RESUMEN

We attempted to clarify the mechanism of the mucosal adjuvanticity of recombinant cholera toxin B subunit (rCTB), which is inherently uncontaminated with the holotoxin produced by Bacillus brevis and has a powerful mucosal adjuvant activity, on cytokine responses compared with that of cholera toxin (CT). rCTB had no ability to stimulate cyclic AMP formation in mouse peritoneal macrophages (Mphi). Cytokine production by non-immunized Mphi cultured with rCTB or CT and by the spleen cells of mice co-immunized intranasally with ovalbumin (OVA) and rCTB or CT was examined. rCTB alone did not induce interleukin (IL)-1alpha/beta or IL-6 production by Mphi, but combination of rCTB with lipopolysaccharide (LPS) enhanced both IL-1alpha/beta production. Conversely, CT plus LPS suppressed IL-1alpha/beta production more than LPS alone. Both rCTB and CT suppressed IL-12 secretion induced by interferon gamma (IFN gamma) plus LPS. IL-2, IL-4, IL-5, and IL-10 were secreted by mouse spleen cells restimulated with OVA after intranasal co-administration of OVA together with rCTB, and in response to CT, the same cytokines were secreted. The different effect of rCTB on Mphi from that of CT may mean a difference between the mechanisms of rCTB and CT during the early stage of an immune response.

Asunto(s)

Adyuvantes Inmunológicos , Bacillus , Toxina del Cólera/inmunología , Inmunidad Mucosa/inmunología , Interleucinas/inmunología , Macrófagos Peritoneales/inmunología , Administración Intranasal , Animales , Toxina del Cólera/aislamiento & purificación , AMP Cíclico/metabolismo , Inmunización , Interleucinas/biosíntesis , Interleucinas/metabolismo , Lipopolisacáridos/inmunología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Subunidades de Proteína , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Bazo/citología , Bazo/inmunología

RESUMEN

A-ring diastereomers of 1alpha,25-dihydroxy-22-oxavitamin D3 (OCT) (2), 3-epi-1alpha,25-dihydroxy-22-oxavitamin D3 (3-epiOCT) (3) and 1,3-diepi-1alpha,25-dihydroxy-22-oxavitamin D3 (1,3-diepiOCT) (4) were synthesized by the convergent method. In vitro binding affinity for rat vitamin D binding protein and calf-thymus vitamin D receptor, differentiation-inducing activity on HL-60 cells, and transcriptional activity of 3-epiOCT (3) and 1,3-diepiOCT (4) were evaluated in comparison with OCT (2), 1-epi-1alpha,25-dihydroxy-22-oxavitamin D3 (1-epiOCT) (5) and 1alpha,25-dihydroxyvitamin D3 (1).

Asunto(s)

Calcitriol/síntesis química , Calcitriol/metabolismo , Animales , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Calcitriol/análogos & derivados , Bovinos , Diferenciación Celular/efectos de los fármacos , Células HL-60 , Humanos , Osteocalcina/efectos de los fármacos , Osteocalcina/genética , Unión Proteica , Ratas , Receptores de Calcitriol/metabolismo , Estereoisomerismo , Esteroide Hidroxilasas/efectos de los fármacos , Esteroide Hidroxilasas/genética , Activación Transcripcional/efectos de los fármacos , Proteína de Unión a Vitamina D/metabolismo

