DDA as an immunological adjuvant.

As compared to other adjuvants, DDA is a moderate or strong adjuvant for humoral responses and a strong adjuvant for CMI, especially DTH responses, against different types of antigens and in both laboratory animals and larger animals. DDA can collaborate with other immunomodulating compounds resulting in further enhanced responses. Mechanisms include interactions with both antigen and components of the host immune system and possibly, multiple beneficial effects contribute to the relatively strong adjuvanticity of DDA. Toxicity of DDA is not known but severe detrimental side effects were not seen. This adjuvant can be applied in experimental vaccines and in commercial vaccines for veterinary purposes, especially if cell-mediated immunity is considered to be important. In immunology, DDA can be of use to study T helper cells responsible for DTH responses (T helper cells type 1) and to characterize T helper cell epitopes on antigens (Snijder et al., 1992).     

Interferon-gamma as an adjuvant in immunocompromised mice.

We have compared interferon-gamma (IFN-gamma) with saponin and interleukin-1 (IL-1) as adjuvants for a blood-stage malaria vaccine in mice with various immunological abnormalities. IFN-gamma was particularly effective in Biozzi low antibody responder mice, mice selectively bred to produce antibody of low affinity, and mice depleted of CD4+ T cells. IFN-gamma and other cytokines may be safe adjuvants for use in human immunodeficiency states.     

On the effect of Al(OH)3 as an immunological adjuvant

Using a mice in a tetanus vaccination system, we have examined the mode of action of the immunological adjuvant, aluminium hydroxide (Al(OH)3), on antibody titers as measured by an ELISA-method. A profound effect of the adjuvant was observed after primary immunizations, increasing titers about 100-fold compared to plain (not adjuvanted) controls. However, no effect on titers could be attributed to a content of Al(OH)3 in the vaccines when used for booster immunizations, as the increase in titers after boosting was either the same for groups given plain or adsorbed toxoids, or even higher in the groups receiving the plain vaccine. Thus, the effect of Al(OH)3 as an adjuvant seems to be exerted mainly on the primary antibody response. Investigating the mode of action by the adjuvant, we adsorbed a certain amount of toxoid onto varying doses of Al(OH)3. An antigen ELISA revealed that all toxoid had been adsorbed to the Al-gel in the preparations. The antibody titers after immunization showed a significantly higher response to toxoid adsorbed to the higher amount of adjuvant, indicating that this effect was most probably due to free, not antigen-bound, Al-particles. Moreover, this effect could be inhibited when BSA was added in excess to the preparation, thereby blocking the free residues on the Al-gel.     

Lithium as an immunologic adjuvant

Lithium, an adenylate cyclase inhibitor, stimulates a variety of in vitro indices of immune function, including proliferation of lymphocytes in response to mitogens, rosette formation by T-cells and phagocytosis by macrophages. Lithium enhances these immunologic responses at concentrations comparable to those achieved in patients receiving lithium for treatment of manic-depressive disorders.Lithium may prove to have important therapeutic applications as an immune adjuvant, particularly in immune deficiency states associated with excessive C-AMP production.     

Poly(gamma-glutamic acid) nanoparticles as an efficient antigen delivery and adjuvant system: potential for an AIDS vaccine

Antigen delivery systems using polymeric nanoparticles are of special interest as stable protein-based antigen carriers. In the present study, novel biodegradable poly(gamma-glutamic acid) (gamma-PGA) nanoparticles were examined for their antigen delivery and immunostimulatory activities in vitro and in vivo. The uptake of ovalbumin by dendritic cells was markedly enhanced by gamma-PGA nanoparticles, and the ovalbumin was gradually released from gamma-PGA nanoparticles into the cells. In addition, gamma-PGA nanoparticles appeared to have great potential as an adjuvant, because they could induce the maturation of dendritic cells. Although not only ovalbumin-encapsulating nanoparticles (OVA-NPs) but also a simple mixture of ovalbumin and nanoparticles induced dendritic cell maturation, the only dendritic cells exposed to OVA-NPs could strongly activate antigen-specific interferon (IFN)-gamma-producing T cells. Subcutaneous immunization of mice with human immunodeficiency virus type 1 (HIV-1) p24-encapsulating nanoparticles activated antigen-specific IFN-gamma-producing T cells in spleen cells and induced p24-specific serum antibodies, as compared to immunization with p24 alone. Like ovalbumin, a mixture of p24 and nanoparticles also induced antigen-specific serum antibodies but did not activate IFN-gamma-producing T cells in spleen cells, suggesting that nanoparticles play a critical role in inducing cellular immune responses. Furthermore, gamma-PGA nanoparticles had a capacity comparable to that of the complete Freund's adjuvant (CFA) in inducing p24-specific serum antibody. However, unlike CFA, they predominantly activated p24-specific IFN-gamma-producing T cells. Thus, gamma-PGA nanoparticles encapsulating various antigens may have great potential as novel and efficient protein-based vaccines against infectious diseases, including HIV-1 infection.     

