A rapid and sensitive assay for pertussis toxin based on pigment aggregation within the melanophores of an isolated fish scale

The noradrenaline-induced pigment aggregation within the melanophores of the cuckoo wrasse ( Labrus ossifagus L.) has been shown to be mediated by postsynaptic alpha₂-adrenoceptors which in turn act via an inhibitory control of adenylate cyclase. In a previous paper it was shown that pertussis toxin (PT) caused a blockade of noradrenaline-induced pigment aggregation. Based on these findings, an assay for PT has been developed. The method involves toxin titration directly on isolated fish scales from L. ossifagus , takes about 2 h to perform and needs no sophisticated equipment. The method allows detection of femtogram quantities of toxin per ml. The effect of purified PT was neutralized by antiserum. Some preliminary results indicate that it is possible to detect PT-like activity in sputum samples from patients with suspected whooping cough, whereas no PT-like activity could be detected in samples from healthy volunteers.     

Verification of components of acellular pertussis vaccines that have been distributed solely, been in routine use for the last two decades and contributed greatly to control of pertussis in Japan.

An ideal acellular pertussis vaccine is now under investigation worldwide. We have had acellular pertussis vaccines available for the last 22 years, which contributed greatly to the control of pertussis in Japan, although it has not been known whether they are one of ideal acellular pertussis vaccines or not. Moreover, the formulations of acellular pertussis vaccines that we have been using have not been widely recognized. Serum samples were taken from recipients of the T type, B type, and two-component acellular pertussis vaccine and assayed by ELISA for anti-PT, anti-FHA, and anti-69 kD OMP antibody levels and by the agglutination test. Although it was shown that T type vaccine contained four components (PT, FHA, 69 kD OMP, agglutingen), B type vaccine contained three components (PT, FHA, 69 kD OMP) and the two-component vaccine contained PT and FHA, it was concluded that PT and FHA were essential and common antigens contained in all three acellular pertussis vaccines in Japan. The national monitoring system for adverse effects of routine immunization demonstrated low reactogenicity of DTaP in Japan. This resulted in high acceptance rates of DTaP and in virtual control of pertussis.     

Humoral reaction to Bordetella pertussis antigens: pertussis toxin, filamentous hemagglutinin and lipopolysaccharide in children with clinical symptoms of whooping cough. II. Occurence and level of B. pertussis antigens in children with suspected whooping cough

The aim of this study was to determine and evaluate IgG, IgM and IgA levels to pertussis toxin (PT), filamentous hemagglutinin (FHA) and endotoxin (LPS) of B. pertussis in children with clinical symptoms of whooping cough. The serum samples obtained from 265 children (age range: 2 months-16 years) suspected of pertussis were examined by indirect haemagglutination (IH) and ELISA tests. Higher antibody level was most frequently observed in IgA class to PT, FHA and LPS in 45.3%, 35.1% and 66% of pertussis patients sera respectively. The least positive results were obtained in IgM class to PT and FHA (in 9.8% and 2.6% of children sera respectively) but in the case of LPS applied as the antigen in ELISA, higher IgM level was determined in 46.8% of pertussis patients sera. The four times increase of antibody level to LPS determined by IH was observed in 86.7% of children suspected of pertussis. Humoral response to B. pertussis infection is mainly connected with higher IgA level to PT, FHA, LPS and IgM to LPS in children with clinical symptoms of whooping cough.     

Human cellular immune responses to Bordetella pertussis infection

Abstract We have compared the responses of peripheral blood leucocytes from three groups (i) patients suffering from pertussis (whooping cough), (ii) clinical staff caring for those patients and laboratory staff working with Bordetella pertussis , and (iii) staff with no known recent contact with B. pertussis . In vitro stimulation with filamentous haemagglutinin (FHA) caused significant increases in proliferation of only the patient group's lymphocytes. In vitro stimulation with pertussis toxin (PT) caused a large increase in proliferation of lymphocytes from all three groups and in the patient group the increase in proliferation was related to the dose of PT. Interleukin 2 (IL-2) production by leucocytes from all three groups was significantly increased following challenge with FHA or PT. The increases in IL-2 production were greatest in lymphocytes from patients with pertussis. Challenge with toxoided pertussis toxin had no effect on either proliferation or IL-2 production in any of the groups.     