RESUMEN

Recombinant cholera toxin B subunit (rCTB) produced by Bacillus brevis carrying pNU212-CTB has been previously found to be a potent mucosal adjuvant to aluminium-non-adsorbed tetanus toxoid (nTT) and diphtheria toxoid (nDT) co-administered intranasally, and the possibility of needle-free inoculation of these vaccines with rCTB has been suggested. In this paper we examined the potentiality of rCTB as a mucosal adjuvant to aluminium-non-adsorbed yeast-derived recombinant hepatitis B surface antigen (rHBs) being a particulate antigen when administered intranasally with rCTB. In-house ELISA showed that a mixture of rHBs (1 or 5 microg) and rCTB (10 microg) elevated not only systemic responses but also mucosal immune responses at the nasal cavity, the lung, the saliva, the small intestine and the vagina against rHBs, and these could be further increased with higher doses of antigen. With antibody isotypes of IgG, there were equally high levels of serum HBs-specific IgG1, IgG2a and IgG2b antibodies and induction of mixed Th1- and Th2-type responses was considered to occur in combination of rHBs and rCTB. Serum anti-HBs titres in almost all mice obtained from sandwich EIA using a commercial kit were higher than 1000 milli-international units ml(-1) (mIU ml(-1)). These results show that rCTB is also very effective as a mucosal adjuvant for a particulate antigen like rHBs, as well as soluble antigens like nTT and nDT reported previously, suggesting the possibility of intranasal immunization with rHBs plus rCTB in humans.

Asunto(s)

Vacunas contra Hepatitis B/administración & dosificación , Adyuvantes Inmunológicos/administración & dosificación , Administración Intranasal , Animales , Toxina del Cólera/administración & dosificación , Femenino , Anticuerpos contra la Hepatitis B/biosíntesis , Anticuerpos contra la Hepatitis B/sangre , Antígenos de Superficie de la Hepatitis B/administración & dosificación , Inmunidad Mucosa , Inmunoglobulina A/biosíntesis , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Vacunas Sintéticas/administración & dosificación

RESUMEN

24-Hydroxylated derivatives were synthesized in 24(R) and 24(S) forms by the convergent method as analogs related to 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3). In the convergent synthesis, the A-ring fragment, synthesized from diethyl D-tartarate, and the C/D-ring fragments in 24(R) and 24(S) forms (vitamin D numbering), obtained from vitamin D(2) via the Inhoffen-Lythgoe diol, were coupled in moderate yields to give 1alpha,24(R),25-trihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3) and 1alpha,24(S),25-trihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3). In preliminary biological evaluations, 24-hydroxylation of 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3) caused weakened affinity to vitamin D binding protein in vitro and less calcemic activity in vivo compared to the parent compound. While the affinity to vitamin D receptor in 24(R) epimer was sustained, the affinity in 24(S) epimer was less than that of the parent compound.

Asunto(s)

Calcitriol/síntesis química , Colecalciferol/análogos & derivados , Animales , Calcitriol/análogos & derivados , Calcitriol/metabolismo , Calcitriol/farmacología , Calcio/metabolismo , Proteínas Portadoras/metabolismo , Pollos , Colecalciferol/síntesis química , Colecalciferol/metabolismo , Ratones , Ratones Endogámicos BALB C , Unión Proteica , Ratas , Receptores de Calcitriol/metabolismo , Estereoisomerismo , Relación Estructura-Actividad , Vitamina D/análogos & derivados

RESUMEN

Mucosal immune responses are known to play important roles in the establishment of protective immunity to microbial infections through mucosa. We examined the toxic effects of recombinant cholera toxin B subunit (rCTB) secreted by Gram-positive bacterium Bacillus brevis as a mucosal adjuvant. Incubation of guinea-pig peritoneal macrophages with cholera toxin (CT) or aluminium hydroxide gel (Al-gel) released a significantly higher activity of lactate dehydrogenase than did commercial natural CTB (CTB) or rCTB. Intraintestinal or intramuscular administration of CT, CTB or Al-gel caused severe histopathological reactions. CT also caused infiltration of neutrophils and irregular arrangement or partial loss of the respiratory epithelium. In addition, CT and CTB elicited vascular permeability-increasing effects. rCTB elicited no toxic effects to macrophages and no vascular permeability-increasing effects. Moreover, it is noticeable that no distinct local histopathological reactions were observed in the nasal cavity, the small-intestinal loop or the muscle given rCTB. These results suggest that, from a safety standpoint, rCTB is a useful candidate as mucosal vaccine adjuvant.

