The fungal biopesticide Metarhizium anisopliae has an adjuvant effect on the allergic response to ovalbumin in mice

The parasitic fungus, Metarhizium anisopliae, is non-pathogenic to humans and licensed for indoor control of cockroach infestation. An important reason for the elimination of this vermin is that sensitisation to cockroaches is associated with asthma. Previously M. anisopliae has been shown to cause allergic- and asthma-like responses in mice and in the present study we have examined the adjuvant activity of M. anisopliae on the allergic response to the model allergen ovalbumin (OVA) in a mouse model. Levels of OVA-specific IgE, IgG1 and IgG2a in serum were measured and the weight and cell number of the excised popliteal lymph node were determined. Mice primed with mycelium+OVA and boosted with OVA had increased anti-OVA IgE and IgG1 levels compared with mice primed with OVA alone or mycelium. Priming with M. anisopliae (as mycelium or MACA) increased weight or cell number of the excised PLNs. These results suggest that M. anisopliae has the ability to increase an allergic response to an allergen and consequently, may worsen allergy in susceptible individuals.     

Update on antifungals targeted to the cell wall: focus on beta-1,3-glucan synthase inhibitors

Currently available antifungal drugs for serious infections are either fungistatic and vulnerable to resistance (azoles) or fungicidal but toxic to the host (polyenes). Cell wall-acting antifungals are inherently selective and fungicidal, features that make them particularly attractive for clinical development. Three classes of such compounds, targeted respectively to chitin synthase (nikkomycins), beta-1,3-glucan synthase (echinocandins) and mannoproteins (pradimicins/benanomicins), have entered clinical development. While nikkomycins and pradimicins/benanomicins are no longer in development, echinocandins have emerged as potentially clinically useful and three compounds, caspofungin (MK-991, L-743,872), micafungin (FK-463) and anidulafungin (LY-303366) are in late clinical development (Phase II and III).     

The effect of soluble beta-1,3-glucan and lipopolysaccharide on cytokine production and coagulation activation in whole blood

Soluble beta-1,3-glucan has been demonstrated to protect against infection and shock in rats and mice, and clinical studies suggest that administration of soluble glucans to trauma/surgical patients decreases septic complications and improves survival. However, little is known about the precise mechanisms by which glucans influence the state of activation of blood cells, which are responsible for the fulminant cytokine production and the activation of the coagulation system observed in serious gram-negative infection. We studied therefore the effect of an underivatized, soluble yeast beta-1,3-glucan and lipopolysaccharide (LPS), either alone or in combination, on tumor necrosis factor-alpha (TNFalpha), interleukin-6 (IL-6), IL-8 and IL-10 secretion and monocyte tissue factor (TF) expression in human whole blood. As expected, LPS induced the secretion of substantial amounts of all measured parameters, whereas only minor amounts of TNFalpha, IL-6, and IL-10 were induced by beta-glucan itself. However, beta-glucan itself induced the production of significant amounts of IL-8 and TF. Soluble beta-1,3-glucan had a strong synergistic effect on the LPS-induced secretion of IL-8, IL-10, and on monocyte TF activity, but not on TNFalpha and 1L-6 production. On the other hand, soluble beta-glucan strongly primed LPS stimulation of all parameters, including TNFalpha and IL-6. beta-Glucan also induced detectable neutrophil degranulation within 15 min, whereas a response to LPS was first detected after 90 min. In conclusion, soluble beta-1,3-glucan upregulated leukocyte activity, both on its own and in concert with LPS.     

Immunological adjuvant effect of Boswellia serrata (BOS 2000) on specific antibody and cellular response to ovalbumin in mice

In this study, the biopolymeric fraction BOS 2000 from Boswellia serrata was evaluated for its potential ability as adjuvants on the immune responses to ovalbumin (OVA) in mice. Balb/c mice were immunized subcutaneously with OVA 100 μg alone or with OVA 100 μg dissolved in saline containing alum (200 μg) or BOS 2000 (10, 20, 40 and 80 μg) on Days 1 and 15. Two weeks later, OVA specific antibodies in serum; concanavalin A (Con A), OVA stimulated splenocyte proliferation, CD4/CD8/CD80/CD86 analysis in spleen cells and its estimation of cytokines (IL-2 and IFN gamma) from cell culture supernatant were measured. OVA specific IgG, IgG1 and IgG2a antibody levels in serum were significantly enhanced by BOS 2000 (80 μg) compared with OVA control group. Moreover, the adjuvant effect of BOS 2000 (80 μg) on the OVA-specific IgG, IgG1, and IgG2a antibody responses to OVA in mice were more significant than those of alum. BOS 2000 significantly enhanced the Con A and OVA induced splenocyte proliferation in the OVA immunized mice especially at a dose of 80 μg (p<0.001). However, no significant differences were observed among the OVA group and OVA/alum group. At a dose of 80 μg (p<0.001), there was a significant increase in the CD4/CD8 and CD80/CD86 analysis in spleen cells and cytokine (IL-2 and IFN-gamma) profile in the spleen cell culture supernatant was observed. In conclusion, BOS 2000 seems to be a promising balanced Th1 and Th2 directing immunological adjuvants which can enhance the immunogenicity of vaccine.     