The effects of purified components of Bordetella pertussis in the weight gain test for the toxicity testing of pertussis vaccines

The effects of highly purified preparations of three Bordetella pertussis components--pertussis toxin (PT), lipopolysaccharide (LPS) and filamentous haemagglutinin (FHA)--were examined in the mouse weight gain test, a toxicity test for pertussis vaccine. When these components were administered alone, PT enhanced initial weight gains of the mice, LPS produced an initial weight loss and FHA had no detectable effect on the weights of the mice. However, testing the components in combinations revealed that the effect of PT and LPS together was not simply the sum of their individual effects. This combination generally produced lower weights than LPS alone, particularly in the later stages of the test.     

Human cellular immune responses to Bordetella pertussis infection

We have compared the responses of peripheral blood leucocytes from three groups (i) patients suffering from pertussis (whooping cough), (ii) clinical staff caring for those patients and laboratory staff working with Bordetella pertussis, and (iii) staff with no known recent contact with B. pertussis. In vitro stimulation with filamentous haemagglutinin (FHA) caused significant increases in proliferation of only the patient group's lymphocytes. In vitro stimulation with pertussis toxin (PT) caused a large increase in proliferation of lymphocytes from all three groups and in the patient group the increase in proliferation was related to the dose of PT. Interleukin 2 (IL-2) production by leucocytes from all three groups was significantly increased following challenge with FHA or PT. The increases in IL-2 production were greatest in lymphocytes from patients with pertussis. Challenge with toxoided pertussis toxin had no effect on either proliferation or IL-2 production in any of the groups.     

Hemagglutination activities of purified pertussis toxin and filamentous hemagglutinin against erythrocytes from various animals

The hemagglutinating (HA) activities of purified pertussis toxin (PT) and filamentous hemagglutinin (FHA) were evaluated against unfixed and glutaraldehyde-fixed erythrocytes from ox, goose, horse, monkey, sheep, chicken, and rabbit. Both PT and FHA showed HA activities against fixed and unfixed erythrocytes from all the animals studied. The HA titers of FHA were higher than those of PT. The HA activities of FHA and PT were not destroyed completely even after heating these preparations at 56 C for 30 min. A simple test for the assay of PT in culture supernatants of Bordetella pertussis on the basis of HA activity has been described.     

Apoptosis in skin pigment cells of the medaka, Oryzias latipes (Teleostei), during long-term chromatic adaptation: the role of sympathetic innervation

Many teleost fish can adapt their body color to a background color by changing the morphology and density of their skin pigment cells. Melanophore density in fish skin decreases during long-term adaptation to a white background. Although cell death, especially apoptosis, is thought to be involved in these morphological changes, there are no data clearly supporting this mechanism. Using medaka fish, Oryzias latipes, we observed that, on a white background, melanophore size was reduced first and this was followed by a decrease in melanophore density caused by gradual cell death. The process of cell death included loss of cell activity, cell fragmentation, phagocytosis of the fragments, and clearance via the epidermis. Apoptosis was assessed by the appearance of phosphatidylserine on the cell surface of melanophores that had lost motile activity, and DNA fragments involved in cell fragmentation were detected by the TUNEL (TDT-mediated dUTP-biotin nick end-labeling) assay. However, when chemically denervated fish were used, although melanophore size was reduced as expected, cell death was suppressed even on a white background. In skin tissue culture, apoptosis in melanophores was stimulated significantly by norepinephrine, but not by melanin-concentrating hormone. These results indicate that melanophore density decreases by apoptosis, and suggest that sympathetic innervation has an important role in the regulation of apoptosis in melanophores. In analogous fashion, leucophores showed a significant decrease in density with an increase of cell death on a black background. We suggest that apoptosis regulates the balance of pigment cells in the skin of medaka fish to adapt their body color to a particular background.     

