A biphasic serum glucose response in mice to inoculation with pertussis vaccine

The effect of B. pertussis vaccine on the serum glucose level of mice was investigated. The results show that at least two components in the vaccine interfere with glucose metabolism. A heat-stable component which is assumed to be LPS induced hypoglycemia 3-5.5 h after inoculation, especially in LPS-sensitized mice. A heat-labile component which is possibly equivalent with the LPF/HSF/IAP complex, is responsible for persistence of the hypoglycaemia for at least 6 days. If hypoglycaemia contributes to the neurological side effects after pertussis vaccination both components have to be considered as being responsible for these effects.     

Effect of pertussis antibodies on the induction of suppressor cells of humoral immunity in mice

Standard pertussis agglutinative serum as well as antibodies to total and purified protective pertussis antigens, isolated from the serum by immunoadsorption technique, were studied. The treatment of donors with pertussis antibodies affected the T-suppressor formation, induced by high doses of sheep red blood cells, as was shown on the model of syngeneic transfer. Immunomodulating effect of antibodies, complementary to immunogen fraction of pertussis cells, was decreased. It was suggested that the observed antilymphocyte activity of antipertussis antibodies could play a certain role in postvaccination complications after corpuscular pertussis vaccine administration.     

Biological activities of pertussigen from Bordetella pertussis strains of various agglutinogen types

Pertussigen prepared from Bordetella pertussis strains of various agglutinogen types was found to have similar biological activities in mice, and to give precipitin lines of identity in agar diffusion tests. The doses required to induce histamine hypersensitivity ranged from 0.9 to 9.5 ng, and to induce leukocytosis from 124 to 190 ng. When pertussigen was detoxified by treatment with glutaraldehyde, it protected mice from intracerebral challenge with B. pertussis at doses of from 12.7 to 24.4 micrograms. The results showed that all smooth strains of B. pertussis produced pertussigen that was biologically and serologically similar, and that it was produced independently of fimbrial hemagglutinin.     

Chemotactic activity of pertussis toxin on murine lymphocytes. Its potential role on lymphocyte accumulation in the lung

The effects of pertussis toxin on lymphocyte migration were studied in vitro. In this study pertussis toxin significantly stimulated lymphocyte migration at concentrations of 0.1 and 1 microgram ml-1 using a microchamber and the leading-front method. Checkerboard analysis demonstrated that pertussis toxin causes directed migration of lymphocytes (chemotaxis). Heat-treatment pertussis toxin abolished its capacity to cause this migration. When murine lymphocytes were preincubated with different concentrations of pertussis toxin, an inhibition of chemotaxis at the dosages of 0.1 and 1 microgram ml-1 was observed. On the other hand, lymphocytes derived from mice treated with pertussis toxin were not inhibited after subsequent exposure to pertussis toxin in vitro. Since lymphocyte accumulation in the lungs of mice treated with pertussis toxin has been well demonstrated, the results of our study could suggest a chemotactic activity of pertussis toxin in determining accumulation of lymphocytes in this organ.     

Chemotactic activity of pertussis toxin on murine lymphocytes. Its potential role on lymphocyte accumulation in the lung

Abstract The effects of pertussis toxin on lymphocyte migration were studied in vitro. In this study pertussis toxin significantly stimulated lymphocyte migration at concentrations of 0.1 and 1 μg ml⁻¹ using a microchamber and the leading-front method. Checkerboard analysis demonstrated that pertussis toxin causes directed migration of lymphocytes (chemotaxis). Heat-treatment of pertussis toxin abolished its capacity to cause this migration. When murine lymphocytes were preincubated with different concentrations of pertussis toxin, an inhibition of chemotaxis at the dosages of 0.1 and 1 μg ml⁻¹ was observed. On the other hand, lymphocytes derived from mice treated with pertussis toxin were not inhibited after subsequent exposure to pertussis toxin in vitro. Since lymphocyte accumulation in the lungs of mice treated with pertussis toxin has been well domenstrated, the results of our study could suggest a chemotactic activity of pertussis toxin in determining accumulation of lymphocytes in this organ.     