Combined treatment with Ren-shen-yang-rong-tang (Japanese name: Ninjin-youei-to) plus prednisolone on adjuvant-induced arthritis in Lewis rat

The effects of combined treatment with 250 mg/kg Ren-shen-yang-rong-tang (Japanese name: Ninjinyouei-to, NYT) plus 4 mg/kg, an average dosage, or 0.2 mg/kg, a suboptimal dosage, of prednisolone (PSL) on adjuvant-induced arthritis in Lewis rats were investigated using two treatment schedules. The agents were administered orally every day from day 0 to 21 (schedule A), or from day -7 to 21 (schedule B) after adjuvant injection. PSL treatment (4 mg/kg) obviously inhibited paw swelling due to non-immune inflammation, diminished the weights of the thymus, spleen, adrenals and iliac lymph nodes, and suppressed the increment of serum interleukin (IL)-6 concentration in both schedules compared to controls. NYT treatment alone inhibited paw swelling due to immune inflammation, diminished the weight of the adrenals and decreased IL-6 concentration only in schedule A. Combined treatment with NYT plus PSL (4 mg/kg) showed: (1) a superior effect to that of PSL on paw swelling in the uninjected hind foot, especially in schedule B, (2) a tendency to diminish adrenal weight in schedule A and the weights of all four organs in schedule B compared with PSL treatment alone, and (3) a suppressive effect on IL-6 concentration weaker than that of PSL alone in schedule B. The suppressive effect of combined treatment with NYT plus PSL (0.2 mg/kg) on paw swelling was significantly stronger compared with either NYT or PSL treatment alone in schedule B. Although this dose of PSL had no influence upon the IL-6 concentration, the combined treatment or NYT alone increased the IL-6 concentration. Mechanisms underlying the synergistic suppressive effects on paw swelling are discussed.     

Chitosan as an adjuvant for poliovaccine

The use of inactivated poliomyelitis vaccine is very important for eradicating poliomyelitis. However, this vaccine is not available readily in underdeveloped countries due to the high cost. Adjuvants can improve the immunogenicity of a vaccine and reduce the antigen dose required for vaccination, thus lowering the cost of the vaccine. Chitosan glutamate solution and a chitosan sulfate micro/nanoparticle suspension were tested as adjuvants for Imovax-inactivated poliovaccine and for inactivated monovalent poliovirus type 1, 2, and 3 vaccines obtained by inactivation of the attenuated Sabin poliovirus strains. Inactivated vaccines admixed with either chitosan glutamate or chitosan sulfate micro/nanoparticles and administered to mice showed significantly enhanced immunogenicity to poliovirus type 1, 2, and 3 strains compared to the respective vaccines administered without chitosan. Chitosan preparations increased the immunogenicity of 1:2 and 1:4 diluted inactivated Sabin strain preparations in mice 8- to 16-fold, so that the neutralizing antibody titers after vaccination with adjuvanted diluted vaccine were equal to those obtained after vaccination with undiluted vaccine administered without chitosan. Neutralizing antibodies could be detected in sera of rats vaccinated with undiluted, 1:10, and 1:100 diluted Imovax vaccine admixed with chitosan sulfate micro/nanoparticles, although in the control group, vaccination only with the undiluted vaccine resulted in antibody production. These results show that the chitosan glutamate solution and chitosan sulfate micro/nanoparticle suspension can significantly improve the immunogenicity of various poliovaccines, and reduce the effective antigen dose.     

Combination chemotherapy as an adjuvant treatment in operable breast cancer.