A quantitative analysis for the ADP-ribosylation activity of pertussis toxin: an enzymatic-HPLC coupled assay applicable to formulated whole cell and acellular pertussis vaccine products.

The majority of the biological effects of pertussis toxin (PT) are the result of a toxin-catalyzed transfer of an adenosine diphosphate-ribose (ADP-ribose) moiety from NAD(+)to the alpha-subunits of a subset of signal-transducing guanine-nucleotide-binding proteins (G-proteins). This generally leads to an uncoupling of the modified G-protein from the corresponding receptor and the loss of effector regulation. This assay is based on the PT S1 subunit enzymatic transfer of ADP-ribose from NAD to the cysteine moiety of a fluorescent tagged synthetic peptide homologous to the 20 amino acid residue carboxyl-terminal sequence of the alpha-subunit of the G(i3)protein. The tagged peptide and the ADP-ribosylated product were characterized by HPLC/MS and MS/MS for structure confirmation. Quantitation of this characterized ADP-ribosylated fluorescently tagged peptide was by HPLC fluorescence using Standard Addition methodology. The assay was linear over a five hr incubation period at 20 degrees C at PT concentrations between 0.0625 and 4.0 microg/ml and the sensitivity of the assay could be increased several fold by increasing the incubation time to 24 h. Purified S1 subunit of PT exhibited 68.1+/-10.1% of the activity of the intact toxin on a molar basis, whereas the pertussis toxin B oligomer, the genetically engineered toxoid, (PT-9K/129G), and several of the other components of the Bordetella pertussis organism possessed little (<0.6%) or no detectable ribosylation activity. Commonly used pertussis vaccine reference materials, US PV Lot #11, BRP PV 66/303, and BRP PV 88/522, were assayed by this method against Bordetella pertussis Toxin Standard 90/518 and demonstrated to contain, respectively, 0.323+/-0.007, 0.682+/-0.045, and 0.757+/-0.006 microg PT/ml (Mean+/-SEM) or in terms of microg/vial: 3.63, 4.09 and 4.54, respectively. A survey of several multivalent pertussis vaccine products formulated with both whole cell as well as acellular components indicated that products possessed a wide range of ribosylation activities. The pertussis toxin S1 subunit catalyzed ADP- ribosylation of the FAC-Galpha(i3)C20 peptide substrate and its subsequent quantitation by HPLC was demonstrated to be a sensitive and quantitative method for measuring intrinsic pertussis toxin activity. This methodology not only has the potential to be an alternative physicochemical method to replace existing bioassay methodology, but has the added advantage of being a universal method applicable to the assay of pertussis toxin in both whole cell and acellular vaccines as well as bulk and final formulated vaccine products. Acceptance of this method by regulatory agencies and industry as a credible alternative to existing methods would, however, require validation in an international collaborative study against the widely accepted bioassay methods.     

Effect of bacterial toxins on the GTPase activity of transducin from bovine rod outer segments

The effects of choleragen- and pertussis toxin (PT)-induced ADP-ribosylation on the GTP-binding protein transducin (TD) from retinal rod outer segments (ROS) have been studied. It has been shown that both toxins cause inhibition of the TD GTPase activity. PT inhibited the GTPase by 30-40% in "native" ROS and by 70-80% in homogeneous TD. Choleragen, in contrast with PT, had no effect on the GTPase activity of homogeneous TD, but was as effective as PT in membrane preparations. The effects of both toxins on the GTPase activity of TD were found to be dependent on the chemical structure of the guanyl nucleotide present in the vehicle. The data obtained suggest that PT and choleragen differ in their specificity for the TD-guanyl nucleotide complex. The former can interact with free TD as well as with the TD-GDP complex, while the latter affects only the TD-GTP complex.     