Passive protection of mice with the 19 S and 7 S fractions of anti-pertussis sera in experiment.

Protective activity of anti-persussis rabbit and mouse sera and 19 S and 7 S fractions obtained from these sera was investigated in the test of passive protection of mice on a model of pertussis meningoencephalitis. The method of simultaneous intracerebral administration of the serum or fraction with live culture of a virulent B. pertussis strain was used. Hyperimmune rabbit serum containing mercaptoethanol-resistant agglutinins in a high titre was found to have the most pronounced protective effect. Serum of mice, collected 14 days after single immunization of the animals, did not show any protective properties. A small amount of protective activity was observed in the serum collected on the 30th day after a single administration of the vaccine. A sharp increase in the protective activity of the serum was observed after double immunization of mice. Correlation was found between the increase in the titre of agglutinins (in particular of 7 S antibodies) and the protective activity of the serum. Protective properties of 19 S and 7 S fractions isolated from immune rabbit and mouse sera by the method of gel filtration were investigated. Both fractions were found to possess protective properties, but fraction 7 S was more active than fraction 19 S.     

Regulation of inflammatory responses to Bordetella pertussis by N(G)-monomethyl-L-arginine in mice intranasally infected

To investigate effect of MMLA, an inhibitor of nitric oxide (NO) production, on regulation of inflammatory responses to Bordetella pertussis infection, mice were infected intranasally, and treated with various concentrations of MMLA. Ten days after infection, mice treated with MMLA at dosage of 100 mg/kg, given intraperitoneally in a single dose or for 5 consecutive days, showed at histopathologic examination, a significant decrease of intensity of inflammation (scores, 0.6 +/- 0.2 and 0.9 +/- 0.5 respectively). A decrease of cellular accumulation of neutrophils and lymphocytes in the bronchoalveolar lavage (BAL) fluid was observed in infected mice treated with MMLA, especially at dosage of 10 mg/kg, given in a single dose intraperitoneally. In addition, BP-infected mice treated with MMLA (100 mg/kg, intraperitoneally) for 5 consecutive days showed higher mortality rate than untreated mice infected with B. pertussis, and the number of B. pertussis in lungs of mice treated with MMLA was significantly increased. However, MMLA treatment of infected mice had some effect on levels of IFN-gamma and nitrite/nitrate (end-stable products of NO) in the BAL fluid. This study indicates that NO may play a role either as microbiocidal agent or as a modulator of immune regulation, inasmuch as it may upregulate tissue inflammatory response to B. pertussis.     

Pertussis toxin treatment In vivo reduces surface expression of the adhesion integrin leukocyte function antigen-1 (LFA-1)

Pertussis toxin treatment in macaques inhibits lymphocyte extravasation from the blood and leads to transient lymphocytosis and leukocytosis. We examined lymphocyte adhesion molecules known to be involved in the extravasation process to find possible mechanisms for the effects of pertussis toxin treatment. The two subunits of LEA-1, CD11a and CD18, showed decreased surface expression on lymphocytes from pertussis toxin treated animals compared to untreated animals. The adhesion molecule CD44, and the alpha subunit of the integrin VLA-4 (CD49d) were not decreased by pertussis toxin treatment. Lower surface expression of CD 11 a and CD 18 was observed on all lymphocyte subsets and was correlated inversely with the extent of lymphocytosis. The magnitude of lymphocytosis after pertussis toxin treatment was higher in SIV-infected macaques than in uninfected animals. However, changes in LFA-1 levels were similar in both groups. These data show that LEA-1 surface levels are affected by pertussis toxin in vivo and this change may account in part, for the ability of pertussis toxin to induce lymphocytosis.     