Di-(2-ethylhexyl) phthalate is without adjuvant effect in mice on ovalbumin

It has been suggested that one possible contributor to the increasing prevalence of IgE-mediated allergic diseases in Europe and the US is exposure to chemicals that may act as adjuvants. It has been reported previously that certain commonly used phthalate plasticizers, such as di-(2-ethylhexyl) phthalate (DEHP), are able to modify immune responses induced in mice by the common hens' egg allergen ovalbumin (OVA). However, the significance of these observations for human health is unclear, not least because the relevant studies have been conducted exclusively using subcutaneous administration of phthalates. We have therefore investigated the ability of DEHP when applied topically to affect anti-OVA antibody responses induced by subcutaneous exposure to OVA in BALB/c strain mice. Doses of DEHP (50mg) were used that resulted in a marked (approximately 30%) increase in liver weight. Dose-responses were conducted in order to identify doses of OVA that were sub-optimal for both anti-OVA IgG1 and IgE antibody responses: 1microg and 0.05microg, respectively. Under these conditions of exposure, topical administration of DEHP was without impact on antibody responses, regardless of whether DEHP was applied local or distant to the site of OVA immunization. Topical application of concentrations of DEHP that provoked marked systemic effects was without effect on the induction of immune responses.     

Haemolytic activities and adjuvant effect of Anemone raddeana saponins (ARS) on the immune responses to ovalbumin in mice

In this study, saponins (ARS) extracted from the rhizoma of Anemone raddeana were evaluated for their haemolytic activities and its potential ability as adjuvant on the cellular and humoral immune responses of ICR mice against ovalbumin. The haemolytic activity of ARS was determined using 0.5% rabbit red blood cell. ARS showed a slight haemolytic effect, with its haemolytic percents being 16.50 and 3.56% at the concentrations of 500 and 250 microg/ml, respectively. ICR mice were immunized subcutaneously with OVA 100 microg alone or with OVA 100 mug dissolved in saline containing Alum (200 microg), QuilA (10 and 20 microg) or ARS (50, 100 or 200 microg) on Days 1 and 15. Two weeks later (Day 28), concanavalin A (Con A)-, lipopolysaccharide (LPS)- and OVA-stimulated splenocyte proliferation and OVA-specific antibodies in serum were measured. ARS significantly enhanced the Con A-, LPS-, and OVA-induced splenocyte proliferation in the OVA-immunized mice especially at a dose of 100 microg (P<0.01 or P<0.05). The OVA-specific IgG, IgG1 and IgG2b antibody levels in serum were also significantly enhanced by ARS compared with OVA control group (P<0.01 or P<0.05). Moreover, no significant differences (P>0.05) were observed between enhancing effect of ARS and QuilA on the OVA-specific IgG2b antibody responses to OVA in mice. The results suggest that ARS showed a slight haemolytic effect and enhanced significantly a specific antibody and cellular response against OVA in mice.     

Haemolytic activity and adjuvant effect of soyasaponins and some of their derivatives on the immune responses to ovalbumin in mice