Optimal conditions for the toxoiding of pertussis toxin with 1-ethyl-3(3-dimethylaminopropyl) carbodiimide · HCl

Abstract The optimal conditions for toxoiding a pertussis toxin (PT) preparation with 1-ethyl-3(3-dimethylaminopropyl) carbodiimide · HCl (EDAC) were determined. The prime factor affecting the toxoiding of PT was the EDAC to protein ratio. A ratio of 40–80 : 1 EDAC to protein by weight was optimal for abolishing the acute toxicity, histamine-sensitising and leucocytosis-promoting activities associated with PT, whilst maintaining the antigenicity of the vaccine antigens. An EDAC-toxoid also manifested no late histamine-sensitising activity. Duration of exposure to EDAC, temperature and pH value of the reaction were found not be be critical for toxoiding. The data indicated that the use of EDAC for toxoiding PT in a B. pertussis extract is a simple and reproducible procedure and should be considered as a method for the production of acellular pertussis vaccines.     

Growth of the fungal pathogen Candida in parotid saliva of patients with burning mouth syndrome

Subclinical Candida infection has been suggested as one of the aetiological factors in patients with burning mouth syndrome (BMS). In order to investigate the possible factors which contribute to the relatively high isolation rate of Candida in BMS, parotid saliva samples (20 in toto) from patients with this condition were collected and the growth of Candida in each sample dynamically observed using a computerized turbidometric assay system. A total of thirteen parotid saliva samples obtained from healthy individuals served as normal controls. The results showed no significant growth differential within the test and control saliva samples, when a single isolate each of Candida albicans and Candida tropicalis were cultured for 24 h, at 37 degrees C. A single isolate of Candida glabrata tended to grow better in the saliva from BMS patients than the controls. These results indicate that the composition of saliva may be a contributory factor for the high isolation rate of Candida in saliva of BMS patients.     

The effect of pertussis toxin on alpha-2-adrenoceptor-mediated pigment migration in fish melanophores

The aggregation of melanin-granules within fish pigment cells (melanophores) can be elicited either by electrical stimulation of intrinsic nerves or by the addition of adrenergic agonists. The pigment aggregation seems to be mediated by alpha-2-adrenoceptors. In this investigation we have used various agonists and antagonists (noradrenaline, (+)- and (-)-adrenaline, isoprenaline, yohimbine and prazosin) to further characterize the pigment-aggregating receptor of Labrus ossifagus. All the results obtained support the notion of alpha-2-adrenoceptor-mediated pigment aggregation. The pertussis toxin, islet-activating protein (IAP), is known to inhibit the alpha-2-adrenoceptor-mediated signal transduction in mammals. We have used IAP to investigated whether fish melanophore alpha-2-adrenoceptors are also inhibited by this toxin. We found that IAP inactivated the alpha-2-adrenoceptor-mediated pigment aggregation in a dose-dependent manner. The inhibitory IAP-effect had a remarkably short onset-time in the melanophores (maximal effect was obtained within 10 min of incubation). Interestingly, binding of an agonist (noradrenaline) to the receptors prevented IAP from exerting its inhibitory action, whereas binding of an antagonist (yohimbine) gave no protection against the IAP-inactivation. In conclusion, the pigment-aggregating receptors of melanophores of L. ossifagus are very similar to the mammalian alpha-2-adrenoceptors. It is possible to inactivate the melanophore receptor system with IAP and this inactivation has a remarkably short onset-time. Stimulation of the alpha-2-adrenoceptors prevents IAP from inactivating the receptor system.     

Bordetella pertussis infection exacerbates influenza virus infection through pertussis toxin-mediated suppression of innate immunity