Prolonged cyclic combination chemotherapy with cyclophosphamide, methotrexate and fluorouracil was evaluated as adjuvant treatment to radical mastectomy in primary breast cancer with positive axillary lymph nodes. After 27 months of study, treatment occurred in 24 per cent of 179 control patients and in 5.3 per cent of 207 women given combination chemotherapy (P less than 10(-6)), the advantage appearing statistically significant in all subgroups of patients. Patients with four or more positive axillary nodes had a higher per cent of relapses than those with fewer nodes. The initial new clinical manifestations occurred in distant sites in 81.5 per cent of relapsed patients. Long-term chemotherapy produced an acceptable toxicity, thus allowing the administration of a high percentage of drug dosage. These results should be considered with caution, since, at present, the effect of this therapy on survival and possible long-term side effects remain unknown.     

Probiotics as an adjuvant to detoxification protocols

Autism is a developmental disease characterized by a spectrum of symptoms ranging from decreased verbal skills and social withdrawal, to repetitive behavior and violent outbursts. Genetic analysis has yielded a few potentially interesting genes, however no clear linkage has been established. For this reason, it has been suggested that the etiology of autism may involve multiple loci. This, in large part, explains why so many different theories abound. One such theory is that of mercury poisoning. Environmentally acquired mercury, either through some causal contact or through vaccination, has been postulated as the culprit. Mercury is thought to be exerting its neurological effect on the brain. The standard treatment has been to apply chelating agents in an attempt to extricate the mercury. One missing component in the treatment is the utilization of the body's own detoxification mechanisms. Arguably the largest detoxification component of the body, the endogenous enteric bacteria are an enormous reservoir, which can be constantly and safely replenished. This paper discusses the use of high-dose probiotics as an adjuvant for detoxification protocols with an emphasis on use in autistics.     

Effect of a traditional Chinese herbal medicine ren-shen-yang-rong-tang (Japanese name: ninjin-youei-to) on hematopoietic stem cells in mice

Cell dynamics after intraperitoneal (i.p.) and oral administration of a traditional herbal medicine, ren-shen-yang-rong-tang (Japanese name: ninjin-youei-to, NYT), were investigated. When NYT was injected i.p. into C3H/He mice, numbers of spleen and peritoneal cells significantly increased in a dose-dependent manner and showed high levels from 4 to 21 days. Two peaks in the total cell number were observed on days 7 and 14 in the peritoneal cavities and spleen of C3H/He mice administered NYT. A marked accumulation of PMN cells in the peritoneal cavity and spleen was detected at 7 days after injection. The numbers of macrophages and lymphocytes also increased by i.p. administration of NYT. The thymus cell number decreased transiently between 4 and 7 days and thereafter returned to the control level. No significant change in the cell number of lymph nodes was observed. Such cellular accumulation was also detected in C3H/HeJ mice, a nonresponder strain to bacterial endotoxin, and athymic nude mice. The activity of colony-forming units in the spleen (CFU-S) of C3H/He as well as C3H/HeJ mice was markedly augmented by i.p. administration of NYT. NYT induced significant CSF production as detectable by its activity in the sera. In addition, oral administration of NYT for 10 days induced a significant increment of peripheral leukocytes and spleen cells and enhanced CFU-S activity of bone marrow cells as induced by i.p. administration, indicating that NYT acts on hematopoietic stem cells capable of differentiating to lymphocytes, macrophages and PMN cells into the periphery.     

Identification of Dioscorea batatas (sanyak) allergen as an inhalant and oral allergen

Dioscorea batatas is widely used in Asia as a herbal medicine or food product with potential health benefits. There have been several reports of occupational asthma caused by inhalation of D. batatas dust. However, there has been no report of systemic allergic reactions after oral administration of D. batatas. Two patients with D. batatas allergy were enrolled. One had experienced severe urticaria and angioedema after indigestion, and the other had been exposed to D. batatas dust and was diagnosed as having occupational asthma. Both patients had high serum-specific IgE and IgG4 antibodies to D. batatas. And IgE immunoblot demonstrated that both sera bound to a 27-kDa protein with an IgE-binding motif, which was revealed by 2-D-electrophoresis to have the sequence Asn-Val-Glu-Asp-Glu-Phe-Ser-X-Ile- Glu-Gly-Asn-Pro-X-X-Pro-Glu-Asn-X-Gly (pI 6.40, 6.04). In conclusion, discorin from D. batatas (DB3S) was identified as the major allergen of D. batatas in patients sensitized via an oral or inhalant route.     