Immunological adjuvants are agents that enhance specific immune responses to vaccines. At present more studies are needed to identify a suitable adjuvant for a particular vaccine with maximum safety and efficacy. In this study, six soyasaponins (Aa, Ab, Af, Ba, Bb, and Bb′) and three soyasaponin Ab-derivatives (AbDs) were selected to evaluate their toxicities and adjuvant activities. The haemolytic activity assay was performed to evaluate the toxicity of the tested soyasaponins and AbDs. Immunoadjuvant activity was investigated in vivo and in vitro using a splenocyte proliferation assay and sera indirect ELISA. Our results demonstrated that soyasaponins and AbDs showed a slight haemolytic effect to 0.5% red blood cell. Except for the Af and Ba groups, other soyasaponins and AbDs groups stimulated by concanavalin A (ConA) and lipopolysaccharide (LPS) showed a greater proliferative response at appropriate doses (0.01–10 μg/ml) compared with the control and Alum groups. Anti-OVA IgG, IgG1, IgG2a, and IgG2b were significantly enhanced by the soyasaponins (Ab, Ba, Bb, and Bb′), QS and AbDs groups (p < 0.05 or p < 0.01). In addition, three AbDs indicated a tendency of immunoadjuvant potential improvement after structural modification. Moreover, Ab-D2 showed adjuvant activity at the lowest injection dose among the three AbDs. In conclusion, these results suggested that soyasaponins together with their derivatives may represent viable candidates for effective vaccine adjuvants due to their higher immune response and lower or non-haemolytic effects.     

The echinocandins: antifungals targeted to the fungal cell wall

The greater incidence of AIDS and use of myelosuppressive therapy has increased the demand for effective antifungal therapy. Currently availiable therapy has limitations associated with toxicity and resistance potential. The ideal agent is selectively targeted to the fungal pathogen. The echinocandins, a family of cyclic lipopeptides, interfere with cell wall formation by noncompetitive inhibition of β-glucan synthase. Since mammalian cells lack a cell wall, this mode of action is likely responsible for the potent fungicidal activity and non-toxic properties of the echinocandin lipopeptides. New analogues of the echinocandins were prepared at the Lilly Laboratories by removal of the native fatty acid acyl group (“side chain”) from the cyclic peptide followed by its replacement with a new synthetic side chain. This scheme gave several analogues with improved therapeutic potential. A new analogue, LY303366, having a side chain containing a substituted terphenyl group, had improved potency against Candida albicans, Pneumocystis carinii, Histoplasma capsulatum and Aspergillus species, had oral bioavailability as well as an extended elimination half life. Newer developments in the discovery and modification of the pneumocandins by the Merck Laboratories are reported. New semi-synthetic water-soluble pneumocandin analogues L-705,589, L-731-373 and L-733,560 also have activity against disseminated aspergillosis.     

Airway inflammation and adjuvant effect after repeated airborne exposures to di-(2-ethylhexyl)phthalate and ovalbumin in BALB/c mice

BACKGROUND: Epidemiological studies have suggested an association between exposure to phthalate plasticizers, including di-(2-ethylhexyl)phthalate (DEHP), and increased prevalence of asthma, rhinitis or wheezing. Furthermore, studies in mice have demonstrated an adjuvant effect from DEHP after parenteral administration with the model allergen ovalbumin (OVA). OBJECTIVE: Exposures to DEHP were investigated for adjuvant effects and airway inflammation in a mouse inhalation model. METHODS: BALB/cJ mice were exposed to aerosols of 0.022-13 mg/m(3) DEHP and 0.14 mg/m(3) OVA 5 days/week for 2 weeks and thereafter weekly for 12 weeks. Mice exposed to OVA alone or OVA+Al(OH)(3) served as control groups. Finally, all groups were exposed to a nebulized 1% OVA solution on three consecutive days. Serum, bronchoalveolar lavage (BAL) fluid, and draining lymph nodes were collected 24h later. RESULTS: In the OVA+Al(OH)(3) group, significantly increased levels of OVA-specific IgE and IgG1 in serum as well as of eosinophils in BAL fluid were observed. DEHP affected OVA-specific IgG1 production in a concentration-dependent manner, whereas little effect was seen on IgE and IgG2a. Dose-dependent increases in inflammatory cells were observed in BAL fluids, leading to significantly higher lymphocyte, neutrophil and eosinophil numbers in the OVA+13 mg/m(3) DEHP group. Ex vivo cytokine secretion by cultures of draining lymph nodes suggested that DEHP has a mixed Th1/Th2 cytokine profile. CONCLUSION: Airborne DEHP is able to increase serum IgG1 and lung inflammatory cell levels, but only at very high concentrations. Realistic DEHP levels do not have an adjuvant effect or induce allergic lung inflammation in the present mouse model.     