Pertussis (whooping cough) is frequently complicated by concomitant infections with respiratory viruses. Here we report the effect of Bordetella pertussis infection on subsequent influenza virus (PR8) infection in mouse models and the role of pertussis toxin (PT) in this effect. BALB/c mice infected with a wild-type strain of B. pertussis (WT) and subsequently (up to 14 days later) infected with PR8 had significantly increased pulmonary viral titers, lung pathology and mortality compared to mice similarly infected with a PT-deficient mutant strain (ΔPT) and PR8. Substitution of WT infection by intranasal treatment with purified active PT was sufficient to replicate the exacerbating effects on PR8 infection in BALB/c and C57/BL6 mice, but the effects of PT were lost when toxin was administered 24 h after virus inoculation. PT had no effect on virus titers in primary cultures of murine tracheal epithelial cells (mTECs) in vitro, suggesting the toxin targets an early immune response to increase viral titers in the mouse model. However, type I interferon responses were not affected by PT. Whole genome microarray analysis of gene expression in lung tissue from PT-treated and control PR8-infected mice at 12 and 36 h post-virus inoculation revealed that PT treatment suppressed numerous genes associated with communication between innate and adaptive immune responses. In mice depleted of alveolar macrophages, increase of pulmonary viral titers by PT treatment was lost. PT also suppressed levels of IL-1β, IL-12, IFN-γ, IL-6, KC, MCP-1 and TNF-α in the airways after PR8 infection. Furthermore PT treatment inhibited early recruitment of neutrophils and NK cells to the airways. Together these findings demonstrate that infection with B. pertussis through PT activity predisposes the host to exacerbated influenza infection by countering protective innate immune responses that control virus titers.     

Development of acellular pertussis vaccines.

In 1974, the authors reported the isolation and characterization of protective antigens of Bordetella pertussis in mice. With this information, an acellular pertussis vaccine was developed, composed mainly of pertussis toxin (PT) and filamentous haemagglutinin (FHA). Substances causing side effects, especially lipopoly sacahoride (LPS) or endotoxin that cause fever, were removed, and detoxification of the PT by formaldehyde with retention of potency was achieved. In 1981, an acellular pertussis vaccine called the "Adsorbed Purified Pertussis Vaccine" was approved in Japan, in place of the whole-cell pertussis vaccine. The acellular pertussis vaccine has been widely accepted as safer and more efficacious in Japan. Since 1981, intense surveillance has shown that there are only rare adverse reactions and that pertussis has virtually been eliminated in Japan. Evaluation of active immunization with highly purified and pharmacologically inert PT and FHA and passive immunization with polyclonal and monoclonal antibodies, provide quantitative data about the vaccine-induced immunity in mice. Finally, it was discovered that the PT toxoid in the vaccine is the major and essential protective antigen. The toxoid of PT should be sufficient for protection against pertussis.     

Control of chromatophore movements in dermal chromatic units of blue damselfish--I. The melanophore

Mechanisms controlling pigment movements in the melanophore of the blue damselfish, Chrysiptera cyanea, were studied. Histological observations revealed that the melanophore had three-dimensionally developed processes to envelop overlying small iridophores, and thus participated in the construction of a simple dermal chromatophore unit. Nervous stimulation, catecholamines and melatonin brought about melanosome aggregation in the melanophore. The actions of the nervous stimulation and catecholamines were antagonized by alpha adrenolytic agents. A beta adrenergic agonist, metaproterenol, adenosine and adenine nucleotides, and alpha-MSH acted as pigment-dispersing agents. These results indicate that the melanophore of the present material is controlled quite orthodoxly by adrenergic nerves and endocrines, notwithstanding the fact that it has quite a unique morphology among fish species, and that its motile rate is remarkably high.     

The indirect hemagglutination reaction in the diagnosis of trichinosis

An indirect hemagglutination test (IHAT) for trichinosis using a Melcher's antigen was evaluated and compared with a precipitin test (PT) and a bentonite flocculation test (BFT). One hundred and forty eight serum samples from patients from the whole country confirmed or suspected to have trichinosis by clinical or epidemiological evidences were studied: 117 (79.1%) samples resulted positive by IHAT, 138 (93.2%) by PT and 65 (43.9%) by BFT. Sixty three serum samples from patients with strong clinical suspect of trichinosis presented the PT and the BFT positive and were compared with the IHAT for sensitivity study. IHAT was positive in 60 (95.2%) serum samples. In order to determine the specificity of IHAT 25 serum samples from healthy volunteers and 124 serum samples from individuals with other parasitoses, such as cysticercosis (48), hydatidosis (45) and fascioliasis (31) were studied. The specificity, using a titre > or = 1:16 as a possible diagnostic value was 96%. The use of IHAT with RP and BFT in the diagnosis of human trichinosis is discussed.     