Optimizing oral vaccines: induction of systemic and mucosal B-cell and antibody responses to tetanus toxoid by use of cholera toxin as an adjuvant.

Cholera toxin (CT) is an effective mucosal antigen and acts as an adjuvant when given orally with various antigens; however, few studies have compared the levels of antibody responses to CT and coadministered protein in systemic and mucosal tissues. In this study, we used tetanus toxoid (TT) for assessment of immune responses. Time course and dose-response studies established that 250 micrograms of TT given orally with 10 micrograms of CT three times at weekly intervals induced high serum and gastrointestinal tract anti-TT and anti-CT antibody responses. Oral immunization with TT alone induced no detectable mucosal immunoglobulin A (IgA) antibodies in fecal extracts and only weak serum IgG anti-TT responses. The coadministration of CT and TT induced peak serum IgG anti-TT responses following two oral doses that remained constant after the third oral immunization, while optimal mucosal IgA responses were seen after the third oral immunization. The serum anti-TT response obtained with CT and TT proved protective against TT challenge (100 minimum lethal doses), whereas mice orally given CT or TT alone died. Antigen-specific B-cell responses were assessed with an isotype-specific Elispot assay of isolated lymphoid cells from the spleen, Peyer's patches, and the small intestinal lamina propria. Interestingly, approximately fourfold-higher numbers of IgA anti-CT than of anti-TT antibody-producing (spot-forming) cells occurred in lymphocytes from the lamina propria of mice orally immunized with both TT and CT. The adjuvant CT did not induce polyclonal B-cell responses in mice given CT by the oral route, since no significant differences in total numbers of B cells producing IgA, IgG, or IgM were found compared with the numbers in mice given TT alone. The results clearly indicate that serum and mucosal antibody responses develop with different kinetics and that protective TT-specific antibody responses are generated in the systemic compartment when TT is administered with CT via the oral route.     

Rotarix (RIX4414): an oral human rotavirus vaccine

Rotavirus is the most common cause of severe gastroenteritis in children younger than 3 years of age worldwide. New rotavirus vaccine candidates were required to confer early protection against the most common rotavirus serotypes and to be well tolerated and not associated with intussusception. RIX4414 is a human-attenuated G1(P8) oral rotavirus vaccine administered in two doses at approximately 6-24 weeks of age. The first dose may be administered from the age of 6 weeks. There should be an interval of at least 4 weeks between doses and the vaccination course should preferably be given before 16 weeks of age and must be completed, according to the manufacturer, by the age of 24 weeks. In a worldwide development program involving more than 70,000 children in six Phase I-III field trials, this vaccine proved to be nonreactogenic, well tolerated and not associated with intussusception. The vaccine provides over 85-96% protection against moderate-to-severe gastroenteritis caused by G1 and non-G1 serotypes, as demonstrated in Latin American and European clinical trial settings, respectively; and reduces gastroenteritis-related hospitalizations by more than 40% in Latin America and by 75% in European settings.     

Functional maturation of immature B cells accumulated in the periphery by an intraperitoneal administration of a traditional Chinese medicine, xiao-chai-hu-tang (Japanese name: shosaiko-to)

We found that an intraperitoneal (ip) injection of a traditional Chinese herbal medicine, xiao-chai-hu-tang (Japanese name: shosaiko-to), induced accumulation of B lymphocytes (sIgM+) in the peritoneal cavity and spleen. 1) Cell surface marker analysis by a fluorescence-activated cell sorter (FACS) demonstrated that the accumulated B cells on day 4 or 7 after shosaiko-to administration (early phase) were composed mainly of sIgM+IgD- cells and suggested that these B cells maturated into sIgM+IgD+ cells on days 10 or 14 (late phase). Relative decrease of IgM+IgD+ cells at early phase was more profound in peritoneal cells (PC) than in spleen cells. 2) With respect to spleen lymphocytes, antibody responses to a thymus-independent (TI) antigen of type 2 (trinitrophenylated Ficoll) and a thymus-dependent (TD) antigen (sheep erythrocyte) were enhanced at late phase but not at early phase. In contrast, responses to trinitrophenylated lipopolysaccharide (TNP-LPS) as a TI-1 antigen and LPS as a B cell mitogen or a polyclonal B cell activator were enhanced markedly at early phase but declined at late phase. 3) With respect to peritoneal lymphocytes, responses to LPS were suppressed at early phase but recovered at late phase. Enhanced responses to TI and TD antigen at late phase in spleen lymphocytes and suppressed response to LPS at early phase in peritoneal lymphocytes may be explained by increases of IgM+IgD+ mature B cells and IgM+IgD- immature B cells, respectively, at those times. Enhanced responses to TI-1 or LPS in spleen lymphocytes at early phase may be explained by elevated sensitivity of IgM+IgD+ cells which reside in the spleen before shosaiko-to administration and receive the direct stimulation by shosaiko-to, or by acquired responsiveness of IgM+IgD- cells which migrate after stimulation with shosaiko-to.     