The effect of 5,7-dihydroxytryptamine treatment on the response to ethanol in mice

In order to assess the role of the serotonergic system in the development of tolerance to ethanol in the mouse, serotonin neurons in the CNS were lesioned with an intracerebroventricular injection of the neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT). Mice injected with 5,7-DHT responded to an acute dose of ethanol with a longer sleep time and greater fall in body temperature than CSF-treated mice. The increased response to acute administration of ethanol was accompanied by higher circulating levels of ethanol in mice pretreated with 5,7-DHT. When mice were fed an ethanol-containing liquid diet for five days, a higher mortality rate was observed in the 5,7-DHT group compared to the CSF pretreated group of mice. When the groups of mice were tested for tolerance 24 hours after withdrawal, the 5,7-DHT group was less tolerant than the CSF group. Therefore, damage to the serotonin neurons results in altered ethanol disposition, altered initial sensitivity to ethanol, and an inhibition in the development of tolerance in the mouse.     

Inhibitory effect of azelastine on allergic itch-associated response in mice sensitized with mosquito salivary glands extract

We examined whether azelastine would inhibit itch-associated responses of mice to mosquito allergy. Repeated injections of mosquito salivary gland extract increased scratching and sensory nerve activity. Azelastine inhibited the increased scratching and nerve activity, while terfenadine was without effects. Dexamethasone did not affect the increased scratching. Azelastine suppressed high K(+)-induced increase in intracellular free Ca(2+) in primary cultures of mouse sensory neurons. Direct inhibition by azelastine of sensory neurons may be at least involved in the anti-pruritic effect of azelastine. Histamine, substance P, and leukotriene B(4) may not play a key role in the itching of mosquito allergy.     

The effect of lead acetate on the immune response in mice

Female BDF₁ mice were exposed to lead in the drinking water at concentrations ranging from 0 to 1000 ppm lead for 3 weeks. Immunological studies demonstrated that lead suppressed macrophage-dependent immune responses. The functional activity of the macrophage was evaluated by the Mishell-Dutton, in vitro, antibody production technique. Lead suppressed the immune response when the macrophage-dependent antigens, sheep red blood cells or dinitrophenyl-Ficoll, were utilized, but the response to the macrophage-independent antigen, Escherichia coli lipopolysaccharide, was not suppressed. The macrophage substitute, 2-mercaptoethanol, caused restoration of the lead-suppressed immune response. The immune responses seen in the four combinations of adherent/nonadherent and lead-exposed/non-lead-exposed cell cultures, were suppressed only in cultures containing lead-exposed adherent cells. The immunosuppressive effects of lead were produced at relatively low lead exposures as indicated by the blood lead concentrations. Weight gains and water consumption were not affected by these lead exposures. The low level lead exposure effects manifested by immunosuppression indicate that immune dysfunction is a sensitive indicator of lead exposure.     

The effect of emetine on the immune response of mice

Emetine (33 mg/kg body weight) administered intraperitoneally blocked the immune response of mice to 10⁹ sheep red blood cells (SRBC). The inhibition was almost complete when the drug was administered simultaneously or 24 hr after immunization, while partial inhibition was caused by treatment at 48 and 72 hr. Incorporation of ¹⁴C-leucine and ³H-thymidine by spleen cells isolatedd 4 hr after emetine injection of the mice was strongly decreased. Incorporation was approaching the control level in cells isolated 72–96 hr after emetine administration. However, the incorporation of labeled precursors was less than after SRBC treatment only, even after 72–96 hr.Emetine apparently blocked the development of immune response at an early stage and, in contrast to macromole synthesis, the inhibition of the antibody response was irreversible.     

Inhibitory effects of water-soluble low-molecular-weight beta-(1,3-1,6) d-glucan purified from Aureobasidium pullulans GM-NH-1A1 strain on food allergic reactions in mice

There have been a number of reports showing that the crude beta-glucan fraction prepared from various kinds of Basidiomycetes mushrooms acts as anti-cancer and anti-allergic reagent through stimulation of IFN-gamma production. It has been reported, however, that the exposure of the airway to beta-(1,3) d-glucan, contained in house dust, indoor moulds and some bacteria, potentiates the airway allergic response. It seems likely that the discrepant effects on immune function may be related to such factors as differences of the administration route, average molecular weight and water solubility. We isolated a new low-molecular-weight (about 100 kDa) beta-glucan from Aureobasidium pullulans 1A1 strain of black yeast, and found that it had low viscosity and was water-soluble. In this study, we examined the effects of water-soluble low-molecular-weight beta-(1-->3) and 50-80% branched beta-(1-->6) glucan (LMW-beta-Glucan) isolated from A. pullulans on the ova-albumin (OVA)-treated allergic reaction in mice. Feeding standard laboratory diets containing 0.5 and 1% LMW-beta-Glucan significantly inhibited the OVA-specific IgE elevation compared to that in OVA-sensitized mice fed standard laboratory diet alone (control). Furthermore, feeding standard laboratory diets containing 0.5 and 1% LMW-beta-Glucan inhibited the reduction of IL-12 and IFN-gamma production from splenocytes and the reduction of CD8- and IFN-gamma-positive cell number in the small intestine of the OVA-sensitized mice. These findings suggest that anti-food allergic action of LMW-beta-Glucan may be due to the inducing IFN-gamma production in the small intestine and splenocytes.     

