Genomic alterations accompanying tumour evolution in colorectal cancer: tracking the differences between primary tumours and synchronous liver metastases by whole-exome sequencing.

BACKGROUND: Colorectal cancer (CRC) patients with metastatic disease can become cured if neoadjuvant treatment can enable a resection. The search for predictive biomarkers is often performed on primary tumours tissue. In order to assess the effectiveness of tailored treatment in regard to the primary tumour the differences in the genomic profile needs to be clarified. METHODS: Fresh-frozen tissue from primary tumours, synchronous liver metastases and adjacent normal liver was collected from 21 patients and analysed by whole-exome sequencing on the Illumina HiSeq 2500 platform. Gene variants designated as 'damaging' or 'potentially damaging' by Ingenuity software were used for the subsequent comparative analysis. BAM files were used as the input for the analysis of CNAs using NEXUS software. RESULTS: Shared mutations between the primary tumours and the synchronous liver metastases varied from 50 to 96%. Mutations in APC, KRAS, NRAS, TP53 or BRAF were concordant between the primary tumours and the metastases. Among the private mutations were well-known driver genes such as PIK3CA and SMAD4. The number of mutations was significantly higher in patients with right- compared to left-sided tumours (102 vs. 66, p = 0.004). Furthermore, right- compared to left-sided tumours had a significantly higher frequency of private mutations (p = 0.023). Similarly, CNAs differed between the primary tumours and the metastases. The difference was mostly comprised of numerical and segmental aberrations. However, novel CNAs were rarely observed in specific CRC-relevant genes. CONCLUSION: The examined primary colorectal tumours and synchronous liver metastases had multiple private mutations, indicating a high degree of inter-tumour heterogeneity in the individual patient. Moreover, the acquirement of novel CNAs from primary tumours to metastases substantiates the need for genomic profiling of metastases in order to tailor metastatic CRC therapies. As for the mutational status of the KRAS, NRAS and BRAF genes, no discordance was observed between the primary tumours and the metastases.

Value of endometrial thickness measurement for diagnosing focal intrauterine pathology in women without abnormal uterine bleeding.

OBJECTIVE: To assess the diagnostic value of transvaginal sonographic (TVS) measurement of endometrial thickness for diagnosing focal intrauterine pathology in women without abnormal uterine bleeding (AUB). METHODS: A random selection from the Danish Civil Registration System was made: 1660 women aged 20-74 years were invited to participate and 686 women were eligible and accepted inclusion (429 pre- and 257 postmenopausal). The women underwent TVS measurement of endometrial thickness and saline contrast sonohysterography (SCSH). Hysteroscopic resection with histopathology (gold standard) was performed when focal intrauterine pathology was suspected at SCSH. We excluded women with AUB (n = 237), failure of SCSH (n = 50), a scan that was not in the follicular phase (n = 11), hysteroscopy contraindicated (n = 2), and users of sequential hormone therapy (n = 9) or selective estrogen receptor modulators (n = 2). Thus, 375 women without AUB were included (217 pre- and 158 postmenopausal). Receiver-operating characteristics (ROC) curves for endometrial thickness and focal lesion were analyzed. RESULTS: Focal intrauterine pathology was confirmed in 41 women (35 with polyps, five with submucosal myomas and one with polypoidal growing cancer). For premenopausal women, the area under the ROC curve (AUC) was 0.79 (95% CI, 0.68-0.89) and for postmenopausal women it was 0.84 (95% CI, 0.76-0.92). For premenopausal women, the best negative likelihood ratio (LR- = 0.11) was obtained at an endometrial thickness of 5.2 mm, with a negative predictive value (NPV) of 99% and a positive predictive value (PPV) of 10%. For postmenopausal women the best LR- (0.08) was obtained at an endometrial thickness of 2.8 mm, with a NPV of 99% and a PPV of 26%. CONCLUSIONS: In women without AUB, TVS measurement of endometrial thickness is a poor diagnostic test, but is apparently efficacious in excluding focal intrauterine pathology, especially in postmenopausal women. The 4-5-mm threshold conventionally used to exclude endometrial malignancy in women with postmenopausal bleeding is not transferable to women without AUB for excluding focal intrauterine pathology.