Asunto(s)

Adyuvantes Inmunológicos/toxicidad , Bacillus/metabolismo , Toxina del Cólera/toxicidad , Mucosa Intestinal/inmunología , Macrófagos Peritoneales/inmunología , Mucosa Nasal/inmunología , Fragmentos de Péptidos/toxicidad , Adyuvantes Inmunológicos/administración & dosificación , Administración Intranasal , Hidróxido de Aluminio , Animales , Síndrome de Fuga Capilar/etiología , Permeabilidad Capilar/efectos de los fármacos , Células Cultivadas , Toxina del Cólera/administración & dosificación , Toxina del Cólera/genética , Toxina del Cólera/inmunología , Femenino , Cobayas , Inflamación/etiología , Inyecciones , Inyecciones Intramusculares , Mucosa Intestinal/patología , Intestino Delgado/patología , L-Lactato Deshidrogenasa/metabolismo , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos BALB C , Músculo Esquelético/patología , Cavidad Nasal/patología , Mucosa Nasal/patología , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Conejos , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/toxicidad , Seguridad

RESUMEN

Nasal mucosal immunization is very attractive for vaccination to prevent various bacterial and viral infectious diseases because of induction of systemic and mucosal immune responses. The aim of the present study was to investigate the possibility of changing the immunization procedure of diphtheria toxoid (DT) from intramuscular or subcutaneous injection to intranasal administration. Intranasal immunization with aluminium-non-adsorbed diphtheria toxoid (nDT) together with recombinant cholera toxin B subunit (rCTB, 10 microg) induced, at a concentration of 5 Lf, high levels of serum DT-specific IgG antibody responses and high or moderate levels of the specific IgA antibody responses in all mice and only a slight level of the specific IgE antibody responses in some mice. Furthermore, sufficiently high diphtheria antitoxin titres more than 0.1 international units (IU) ml(-1) were obtained from mice which showed high levels of serum DT-specific IgG antibody responses. Under the same experimental conditions, induction of significant levels of mucosal DT-specific IgA antibody responses occurred in the nasal cavity, the lung, the saliva and vaginal secretions and the small and large intestines of all mice, although there were different titres between individual mice. Similar results were also obtained with rCTB-specific serum IgG and IgA and mucosal IgA antibody responses; serum rCTB-specific IgE antibody titres were not detected. These results show that intranasal administration of nDT with rCTB must be a very useful means for vaccination against diphtheria.

Asunto(s)

Adyuvantes Inmunológicos/administración & dosificación , Anticuerpos Antibacterianos/biosíntesis , Toxina del Cólera/inmunología , Toxoide Diftérico/administración & dosificación , Toxoide Diftérico/inmunología , Mucosa Nasal/inmunología , Adyuvantes Inmunológicos/química , Administración Intranasal , Adsorción , Aluminio/administración & dosificación , Aluminio/química , Animales , Anticuerpos Antibacterianos/sangre , Especificidad de Anticuerpos , Toxina del Cólera/administración & dosificación , Toxina del Cólera/química , Difteria/inmunología , Difteria/prevención & control , Antitoxina Diftérica/sangre , Antitoxina Diftérica/inmunología , Toxoide Diftérico/química , Relación Dosis-Respuesta Inmunológica , Femenino , Inmunidad Mucosa/inmunología , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/química