Host mediated anti-tumor effect of Nocardia rubra cell wall skeleton on syngeneic tumor in mice

The mode of action of Nocardia rubra cell wall skeleton (N-CWS) on Meth A fibrosarcoma (Meth A) was studied in BALB/c mice. N-CWS suppressed or regressed the intradermal growth of syngeneic Meth A cells in normal BALB/c and athymic BALB/c mice. The intradermally and subcutaneously infiltrated cells harvested from injection sites of N-CWS in normal mice showed in vitro cytotoxic activity against Meth A cells. Pretreatment of normal BALB/c mice with immunosuppressing agents such as hydrocortisone, carrageenan, or silica particles significantly reduced the anti-tumor effect of N-CWS. The growth of Meth A cells, rechallenged into BALB/c mice in which Meth A cells had once been suppressed or regressed by N-CWS treatment, was also inhibited, but not in similarly treated athymic nude mice. This resistant mechanism was shown to be dependent out cellular components but not on humoral components by the Winn Assay. The present results suggest that N-CWS exerts its anti-tumor activity by mediation of the immune system of the host and that the main effector cells in the early stage of tumor rejection are macrophages; T cells may also be involved in the later stage.     

The effect of the Melissa officinalis extract on immune response in mice

The effect of an extract from Melissa officinalis on immune response in mice was analysed using the cytotoxicity test in three dilutions (undiluted extract and extract diluted 10 and 100 times) and hemagglutination and rosette tests with various routes of administration (oral and subcutaneous). The immunostimulating activity of the extract was compared with that of a synthetic compound--levamisole, which influence on the immune system is well known. The present results confirm the effect of water extracts from leaves of Melissa on the immune system, in both humoral and cellular response.     

Protective effect of Nocardia rubra cell wall skeleton on experimental infection in normal and immunosuppressed mice

The protective effect of Nocardia rubra cell wall skeleton (N-CWS) on experimental infections was investigated in normal and immunosuppressed mice. Pretreatment with N-CWS provided protection against acute systemic infections due to Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens and Listeria monocytogenes in normal mice. N-CWS administered before or after challenge also showed protective activity against Herpes simplex virus infection in normal mice. N-CWS was the most effective against extracellular parasitic Pseudomonas aeruginosa infection when administered 1 day before challenge, but displayed the most potent protective activity against infection with Listeria monocytogenes, a facultative intracellular parasite, when administered 7 to 14 days before challenge. In mice with subcutaneous Pseudomonas aeruginosa infection, N-CWS suppressed abscess formation and decreased the viable cell count in the resultant abscess foci when administered either intraperitoneally or subcutaneously to the infected site. In addition, treatment with N-CWS markedly restored host defense ability against pseudomonal infection in mice immunosuppressed with cyclophosphamide, hydrocortisone and X-ray irradiation.     

Effects of kampo-hozais on the induction of oral tolerance to ovalbumin in mice.

We studied here the effect of 3 kinds of kampo-hozais (Shofu-san, Ji-zuso-ippo and Unsei-in) on the induction of oral tolerance to ovalbumin (OVA) in C3H/HeN mice by measuring serum levels of OVA-specific antibodies. Oral tolerance was induced by a single administration of OVA (2 or 20 mg, p.o.) 7 d before the immunization. Among these kampo-hozais, mice administered Ji-zuso-ippo for 7 d before feeding of 2 mg OVA had significantly lower levels of anti-OVA IgG and IgG1, and further, Ji-zuso-ippo tended to reduce anti-OVA Igs in 20 mg OVA-fed mice, whereas Ji-zuso-ippo did not alter anti-OVA Igs in non-OVA-fed mice. Unsei-in also slightly augmented the suppression of anti-OVA IgG and IgG1; however, this lowering action of Unsi-in should be due to its immunosuppressive effect. Shofu-san did not alter these anti-OVA Ig levels in serum. These results suggested that Ji-zuso-ippo could augment the induction of oral tolerance.