Pertussis toxin treatment in vivo reduces surface expression of the adhesion integrin leukocyte function antigen-1 (LFA-1)

Pertussis toxin treatment in macaques inhibits lymphocyte extravasation from the blood and leads to transient lymphocytosis and leukocytosis. We examined lymphocyte adhesion molecules known to be involved in the extravasation process to find possible mechanisms for the effects of pertussis toxin treatment. The two subunits of LFA-1, CD11a and CD18, showed decreased surface expression on lymphocytes from pertussis toxin treated animals compared to untreated animals. The adhesion molecule CD44, and the alpha subunit of the integrin VLA-4 (CD49d) were not decreased by pertussis toxin treatment. Lower surface expression of CD11a and CD18 was observed on all lymphocyte subsets and was correlated inversely with the extent of lymphocytosis. The magnitude of lymphocytosis after pertussis toxin treatment was higher in SIV-infected macaques than in uninfected animals. However, changes in LFA-1 levels were similar in both groups. These data show that LFA-1 surface levels are affected by pertussis toxin in vivo and this change may account in part, for the ability of pertussis toxin to induce lymphocytosis.     

微囊藻细胞抽提液对小鼠血液的亚慢性毒性

本文研究了注射含微囊藻毒素的微囊藻细胞抽提掖对小鼠血液以及免疫系统的亚慢性毒性作用。实验分为3个处理组和1个对照组（每组10只昆明小鼠,雌雄各半）,采用腹腔注射的染毒方法对3个处理组进行暴露,剂量分别为2.4、4.8 和 9.6 μg microcystin-LR/kg body weigh,对照组注射等量的生理盐水,连续注射14d。实验结果表明,14d 染毒后,小鼠的肝体比和脾体比都明显增大（p < 0.05）, 同时在9.6 μg/kg处理组,血清丙氨酸转移酶、天冬氨酸转移酶、乳酸脱氢酶和碱性磷酸酶活性与对照相比明显升高,但血清总蛋白、白蛋白和白蛋白/球蛋白比率下降。这些指标的变化说明,含微囊藻毒素的微囊藻细胞提取液对处理组小鼠肝脏造成了损伤,肝组织学观察也印证了这个结果,在处理组小鼠肝组织有明显的水样变性。另外,9.6 μg/kg处理组小鼠血液白细胞数量比对照组明显减少。组织细胞学观察发现,处理组小鼠脾脏也有明显的损伤。该实验结果说明,含微囊藻毒素的微囊藻细胞抽提液对小鼠的血液和免役系统都产生了一定程度的损伤。     

Mice are protected against Bordetella pertussis infection by intra-nasal immunization with filamentous haemagglutinin

Abstract Intra-nasal immunization of mice with purified Bordetella pertussis filamentous haemagglutinin (FHA) or a crude cell sonicate was shown to protect against subsequent B. pertussis aerosol challenge. Immunization with FHA was found to be the most effective and resulted in complete clearance of the bacterial infection from the lungs within 14 days. Serum IgG and lung IgA anti-FHA antibodies were detectable within 4 weeks of the first immunization and anamnestic responses were seen following secondary immunization and subsequent challenge with B. pertussis . Nasal administration of pertussis is a route which induces good systemic serum, as well as local secretory, antibody responses.     

Specificity and detection limit of a dermal temperature histamine sensitization test for absence of residual pertussis toxin in vaccines

Currently, an assay based on fatal sensitization of mice to histamine challenge is widely used for testing absence of residual pertussis toxin in acellular pertussis containing vaccines. For replacement of this lethal end-point assay, an alternative method based on body temperature measurement in mice has been presented, and in this study the specificity and detection limit of a dermal temperature-based assay were assessed. Test preparations containing pertussis toxin were prepared in aluminum-adjuvanted pertussis toxoid vaccine and injected intraperitoneally in histamine sensitive mice. Later the mice were challenged with histamine and the pertussis toxin-induced decrease in dermal temperature recorded. By comparison of mice treated with pertussis toxoid vaccine spiked with pertussis toxin with mice treated with pertussis toxoid vaccine alone, the assay gave a response that specifically could detect presence of pertussis toxin. The acellular pertussis containing vaccine did not interfere with the pertussis toxin-induced temperature response recorded. In tests for presence of pertussis toxin in the pertussis vaccine preparation, the detection limit of the assay was estimated to approximately 5 ng pertussis toxin per human dose of pertussis toxoid. The dermal temperature-based assay was found to be a valid method to be applied in routine quality control of vaccines.     