RESUMEN

Effects of calcium phosphate and aluminium hydroxide adjuvants with different physical properties were examined in guinea pigs for local histopathological reactions, electron-microscopical changes of macrophages and adjuvanticity on total IgG antibody response to subcutaneously administered ovalbumin (OVA) and tetanus toxoid (TT). Calcium phosphate gel (Ca-gel) induced active inflammatory reactions consisting of neutrophils (pseudoeosinophils) and foamy macrophages associated with many multinuclear giant cells for at least 4 weeks. Aluminium hydroxide gel (Al-gel) also elicited granulomatous inflammatory reactions consisting mainly of macrophages with foamy cytoplasm, small lymphocytes and giant cells at the injection sites for up to 8 weeks or longer. Severity of local tissue irritation due to calcium phosphate gel (Ca-gel) was similar to that due to Al-gel except for the duration of the inflammatory reactions. Calcium phosphate suspension (Ca-sus)-induced local reactions completely ceased by the 4th week, while aluminium hydroxide suspension (Al-sus)-induced reactions were seen up to the 8th week. Electron-microscopical observations showed that both Al-gel and Al-sus caused damage of macrophages. The adjuvant activity of Al-gel for OVA or TT was significantly stronger than that of any other adjuvant material, whereas those of Ca-gel and Ca-sus were not seen at a dose of 3 mg calcium phosphate per millilitre. Al-sus-TT at a dose of 3 mg aluminium hydroxide per millilitre induced very low levels of antibody. These results suggest that calcium phosphate adjuvant may not be an useful alternative to Al adjuvant.

Asunto(s)

Adyuvantes Inmunológicos/toxicidad , Hidróxido de Aluminio/toxicidad , Fosfatos de Calcio/toxicidad , Irritantes/toxicidad , Hidróxido de Aluminio/farmacología , Animales , Fosfatos de Calcio/farmacología , Ensayo de Inmunoadsorción Enzimática , Femenino , Cobayas , Inmunoglobulina G/sangre , Macrófagos/efectos de los fármacos , Macrófagos/ultraestructura , Microscopía Electrónica de Rastreo , Ovalbúmina/inmunología , Toxoide Tetánico/inmunología

RESUMEN

Aluminium hydroxide (Al) and calcium phosphate (Ca) have been used for many years as immunological adjuvants for biologicals. We investigated the toxic effects of both adjuvants with different physical properties. Al-gel elicited vascular permeability-increasing and toxic effects to macrophages (M phi), while its haemolytic effect was weak. Ca-gel elicited a significantly stronger haemolytic effect, but no other toxic effect. Incubation of M phi or polymorphonuclear leucocytes with Al-suspension resulted in the largest release of lactate dehydrogenase. Ca-suspension caused haemolysis of about 50% of that caused by Ca-gel.

Asunto(s)

Adyuvantes Inmunológicos/toxicidad , Hidróxido de Aluminio/toxicidad , Fosfatos de Calcio/toxicidad , Vacunas , Animales , Permeabilidad Capilar/efectos de los fármacos , Células Cultivadas , Femenino , Geles , Cobayas , Hemólisis/efectos de los fármacos , L-Lactato Deshidrogenasa/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Suspensiones

RESUMEN

The amino acid sequence of a cytotoxic factor, CTF-I, isolated from the venom of the Japanese habu snake (Trimeresurus flavoviridis) has been determined through automatic phenylisothiocyanate degradation of the PE-protein and derived proteolytic peptides. CTF-I consists of 72 amino acids and contains an Arg-Gly-Asp sequence present in trigramin-like peptides isolated from other snake venoms. The primary structure of another cytotoxic factor, CTF-II, consisting of 75 amino acids, was deduced to comprise that of CTF-1 with an additional Glu-Leu-Leu-sequence at its N-terminal.

Asunto(s)

Venenos de Crotálidos/química , Citotoxinas/química , Secuencia de Aminoácidos , Animales , Citotoxinas/aislamiento & purificación , Péptidos y Proteínas de Señalización Intercelular , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/química , Péptidos/química , Homología de Secuencia de Ácido Nucleico