Prevalence of endometrial polyps and abnormal uterine bleeding in a Danish population aged 20-74 years.

OBJECTIVE: To estimate the prevalence of endometrial polyps and to investigate associated abnormal uterine bleeding in a Danish population aged 20-74 years. METHODS: This was a study of a random selection of women from the Danish Civil Registration System: 1660 women were invited of whom 686 were included (429 pre- and 257 postmenopausal). AUB was assessed by a validated questionnaire. The women underwent transvaginal sonography (TVS) and saline contrast sonohysterography (SCSH). Hysteroscopic resection was performed in cases with suspected focal intrauterine pathology. Full evaluation was performed in 619 women (two failures of TVS and 60 failures of SCSH, in two women SCSH was contraindicated (endometrial cancer), in two women hysteroscopy was contraindicated, and one polyp was lost before histology). World Health Organization histopathological criteria were used for diagnosing true endometrial polyps. RESULTS: On final diagnosis there were 48 women with polyps, eight with submucosal myomas, four with other benign findings and one with polypoidal growing endometrial cancer. Complex hyperplasia without atypia was diagnosed in two women with polyps. The prevalence of endometrial polyps was 7.8% (48/619; 95% CI, 5.6-9.9%). The prevalence was influenced significantly by age (P<0.005); in women below the age of 30 years, the prevalence was 0.9%. Polyps were diagnosed in 5.8% of pre- and 11.8% of postmenopausal women (P<0.01). Thirty-nine (82%) of the women who had histopathologically verified polyps were asymptomatic. In asymptomatic premenopausal women the prevalence of polyps was 7.6%, while it was 13% in asymptomatic postmenopausal women. AUB, in particular intermenstrual bleeding, was more frequent among women without polyps (38%). By ultrasound examination, submucosal myomas were diagnosed in 4.2% (26/622; 95% CI, 2.6-5.8%) and intramural myomas in 11.1% (76/684; 95% CI, 8.8-13.5%) of women. Polyps were diagnosed in 2% of oral-contraceptive and 25% of hormone-therapy users. CONCLUSIONS: The overall prevalence of endometrial polyps was 7.8% and the prevalence increased with age. Polyps were rare (0.9%) in women below the age of 30 years. Surprisingly, AUB was less frequent among women with polyps than among those without polyps.

The effect of formaldehyde, hydrogen peroxide and genetic detoxification of pertussis toxin on epitope recognition by murine monoclonal antibodies.

The effect of detoxification of pertussis toxin (PT) for vaccine usage by either genetic manipulation, hydrogen peroxide or formaldehyde treatment on epitope recognition by a large collection of murine monoclonal pertussis toxin antibodies (PT MAbs) was assessed in a solid-phase and a soluble phase enzyme-linked immunosorbent assay (ELISA). The MAb binding patterns were found to be different in the two assays as the immobilization step appeared to cause conformational alterations in the native as well as the toxoided forms of PT. According to the solid-phase ELISA, genetic, hydrogen peroxide and 0.35% formaldehyde detoxification of PT resulted in reduced epitope binding in 2.9, 31.4 and 78.1% of the MAbs, respectively. In the soluble-phase ELISA, in which the MAbs were allowed to react with the toxoids or native toxin in solution, the percentages of MAbs showing decreased binding activity were 9.1, 50.0 and 71.4%, respectively. Stabilization of native PT and the genetically inactivated PT by 0.035% formaldehyde reduced the epitope binding activity in 50.0 and 8.7% of the MAbs, respectively. Increased antibody binding in the soluble-phase ELISA was observed in some of the toxoids: this ranged from 0% in the 0.35% formaldehyde-treated PT to 13.6% in the hydrogen peroxide-treated and 27.3% in the genetically detoxified PT. Regarding the effects of detoxification on epitopes recognized by PT-neutralizing MAbs in the soluble-phase ELISA, we found that treatment of PT with either 0.035%, 0.35% formaldehyde or hydrogen peroxide induced impairment of epitope binding in 72.7, 81.8 and 45.5% of the MAbs, respectively. In the genetically inactivated PT, the epitopes recognized by the neutralizing MAbs either appeared to remain intact or to show increased MAb binding activity. The epitope-binding patterns of several PT MAbs with mouse-protective properties varied considerably and were shown to be dependent on the detoxification procedure employed. The relevance of epitope alterations on PT as a vaccine component is discussed. The results of the present study may have important implications for future quality assessment of PT for use in acellular pertussis vaccines.