Immunosuppression-induced defective lymphocyte proliferation to murine cytomegalovirus is prolonged following the cessation of immunosuppression

Defective murine cytomegalovirus (MCMV) antigen-specific proliferation, induced by treatment of MCMV-infected mice with either antilymphocyte globulin (ALG), prednisolone, or both agents, was eventually restored following the cessation of immunosuppression. At 100 and 278 days following the end of immunosuppressive therapy splenic lymphocytes from infected and subsequently immunosuppressed mice responded significantly in vitro to soluble MCMV antigen after having lost this response immediately upon initiation of immunosuppression. Circulating specific antibodies and mitogen-induced blast transformation were comparable between infected mice and infected mice that also were immunosuppressed. At 278 days following the cessation of immunosuppression splenocytes from infected mice that had been treated with ALG yielded greatly increased background proliferation. Nylon-wool adherence was used to obtain enriched populations of T cells, and B cells and monocytes from MCMV-infected mice. While T cells alone did not respond in vitro to MCMV antigen, recombining B cells and monocytes with the T cells reconstituted in vitro proliferation. Defective lymphocyte proliferation to MCMV for an extended period of time following the end of immunosuppressive therapy indicated a prolonged inability to respond to an active MCMV infection. Identification of the cellular basis for the proliferation defect might lead to the development of effective immunotherapy.     

Evaluation of immunomodulatory and anti-inflammatory effects and phytochemical screening of Alternanthera tenella Colla (Amaranthaceae) aqueous extracts

Alternanthera tenella Colla extracts are used in Brazilian traditional folk medicine to treat a variety of infectious diseases as well as inflammation and fever. In this work, the immunomodulatory, anti-inflammatory and potential toxic effects of cold (CAE) and hot (HAE) aqueous extracts of A. tenella were investigated in vivo. In addition, we analyzed the phytochemical properties of both extracts. BALB/c mice were immunized in vivo with sheep red blood cells and concomitantly inoculated intraperitoneally (i.p.) with each extract (50, 100 or 200 mg/kg). Specific antibody-producing cells were enumerated using plaque-forming cell assays (PFC) and anti-SRBC IgG and IgM serum levels were measured via enzyme-linked immunosorbent assay. Body and lymphoid organ weights were determined after treatments in order to evaluate toxic effects. Carrageenan-induced paw edema was employed to investigate anti-inflammatory activity in mice inoculated i.p. with CAE or HAE (200 or 400 mg/kg). Phytochemical screening was performed using spectrometric and chromatographic approaches and revealed that CAE possessed higher tannin and flavonoid levels than HAE. PFC numbers were increased after treatment with CAE (100 mg/kg) four days after immunization, as were the serum antibody titers after four and seven days, suggesting immunostimulatory activity through modulation of B lymphocyte functions. Body and organ weights did not show major changes, suggesting that extracts administered to mice did not induce significant toxicity. Both extracts had significant anti-inflammatory activity in the paw edema assay. These results suggested that aqueous extracts from A. tenella contained several chemical compounds that possess positive and/or negative modulator effects on the immune system, which appeared to correlate with tannin and flavonoid levels in those extracts. In summary, these studies provide important insight into the biological activities of A. tenella.     

