastructural changes remained in the hepatocytes and macrophages. In contrast, Al concentration did not increase in the liver at 17 weeks administration.     

Diamine oxidase knockout mice are not hypersensitive to orally or subcutaneously administered histamine

Objective

To evaluate the contribution of endogenous diamine oxidase (DAO) in the inactivation of exogenous histamine, to find a mouse strain with increased histamine sensitivity and to test the efficacy of rhDAO in a histamine challenge model.

Methods

Diamine oxidase knockout (KO) mice were challenged with orally and subcutaneously administered histamine in combination with the β-adrenergic blocker propranolol, with the two histamine-N-methyltransferase (HNMT) inhibitors metoprine and tacrine, with folic acid to mimic acute kidney injury and treated with recombinant human DAO. Core body temperature was measured using a subcutaneously implanted microchip and histamine plasma levels were quantified using a homogeneous time resolved fluorescence assay.

Results

Core body temperature and plasma histamine levels were not significantly different between wild type (WT) and DAO KO mice after oral and subcutaneous histamine challenge with and without acute kidney injury or administration of HNMT inhibitors. Treatment with recombinant human DAO reduced the mean area under the curve (AUC) for core body temperature loss by 63% (p?=?0.002) and the clinical score by 88% (p?<?0.001). The AUC of the histamine concentration was reduced by 81%.

Conclusions

Inactivation of exogenous histamine is not driven by enzymatic degradation and kidney filtration. Treatment with recombinant human DAO strongly reduced histamine-induced core body temperature loss, histamine concentrations and prevented the development of severe clinical symptoms.

     

Modulation of arachidonic acid metabolism by orally administered morniflumate in man

Unlike other classic NSAIDs, some fenamates given at therapeutic concentrations, have been shown to inhibit, bothin vitro andin vivo, the 5-lipoxygenase pathway of arachidonic acid cascade as well as the synthesis of cyclooxygenase products. This dual inhibitory property might represent an improvement in anti-inflammatory therapy. The aim of this work was to characterize the effect of morniflumate, admistered at therapeutic dosages to normal human volunteers, on leukotriene B₄ (LTB₄) and thromboxane (TXB₂) synthesis, both in purified PMNs and in whole blood. PMNs, isolated two hours after a single oral administration of morniflumate and at steady-state condition, fully retain their capacity to release LTB₄ and TXB₂. Since intracellular concentrations of the drug were undetectable, in spite of its elevated concentrations in platelet poor plasma, the results obtained using PMNs suggest a drug loss during the cells purification procedure. In whole blood experiments, morniflumate reduced blood LTB₄ synthesis induced by Ca-ionophore A23187 Bx approximately 50%, both after single dose and at steady state; the degree of inhibition showed a pattern similar to the plasma levels of the bioactive metabolite of morniflumate (M1). The inhibition of serum TXB₂ levels was higher than 85%. Hence, morniflumate is capable of reducing arachidonic acid metabolism acting both on cyclooxygenase and 5-lipoxygenase. This characteristic might provide a better approach in anti-inflammatory therapy.     

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.     

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).     

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.     

Inhibition of allergen-induced bronchoconstriction in sensitized guinea pigs by orally administered allergen

Crude mite extract (CME) was orally administered to guinea pigs sensitized to CME. It was shown that such treatment reduces the bronchoconstrictive response upon allergen provocation. Isolated tracheae taken from guinea pigs orally administered CME allergen showed less contraction in response to CME as compared to those obtained from sensitized but not orally treated animals. The oral administration of allergens seemed to attenuate the bronchial hyperresponsiveness of sensitized animals to a non-specific chemical stimulus (histamine). IgE antibodies titrated by 8 days passive cutaneous anaphylaxis, and IgG1 and IgG2 antibodies measured by ELISA were comparable in the sera obtained from animals before and after CME treatment.     

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.     

Suppression of NSAID-induced small intestinal inflammation by orally administered redox nanoparticles

Patients regularly taking non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin (IND) have a risk of small intestinal injuries. In this study, we have developed an oral nanotherapeutics by using a redox nanoparticle (RNP^O), which is prepared by self-assembly of an amphiphilic block copolymer that possesses nitroxide radicals as side chains of hydrophobic segment via ether linkage, to reduce inflammation in mice with IND-induced small intestinal injury. The localization and accumulation of RNP^O in the small intestine were determined using fluorescent-labeled RNP^O and electron spin resonance. After oral administration, the accumulation of RNP^O in both the jejunum and ileum tissues was about 40 times higher than those of low-molecular-weight nitroxide radical compounds, and RNP^O was not absorbed into the bloodstream via the mesentery, thereby avoiding the adverse effects of nitroxide radicals in the entire body. RNP^O remarkably suppressed inflammatory mediators such as myeloperoxidase, superoxide anion, and malondialdehyde in the small intestines of IND-treated mice. Compared to low-molecular-weight nitroxide radical compounds, RNP^O also significantly increased the survival rate of mice treated daily with IND. On the basis of these results, RNP^O is promising as a nanotherapeutics for treatment of inflammation in the small intestine of patients receiving NSAIDs.     

Agranulocytosis during therapy with orally administered cloxacillin

Agranulocytosis developed in a patient with staphylococcal osteomyelitis after 35 days of treatment with orally administered cloxacillin. The patient had fever, prostration, pharyngitis, and profound leukopenia, which subsequently abated upon withdrawal of the drug. Cloxacillin should be included in the growing list of drugs capable of producing leukopenia and agranulocytosis.