Characterisation of ovine Bordetella parapertussis isolates by analysis of specific endotoxin (lipopolysaccharide) epitopes, filamentous haemagglutinin production, cellular fatty acid composition and antibiotic sensitivity.

Isolates of Bordetella parapertussis, recovered from sheep or man, were characterised by reaction with specific anti-Bordetella lipopolysaccharide monoclonal antibodies, production of filamentous haemagglutinin, fatty acid patterns, and antibiotic sensitivity. Generally, the isolates lay within one of four groups, with separation of the ovine isolates into two groups. Reactions with specific monoclonal antibodies against lipopolysaccharide separated the ovine isolates into these two groupings. Analysis of the cellular fatty acid compositions by cluster analysis differentiated between the human and the ovine strains and also showed variation within the ovine isolates. When the production of filamentous haemagglutinin was analysed in an ELISA system, a similar pattern emerged. Varying concentrations of filamentous haemagglutinin (11-429 ng (mg total protein)-1) were extracted from the human isolates and the one group of ovine isolates with no significant protein detected in the other ovine group. These studies demonstrate variation between and within B. parapertussis isolates recovered from two mammalian sources.

Atherosclerosis in Watanabe heritable hyperlipidaemic rabbits. Evaluation by macroscopic, microscopic and biochemical methods and comparison of atherosclerosis variables.

The spontaneous development of atherosclerotic disease in 38 homozygous and 34 heterozygous Watanabe heritable hyperlipidaemic rabbits was evaluated by qualitative and quantitative light microscopy in aorta, coronary, pulmonary and renal arteries, by naked eye and macroscopic morphometric estimation of aortic atherosclerosis extent and by biochemical analysis of aortic cholesterol content. No noteworthy atherosclerosis was demonstrated within 19 months in heterozygous rabbits. In homozygous rabbits, atherosclerotic lesions were seen from the age of 4 months and progressed with age. All 19-month-old rabbits had severe atherosclerotic disease. As much as 64% of the variation in atherosclerosis extent/severity could be explained by serum cholesterol and age. A highly significant correlation between the various methods for quantitation of atherosclerosis extent and/or severity was demonstrated, suggesting that quantitative microscopy, macroscopic morphometry and determination of aortic cholesterol content may be equally valid as a measure of atherosclerosis in WHHL rabbits and are therefore interchangeable.

Reversible opening of the blood-brain barrier by anti-bacterial antibodies.

The leukocyte adhesion molecule CR3 (CD11b/CD18, Mac-1) promotes leukocyte transmigration into tissues by engaging an unknown cognate ligand on the surface of vascular endothelial cells. Filamentous hemagglutinin (FHA), an adhesin of the bacterium Bordetella pertussis, binds to CR3. We hypothesized that FHA mimics the native ligand for the CR3 integrin on endothelial cells and predicted that anti-FHA antibodies should bind to endothelial cells, interfere with leukocyte recruitment, and induce endothelial permeability. Anti-FHA monoclonal antibodies bound to cerebral microvessels in sections from human brain and upon intravenous injection into rabbits. Antibody binding correlated with the ability to recognize two polypeptides in extracts of human cerebral vessels that were also bound by CD18. In vivo, antibody binding not only interfered with transmigration of leukocytes into cerebrospinal fluid but also induced a dose-dependent reversible increase in blood-brain barrier permeability sufficient to improve delivery of intravenously administered therapeutic agents to brain parenchyma.