Optimal conditions for the toxoiding of pertussis toxin with 1-ethyl-3(3-dimethylaminopropyl) carbodiimide.HCl

The optimal conditions for toxoiding a pertussis toxin (PT) preparation with 1-ethyl-3(3-dimethylaminopropyl) carbodiimide.HCl (EDAC) were determined. The prime factor affecting the toxoiding of PT was the EDAC to protein ratio. A ratio of 40-80: 1 EDAC to protein by weight was optimal for abolishing the acute toxicity, histamine-sensitising and leucocytosis-promoting activities associated with PT, whilst maintaining the antigenicity of the vaccine antigens. An EDAC-toxoid also manifested no late histamine-sensitising activity. Duration of exposure to EDAC, temperature and pH value of the reaction were found not to be critical for toxoiding. The data indicated that the use of EDAC for toxoiding PT in a B. pertussis extract is a simple and reproducible procedure and should be considered as a method for the production of acellular pertussis vaccines.     

Thromboplastin standards

The Prothrombin Time (PT) test is used for monitoring of treatment with Vitamin K-antagonists (VKA). The result of the PT test should be expressed as the International Normalized Ratio (INR). Calculation of INR is based on the availability of International Standards (IS) for thromboplastin and a calibration model. Calibration of a new PT test system is performed with the appropriate IS and fresh plasma samples of healthy (normal) volunteers and patients treated with VKA. The calibration model is based on the assumption of a linear relationship between the log(PT)'s obtained with the new PT system and the reference IS for both normal and patients' samples. Patients' samples for calibration should be selected by rejecting samples beyond the 1.5–4.5 INR range. Outliers should be rejected defined as points with a perpendicular distance greater than three residual standard deviations from the line of relationship. Selection of patients' samples and rejection of outliers result in a reduction of the between-laboratory variation of calibration. In addition to monitoring of VKA, the PT is used for management of patients with chronic liver disease. Likewise, INR_liver should be based on calibration with an IS using samples from patients with chronic liver disease.     

A threshold level of coupled G-proteins is required to transduce neutrophil responses.

Chemoattractant-induced activation of human polymorphonuclear leukocytes involves receptor coupling to guanine nucleotide binding proteins (G-proteins). Treatment of polymorphonuclear leukocytes with pertussis toxin, which ADP-ribosylates neutrophil G-proteins and uncouples G-proteins from receptors, causes a conversion of cells from responders to nonresponders rather than a gradual decrease in the ability of all cells to respond (Omann, G. M., and J. M. Harter. 1991. Cytometry 12:252; Omann, G. M., and M. M. Porasik-Lowes. 1991. J. Immunol. 146:1303). Flow-cytometric methods were used to measure N-formylpeptide-induced cytosolic Ca2+ elevation and actin polymerization over a wide range of ADP-ribosylation levels and showed that although the percentage of responding cells varied markedly, the responding cells were stimulated equivalent to controls. The conditions of pertussis toxin (PT) treatment did not interfere with non-G-protein-mediated pathways as assessed by measurement of phagocytosis, a complex process involving the cytoskeleton. We tested the explanation that the all-or-none effect may have been due to heterogeneous insertion of the catalytic subunit of PT into the cells such that responders had no ADP-ribosylation and nonresponders were completely ADP-ribosylated. Measurement of the binding of fluorescent N-formylpeptides to permeabilized cells, which allows the distinction between completely ribosylated and normal cells, showed that all cells treated with a submaximal concentration of PT had intermediate levels of receptor-coupled G-proteins. Thus, partial ADP-ribosylation had occurred in all cells and the all-or-none insertion of the catalytic subunit of PT was ruled out. Thus, there is a threshold of coupled G-proteins required to transduce responses. The ability of PT to inhibit N-formylpeptide-induced actin polymerization and cytosolic calcium elevation was compared and showed that both responses have essentially the same threshold of G-proteins required to transduce the responses. Thus, the pathways regulating actin polymerization and calcium elevation appear to be coupled with equal efficiency to the G-proteins.