Endotoxin-induced hypersensitivity to histamine in mice. I. Contrasting effects of bacterial lipopolysaccharides and the classical histamine-sensitizing factor of Bordetella pertussis

Pieroni, Robert E. (Massachusetts Department of Public Health, Boston), Edward J. Broderick, and Leo Levine. Endotoxin-induced hypersensitivity to histamine in mice. I. Contrasting effects of bacterial lipopolysaccharides and the classical histamine-sensitizing factor of Bordetella pertussis. J. Bacteriol. 91:2169-2174. 1966.-The capacity of typhoid and possibly of pertussis endotoxins to induce histamine-shock susceptibility in some of the mice that survive graded doses of these endotoxins was confirmed. It was demonstrated, however, that pertussis endotoxin cannot be the main source of the typical histamine sensitization of pertussis vaccine. The following points are made. (i) With typhoid and pertussis endotoxins as inducers of histamine shock, no systematic relation between deaths and induction dose could be found, and 100% mortality could not be achieved. In contrast, with pertussis protective fraction as inducer, there was clear dose-response regression, with 100% mortality possible. (ii) The major part of the histamine-sensitizing activity of pertussis vaccine or its extracts was destroyed by trypsinization or by heating for 30 min at 100 C. These processes do not affect the histamine-sensitizing activity of the endotoxins. The implication for purified pertussis vaccine with high histamine-sensitization capacity is that endotoxin need not necessarily be present. The significance and possible mechanisms of action of endotoxin-induced histamine sensitivity are briefly discussed.     

Renal deposits of lipoprotein-immunoglobulin complexes in Plasmodium chabaudi-infected mice

Lipoprotein metabolism is altered and immunoglobulin-lipoprotein complexes (Ig-Lp) are formed during malaria infection (1-5). Ig-Lp were detected in the sera of Plasmodium chabaudi-infected mice 9 days post-infection (1 or 2 days after parasitemia had peaked at about 50%) and reached a maximum on day 13 (when the parasitemia had decreased to less than 1%). Renal glomerular deposits of IgM were first detected at day 3 and were heavy from day 9 to day 29; deposits of IgG and low density lipoprotein (LDL) were present from days 9 to 62, and were more dense from days 22 to 29; deposits of C3 were observed from day 13 to day 29. Apoprotein B component was found in heparin eluates of kidneys on day 10, 14, and 29. Fractionated Ig-Lp, as well as whole sera from day-13 infected mice, were injected into uninfected mice that developed LDL glomerular deposits only when pre-treated with histamine. LDL glomerular deposits were also observed after i.v. injection of day-29 sera (containing free anti-lipoprotein antibody) into day-7 infected mice, but not when a mixture of day-29 and day-7 sera was injected into normal recipient mice. LDL glomerular deposits, however, were observed when recipient mice were treated with the Plasmodium-derived Insoluble Material (PDIM) 3 days before the injection of the day-29-day-7 sera mixture or day-13 serum. Two hours after the i.v. injection of 125I-Ig-Lp, the radioactivity of the kidneys was higher in histamine-treated, PDIM-treated, and P. chabaudi-infected mice than in controls. The clearance of 125I-Ig-Lp was higher in infected and in PDIM-treated mice than in controls. We suggest that the glomerular deposit of Ig-Lp that occurs during P. chabaudi infection requires an enhancing factor such as PDIM that is released during infection.     

Role of pertussigen (pertussis toxin) on the mouse protective activity of vaccines made from Bordetella species