Identification of B-cell epitopes on the S4 subunit of pertussis toxin.

The main purpose of the present study was to identify B-cell epitopes on the S4 subunit of pertussis toxin (PT) by the synthetic peptide approach. Two strategies were followed: (i) screening of two series of overlapping peptides (12- and 25-residue peptides) covering the entire S4 sequence by a panel of murine monoclonal anti-PT antibodies and various polyclonal anti-PT antisera in an enzyme-linked immunosorbent assay (ELISA), and (ii) analysis of the S4 amino acid sequence by a predictive algorithm followed by synthesis and immunization of mice with the predicted peptides coupled to diphtheria toxoid. The anti-peptide conjugate antisera were tested in an ELISA for cross-reactivity with native PT, B oligomer, and S4. Screening of the free peptides in an ELISA by the PT antisera indicated the presence of six B-cell epitope-containing domains covered by residues 18 to 32, 33 to 46, 39 to 52, 51 to 65, 71 to 84, and 91 to 106. None of the peptides, however, were recognized by the monoclonal anti-PT antibodies in an ELISA. Immunization with six computer-predicted peptides (B1 to B6) and three potential T-cell epitopes (T1 to T3) gave rise to very high antibody responses towards the homologous conjugates. With the exception of the anti-T1/diphtheria toxoid antisera, all anti-peptide conjugate antisera cross-reacted with PT in an ELISA at different levels. None of these anti-peptide conjugate antisera, however, showed any PT-neutralizing effect as measured by the Chinese hamster ovary cell assay and the leukocytosis-promoting activity test. The results of the present study suggest that discontinuous epitopes are predominant in the S4 subunit of native PT.

Identification of murine T-cell epitopes on the S4 subunit of pertussis toxin.

The aim of the present study was to identify murine T-cell epitopes on pertussis toxin subunit S4. Six mouse strains with five different haplotypes at the H-2 locus were immunized with the pertussis toxin B oligomer. Lymph node lymphocytes were isolated and stimulated in an in vitro proliferation assay with pertussis toxin components and 11 overlapping synthetic peptides synthesized on the basis of the primary sequence of S4. In vitro proliferative responses to the synthetic peptides revealed the presence of four distinct murine T-cell epitopes on subunit S4. The recognition of the peptides was major histocompatibility complex restricted. Immunizing four of the six mouse strains with the synthetic peptides showed that the peptides which were demonstrated to contain T-cell epitopes following immunization with the B oligomer were able to induce proliferative responses to detoxified pertussis toxin and pertussis toxin components containing subunit S4. One of the identified murine T-cell epitopes corresponded to one of the major human T-cell epitopes previously identified on subunit S4. It is hoped that this murine model system will facilitate the development of a synthetic immunogen mimicking the protective properties of pertussis toxin.

Quantification of pertussis toxin, filamentous haemagglutinin, 69 kDa outer membrane protein, agglutinogens 2 and 3 and lipopolysaccharide in the Danish whole-cell pertussis vaccine.

The amounts of pertussis toxin (PT), filamentous haemagglutinin (FHA), 69 kDa outer membrane protein (69 kDa OMP) and agglutinogens (AGG) 2 and 3 in extracts from the Danish whole-cell pertussis vaccine were studied in quantitative capture ELISA. With the exception of PT, the most effective extraction of these antigens was by heating the bacteria at 60 degrees C for 30 min in 2 M urea followed by sonication for 45 s. Extraction by 1 M sodium chloride prior to sonication resulted in higher levels of antigenic and biologically active PT. On average, a single human dose of pertussis vaccine (approximately 16 opacity units) was found to contain 5520 ng FHA, 63 ng PT, 1061 ng 69 kDa OMP, 397 ng AGG 2, 534 ng AGG 3 and 4840 ng lipopolysaccharide (LPS). The antigen content of one dose of the Danish pertussis vaccine appears to be low compared with the amounts found in the acellular vaccines currently in use. These findings may have important implications for the evaluation of the protective substances and the immunogenicity of whole-cell as opposed to acellular pertussis vaccines.