Glucan as an adjuvant for a murine Babesia microti immunization trial.

A vaccination protocol against murine Babesia microti infection, using glucan, a beta-1,3-glucopyranose derivative of yeast cell walls, and glutaraldehyde-fixed infected erythrocytes was evaluated. BALB/c mice were immunized intravenously four times at 2-day intervals with 2 X 10(8) fixed infected erythrocytes with and without glucan (0.45 mg). The mice were challenged 2 weeks after the last immunization with 10(8) viable infected erythrocytes. Peak parasitemia was significantly reduced (8.9 +/- 1.0%; P less than 0.001) in glucan-immunized mice as compared with nonimmunized controls (41.2 +/- 1.4%), glucan-treated controls (31.7 +/- 2.5%; P less than 0.05), or mice which received fixed infected erythrocytes without glucan (21.0 +/- 1.2%; P less than 0.01). Humoral and cellular immunity to B. microti was evaluated before challenge by measuring antibody titers and splenocyte blastogenic responses to B. microti antigens. The in vitro cellular response was inversely correlated with mean peak parasitemia (P less than 0.05). These observations demonstrate that glucan is an effective adjuvant in enhancing immunity to murine babesiosis.     

Protective effect of L. donovani antigens using glucan as an adjuvant

Golden hamsters were immunized with various antigen fractions of Leishmania donovani promastigotes. Beta 1,3-glucan was used as an adjuvant in these vaccination experiments. The results indicate that immunization of animals with the microsomal fraction (subcellular fraction III) in combination with glucan confers considerable immune protection against L. donovani infection. The immune protection was confirmed by correspondingly lower parasite burden in the livers and spleens of test animals compared to controls. Additionally, the vaccinated animals showed positive skin test responsiveness after challenge, along with increased antibody titres. Immunization of animals with whole and particulate antigen fractions was also found to afford a high degree of resistance. The other subcellular and soluble antigen fractions conferred very little protection. In these experiments, glucan was found to be a potent adjuvant when injected, intraperitoneally, with Leishmania antigens. Similar doses of parasite extracts given without an adjuvant were able to confer only very little or no protection.     

Suprabasal 40 kd keratin (K19) expression as an immunohistologic marker of premalignancy in oral epithelium.

The authors have studied the expression of keratin 19 in normal oral mucosa and in oral lesions exhibiting a range of histopathologic changes that are thought to precede squamous cell carcinoma. Formalin-fixed, paraffin-embedded sections were pretreated with pronase and stained with a K19-specific antibody by the avidin-biotin immunoperoxidase method. In nonkeratinized mucosa, whether normal or benign hyperplastic, K19 was detectable in the basal cell layer. In keratinized mucosa, whether normal or benign hyperplastic, there was no detectable K19. All lesions from any oral site that exhibited atypia diagnosed from hematoxylin and eosin stained sections as moderate-to-severe dysplasia or carcinoma in situ, whether hyperkeratotic or not, stained strongly for K19 in the basal and suprabasal cell layers. The number of cell layers that were K19-positive correlated with the level in the epithelium to which dysplasia persisted. Suprabasal K19 staining tended to occur in regions of the epithelium in which expression of the terminal differentiation protein involucrin was delayed or absent. Thus, K19 expression may be linked to the retention of stem cell character or a state otherwise uncommitted to terminal squamous differentiation. Suprabasal K19 staining is clearly correlated with premalignant change in oral epithelium and therefore promises to be a useful tool in oral histopathologic diagnosis.