Pertussigen [pertussis toxin (Ptx)] from Bordetella pertussis, when detoxified, induces protection in mice to intracerebral challenge (ic) with virulent B. pertussis. In its native form, minute nonprotective doses promote the development of immunity induced by other antigens of B. pertussis. As little as 4 ng of Ptx, given with a nonprotective dose of 8 X 10(7) killed cells of the phase III Sakairi strain, promoted detectable protection to ic challenge. Native Ptx in doses of 0.4 to 400 ng did not protect mice, and vaccines made from strains not producing Ptx induced only weak protection. The marked enhancing action of Ptx was also observed with 5 micrograms of purified filamentous hemagglutinin and with vaccines made from other species of the Bordetella genus, such as B. parapertussis and B. bronchiseptica, but it was not observed with B. pertussis endotoxin. In addition, Ptx was still effective when given as late as 7 days after the vaccine. Antibodies to surface antigens of the challenge strain were demonstrated in sera of mice immunized with vaccines prepared with the different Bordetella species tested, but antibodies to Ptx were detected only in the sera of mice immunized with the wild-type B. pertussis strains. Glutaraldehyde detoxified Ptx does not have this action. Pretreatment of normal mice with Ptx, also enhanced the protective action of a mouse antiserum to a wild-type strain of B. pertussis. These observations show that antigens other than Ptx are responsible for the protection, and that Ptx acts non-specifically to enhance the mouse protective action of those antigens.     

Histamine-sensitizing Factor, Mouse-protective Antigens, and Other Antigens of Some Members of the Genus Bordetella

The three species of the genus Bordetella-B. pertussis, B. parapertussis, and B. bronchiseptica-have many antigens in common. Studies on representative strains of these species have shown that there are only a few specific antigens in each species. Whole-cell vaccines and extracts from B. pertussis contained specific mouse-protective antigen and a histamine-sensitizing factor. In addition, whole-cell vaccines and some saline extracts protected mice against intracranial challenge with B. bronchiseptica. Cells and a saline extract of B. parapertussis also protected against B. bronchiseptica but not against B. pertussis. Whole cells of B. bronchiseptica protected against B. bronchiseptica, but only one of three saline extracts protected against this challenge. Neither whole cells nor saline extracts from B. bronchiseptica protected against B. pertussis. The antigen in B. pertussis responsible for cross-protection against B. bronchiseptica was less resistant to heat than the protective antigen in B. bronchiseptica. Since histamine-sensitizing factor was not detected in B. bronchiseptica or B. parapertussis cells or extracts, this factor is not required to protect mice against B. bronchiseptica challenge. Whether B. pertussis vaccines protected against B. bronchiseptica by a nonspecific mechanism was not established, but it is clear that the specific antigen responsible for protection against B. pertussis was found only in B. pertussis and not in B. bronchiseptica or B. parapertussis.     

Effect of Bordetella pertussis vaccine on the course of lymphocytic choriomeningitis (LCM) virus infection in suckling mice pretreated with dianhydrodulcitol (DAD)

In earlier experiments Bordetella pertussis vaccine was found to enhance and dianhydrodulcitol (DAD) to weaken cellular immune response to intracerebral LCM virus infection in suckling mice. B. pertussis vaccine proved to inhibit the restrictive effect of DAD produced on the immune response in mice when 2-to-4-days-old animals were pretreated with DAD and subsequently, at the age of 16 to 18 days of life, treated with B. pertussis vaccine then infected with LCM virus. Consequently, B. pertussis vaccine enhanced the immune response previously affected by DAD.     

Effects of concanavalin A on the migration of radioactively labeled lymphoid cells

The effects of the jackbean globulin Concanaalin A (Con A) on the distribution of radioactive ⁵¹Cr-labeled lymph node cells was studied in CBA mice. Lymph node cells treated in vitro with Con A in subagglutinating noncytotoxic doses were unable to “home” to the lymph nodes of syngeneic recipients after intraenous injection. The effect was almost immediate and seemed unrelated to mitogenesis. The inhibitory effect of Con A on lymphocyte migration could be partially reersed by alpha-methyl mannoside; the degree of migratory impairment was related to the amount of Con A bound to the lymphocyte surface at the time of transfer. The membrane site at which Con A binds to the lymphocytes is similar to that which is bound by heterologous antilymphocyte serum but is probably distinct from the theta antigenic site. These data lend support to the hypothesis that surface lymphocyte carbohydrate determinants are involved in the specific lymphocyte “homing” receptor.