Proliferative responses to purified and fractionated Bordetella pertussis antigens in mice immunized with whole-cell pertussis vaccine.

The specificity of the cell-mediated immune response to Bordetella pertussis following immunization of C57B1 mice with a whole-cell pertussis vaccine was assessed in a proliferation assay. A proliferative response of lymph node lymphocytes to the filamentous haemagglutinin, the 69 kDa outer membrane protein and the agglutinogens 2 and 3 was demonstrated. The proliferative cells were T cells of the CD4+ phenotype. In addition, several as yet uncharacterized antigens expressed by B. pertussis were shown to induce a proliferative response, also mediated by T cells of the CD4+ phenotype. Although a range of different immunization schedules and preparations of pertussis toxin were used, no specific proliferative responses to pertussis toxin, which is regarded as a protective antigen of major importance from B. pertussis, were found.

Immunosuppressive effects induced by the polysaccharide moiety of some bacterial lipopolysaccharides.

The immunomodulatory properties of several lipopolysaccharides (LPS) derived from clinical isolates of Pseudomonas aeruginosa, Branhamella catarrhalis, and Bordetella pertussis were evaluated for their capacity to influence the magnitude of the antibody response to type III pneumococcal polysaccharide (SSS-III), which is known to be regulated by suppressor and amplifier T cells (Ts and Ta, respectively). The administration of LPS, two days after immunization resulted in a significant increase in the antibody response. Such enhancement may be due mainly to the ability of the lipid A moiety of LPS to abolish the negative effects of activated Ts, thereby enabling Ta function to be more fully expressed; however, B cell mitogenicity of the LPS molecule also may be involved. By contrast, treatment with LPS at the time of immunization with SSS-III induces significant suppression of the SSS-III-specific antibody response; such suppression is not induced by LPS or lipid A derived from Escherichia coli and Salmonella minnesota, and is independent of the capacity of LPS to activate B cells polyclonally, an activity generally attributed to the lipid A fraction of LPS. Studies conducted with the LPS of P. aeruginosa indicated that the suppression induced is T cell dependent and mediated by the polysaccharide (PS) fraction of LPS; it appears to be due-at least in part-to the capacity of PS to expand or increase the size of the precursor pool of Ts, activated in response to SSS-III. The significance of these findings to the pathogenesis of certain gram-negative infections is discussed.

Proliferative responses and gamma interferon and tumor necrosis factor production by lymphocytes isolated from tracheobroncheal lymph nodes and spleen of mice aerosol infected with Bordetella pertussis.

A group of mice was aerosol infected with live, virulent Bordetella pertussis bacteria. During a period of 7 weeks following the infection, with intervals of 1 week, lymphocytes were isolated from the tracheobroncheal lymph nodes (TBL) and the spleens (SPL) of the infected mice. The in vitro proliferative responses as well as the gamma interferon and tumor necrosis factor production levels of the isolated lymphocytes in response to stimulation with whole killed B. pertussis bacteria were measured as parameters for cell-mediated immunity (CMI). The course of the infection was monitored by counting of CFU in the lungs of the mice. Moreover, antibody responses in serum against a range of B. pertussis antigens were assessed. The results showed that a vigorous proliferative response of the TBL and SPL to stimulation with whole killed B. pertussis bacteria was induced by the infection. The proliferative response of the TBL was significantly higher than the response of the SPL. The proliferative responses were maximal 3 to 4 weeks after the infection and were paralleled by in vitro gamma interferon and tumor necrosis factor production upon specific stimulation. The development of the CMI was observed simultaneously with the clearance of the infection from the lungs. Antibody responses became measurable in the sera only after the infection was cleared. A specific CMI against pertussis toxin, the filamentous hemagglutinin, the 69-kDa outer membrane protein, and the agglutinogens 2 and 3, antigens which are under consideration for inclusion in future acellular pertussis vaccines, was successfully demonstrated in mice 3 weeks after the infection.