RESUMEN

Ovalbumin (OA)-complexed guinea-pig IgG1 and IgG2 antibodies were found to bind to homologous polymorphonuclear leukocytes (PMNs). As these bindings are assumed to be mediated by certain Fc receptors (FcRs) for IgG1 and IgG2, the variety and properties of the FcRs on the cells were investigated by the use of two monoclonal antibodies to guinea-pig macrophage FcRs which were prepared by Shimamura T. et al., 1987 (Molec. Immun. 24, 67-74): VI A2 IgG1 to the FcR for IgG1 and IgG2 (FcR1,2) and VII A1 IgG1 to the FcR for IgG2 (FcR2). PMNs were shown to bind the Fab' of VI A2 IgG1 (VI A2 Fab') by flow cytofluorometry, suggesting that the cells possess a certain FcR which cross-reacts antigenically with macrophage FcR1,2. In fact, VI A2 Fab' inhibited completely the binding of OA-complexed IgG1 antibody to the cells. When the FcR was isolated by affinity chromatography on the F(ab')2 of VI A2 IgG1 coupled to Sepharose, it gave a 55,000 mol. wt band on sodium dodecylsulfate-polyacrylamide gel electrophoresis, as in the case of macrophage FcR1,2. The number of the FcR molecules per PMN cell was estimated to be 2 X 10(4) by measuring the binding of 125I-VI A2 Fab'. The binding of OA-complexed IgG2 antibody to PMNs was also inhibited with VI A2 Fab', but partially. This finding indicates that the FcR bound by VI A2 Fab' may be an FcR1,2 which is able to bind both OA-complexed IgG1 and IgG2 antibodies, and also that PMNs possess another FcR, namely FcR2 which binds IgG2 antibody alone. The Fab' of VII A1 IgG1 (VII A1 Fab'), on the other hand, did not exhibit any inhibitory activity on the bindings of OA-complexed IgG1 and IgG2 antibodies to PMNs. Since no evidence indicating the binding of VII A1 Fab' to PMN cells was obtained by flow cytofluorometry, the FcR2 of PMNs may be antigenically different from its macrophage counterpart. In conclusion, these results indicate that two distinct types of FcR for IgG isotypes exist on guinea-pig PMN cells: FcR1,2 similar to macrophage FcR1,2, and FcR2 distinct from macrophage FcR2.

Asunto(s)

Inmunoglobulina G/clasificación , Neutrófilos/inmunología , Receptores Fc/análisis , Animales , Complejo Antígeno-Anticuerpo/metabolismo , Unión Competitiva , Cobayas , Fragmentos Fab de Inmunoglobulinas/inmunología , Inmunoglobulina G/metabolismo , Peso Molecular , Neutrófilos/metabolismo , Receptores Fc/inmunología , Receptores de IgG

RESUMEN

Hydrolysis of substance P and nine kinds of substance P analogs by angiotensin-converting enzyme highly purified from rat lung was examined by using amino-group fluorometry and high-performance liquid chromatography. The enzyme hydrolyzed substance P and several analogs, notwithstanding that they did not contain free C-terminal residues. The analyses of cleavage products separated by high-performance liquid chromatography indicated that the enzyme hydrolyzed substance P and its analogs mainly at the bond between Phe8-Gly9 and also at another bond, possibly between Gly9-Leu10, to a lesser extent by an endopeptidase action, followed by successive release of dipeptides by a dipeptidyl carboxypeptidase action. The analogs that had D-amino acid residues substituted at the presumed cleavage sites were scarcely hydrolyzed. It was further found that (Pyr6)-fragment (6-11) was hydrolyzed by the enzyme more efficiently than the other fragment-type analogs and was cleaved at a single bond by the endopeptidase activity of the enzyme. Therefore, this fragment was used as a substrate in order to characterized the endopeptidase activity of the enzyme by employing fluorometry. The activity was dependent on chloride ion, and was inhibited by captopril, MK-421, and EDTA. Thus, the endopeptidase activity of the enzyme showed properties similar to those of the dipeptidyl carboxypeptidase activity of the enzyme.

Asunto(s)

Endopeptidasas/metabolismo , Pulmón/enzimología , Peptidil-Dipeptidasa A/metabolismo , Sustancia P/metabolismo , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Hidrólisis , Cinética , Masculino , Ratas , Sustancia P/análogos & derivados