The influence of dietary olive oil and margarine on aortic cholesterol accumulation in cholesterol-fed rabbits maintained at similar plasma cholesterol level.

The present study compares the atherogenicity of a standard diet and diets with 10% olive oil or 10% margarine added, in rabbits maintained at a mean plasma cholesterol level of about 20 mM for 13 weeks. Each group consisted of 15 animals. The distribution of cholesterol in plasma between VLDL, IDL, LDL and HDL was similar in the 3 groups. The thoracic aortic cholesterol accumulation was 16.6 +/- 1.6, 11.4 +/- 1.0 (P < 0.05) and 12.6 +/- 1.7 (P > 0.05) nmol/mg wet weight for the group receiving standard diet, diet with 10% olive oil added and diet with 10% margarine added, respectively. There was no significant difference between groups in the occurrence of the atherosclerotic changes in the proximal and distal parts of coronary arteries, abdominal aorta and renal arteries. The occurrence of atherosclerotic changes in the pulmonary arteries was equal in the groups receiving standard diet and diet with 10% margarine added while it was significantly lower (P < 0.05) in the group receiving diet with 10% olive oil added. The atherosclerotic changes at the aortic orifice of coronary arteries were quanticated morphometrically and were most severe in the group on the standard diet. The results indicate a comparable atherogenic effect of 10% olive oil or margarine addition to standard diet on development of atherosclerosis in rabbits maintained at a similar plasma cholesterol level. The study also suggests that supplementation of olive oil or margarine to standard rabbit diet leads to lower cholesterol accumulation in the thoracic aorta compared with standard diet, an effect not modulated by changes in plasma cholesterol concentrations.

Identification of human T-cell epitopes on the S4 subunit of pertussis toxin.

Ten adult humans were vaccinated with the Japanese acellular pertussis vaccine JNIH-3, containing detoxified pertussis toxin (PT), formaldehyde, and filamentous hemagglutinin. The vaccination induced a specific antibody response to PT and filamentous hemagglutinin, and a Western blot (immunoblot) analysis of the antibody response to PT revealed antibodies to PT subunits S1, S2, S3, S4 and S5. The response of peripheral lymphocytes to PT was assessed in an in vitro proliferation assay. A proliferative response to detoxified PT and PT dimers S2-S4 and S3-S4 was found, and it was further demonstrated that the proliferative response to detoxified PT and dimer S2-S4 was mediated by T cells of the CD4+ phenotype. The specificity of the proliferative response to subunit S4 was analyzed with a range of synthetic peptides synthesized on the basis of the primary sequence of subunit S4. The proliferative response to the peptides revealed two major and one minor T-cell epitope located in the NH2-terminal end of subunit S4.

Induction of polyclonal antibodies to the S1 subunit of pertussis toxin by synthetic peptides coupled to PPD: effect of conjugation method, adjuvant, priming and animal species.

Two peptides, designated L and K, covering a sequence near the NH-terminal end of the S1 subunit of pertussis toxin (PT) were conjugated to the PPD (purified protein derivative) of M. tuberculosis by either glutaraldehyde (GLUT) or succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and injected into groups of mice and guinea pigs. Initially, the effect of priming the animals with BCG vaccine and the use of aluminium hydroxide as adjuvant for the anti-peptide antibody response was studied. The group of BCG-primed mice immunized with adsorbed peptide conjugates showed the highest anti-peptide conjugate antibody response. Based on this finding, groups of BCG-primed mice were immunized four times with either adsorbed peptide L-GLUT, peptide L-SMCC/SPDP or peptide K-SMCC/SPDP conjugates and the fine peptide specificity as well as the PT and S1 cross-reactivity was investigated in ELISA. Mice immunized with peptide L-GLUT showed a significant antibody response to the homologous conjugate, only, whereas the group injected with the peptide L-SMCC/SPDP conjugate gave a significant response to both peptide K and L conjugated by the SMCC-SPDP method. Likewise, mice immunized with the peptide K-SMCC/SPDP conjugate reacted with the homologous and peptide L-SMCC/SPDP conjugate, although only the response to the former conjugate was significantly greater than the response to PPD. All groups showed a strong anti-PPD response. The anti-PT/S1 cross-reactivity of the antisera varied considerably within each group but was found to be highest in the peptide L-GLUT-immunized animals. The results of the present study not only stress the importance of BCG priming and use of aluminium hydroxide adjuvants for the immunogenicity of the peptides in question but also point to the specificity of the conjugation methods employed as low cross-reactivity between the anti-peptide L-GLUT and L-SMCC/SPDP antisera was noted. Moreover, it appeared that the choice of conjugation method may have an effect on the ability of the peptide conjugates to induce an antibody response cross-reacting with the native protein.

The cell mediated and humoral immune response to vaccination with acellular and whole cell pertussis vaccine in adult humans.

The cell mediated immune response (CMI) against pertussis antigens following vaccination with the traditional Danish whole cell pertussis vaccine (WC-P) and the Japanese acellular pertussis vaccine (A-PV) JNIH-3 was studied in four adult human volunteers. Vaccination with the A-PV induced an in vitro proliferative response of peripheral blood lymphocytes to pertussis toxin (PT) subunits S2-S4, S3-S4 and S5 and the filamentous hemagglutinin (FHA), and a better serological response to native PT, detoxified PT (dPT) and FHA than the WC-PV. The induced CMI and serological response were followed over a period of 17 weeks, and were not seen to decline during this period. Further, an in vitro proliferative response to Bordetella pertussis agglutinogen 2 and 3 were demonstrated using lymphocytes from recently and not-so-recently pertussis-vaccinated adults.

Quantitation of pertussis toxin in an enzyme linked immunosorbent assay with improved specificity.

The quantitation of pertussis toxin (PT) in two sandwich ELISAs was tested for specificity. The detection of the captured PT was obtained by using either polyspecific rabbit anti Bordetella pertussis serum (RaBp-ELISA) or a monoclonal anti-PT antibody (McaPT-ELISA). No major differences in the estimation of PT in highly purified preparations were noted using either ELISA variants. In contrast, the quantitation of PT in crude extracts of B. pertussis cultures by the RaBp-ELISA was found to be over-estimated and showed greater variability when compared to the McaPT-ELISA. Comparison of the distribution of PT in the eluate fractions following partial purification by hydroxylapatite chromatography revealed that the results of the McaPT-ELISA were more specific as judged by SDS-PAGE analysis.

The effect of cyclodextrin on lipopolysaccharide production in cultures of Bordetella pertussis.

The effect of adding 500 micrograms of (2,6-0-dimethyl) beta-cyclodextrin (Me-beta-CD) per ml of Stainer-Scholte (SS) medium in two-day shaker flask cultures of Bordetella pertussis on the production of lipopolysaccharide (LPS) was investigated. The amount of LPS per 10(9) cells found in the supernatants of these cultures was either somewhat reduced or unaffected by comparison with the amounts in cultures grown in SS-medium alone. In addition, the time course of LPS release from cultures of B. pertussis strain 3843 cells during a 96-h growth period in normal and Me-beta-CD-enriched SS medium is described. By using the enriched medium bacterial growth, the production of filamentous haemagglutinin (FHA) and of pertussis toxin (Pt) and the levels of haemagglutination and lymphocytosis-promoting activity were enhanced to various degrees. Measurements made on sedimented whole and on sonicated B. pertussis cells grown in the two media showed no differences in LPS content. The reasons for the reduced/unaffected LPS production are discussed. It has been suggested that an interaction between hydrophobic cavities of the Me-beta-CD molecules and the 'lipid A' part of LPS reduces the reactivity of LPS in the Limulus Amoebocyte Lysate (LAL) assay. This possibility, however, was rejected as the reactivity of Me-beta-CD-spiked purified B. pertussis strain 3803 LPS, compared with unspiked samples, remained unchanged.