In search of acceptable alternatives to the murine histamine sensitisation test (HIST): what is possible and practical?

The 'International Workshop on Alternatives to the Murine Histamine Sensitization Test for Acellular Pertussis Vaccines: In Search of Acceptable Alternatives to the Murine Histamine Sensitization Test (HIST): What is Possible and Practical?' was held on 4 and 5 March 2015 in London, United Kingdom. Participants discussed the results of the data generated from an international collaborative study (BSP114 Phase 2) sponsored by the European Directorate for the Quality of Medicines & Health Care (EDQM) to determine if a modified Chinese hamster ovary (CHO) cell-based clustering assay is a suitable alternative to replace HIST. Workshop participants agreed that protocol transferability demonstrated in the collaborative study indicates that a standardised CHO cell assay is adequate for measuring pure PTx in reference preparations. However, vaccine manufacturers would still need to demonstrate that the method is valid to detect or measure residual PTx in their specific adjuvanted products. The 2 modified CHO cell protocols included in the study (the Direct and the Indirect Methods) deserve further consideration as alternatives to HIST. Using the CHO cell assay, an in vitro alternative, for acellular pertussis (aP) vaccine batch release testing would reduce the number of animals used for aP vaccine safety testing. A strategic, stepwise adoption plan was proposed, in which the alternative test would be used for release purposes first, and then, once sufficient confidence in its suitable performance has been gained, its use would be extended to stability testing.

Relations between the occurrence of resistance to antimicrobial growth promoters among Enterococcus faecium isolated from broilers and broiler meat.

From 1995 to 2001, Enterococcus faecium isolates were collected from broiler flocks at slaughter and broiler meat products at retail outlets and were tested for susceptibility to classes of antimicrobials used for growth promotion in broilers in Denmark, namely: evernimicin, glycopeptide, macrolide and streptogramin. By February 1998, all antimicrobial growth promoters (AGPs) were withdrawn from the Danish broiler production. The present study investigates, by logistic regression analyses, the (1) changes in the occurrence of AGP resistance among E. faecium from broilers and broiler meat from the fourth quarter of 1995 to the fourth quarter of 2001 and (2) relations between the occurrence of AGP resistance among E. faecium isolates from Danish broilers and AGP resistance among E. faecium isolates from the broiler meat of Danish and unknown origin collected in the same quarter within the year. In the present study, we showed that after the AGP withdrawal, a significant decline in resistance to avilamycin, erythromycin, vancomycin and virginiamycin was observed among E. faecium from broilers and broiler meat. In addition, a decline in the occurrence of AGP resistance among E. faecium from Danish broilers was associated with a decrease in the predicted probability of isolating an AGP-resistant E. faecium isolate from a randomly selected broiler meat product. In the analyses "relations between the occurrence of AGP resistance among E. faecium isolated from broilers and broiler meat collected in the same quarter" errors in the explanatory variable were expected. Therefore, a simulation study was performed to validate the results from logistic regression analyses. The results obtained by the two methods were similar.

Antibody responses in sheep vaccinated against Staphylococcus aureus mastitis: a comparison of two experimental vaccines containing different adjuvants.

A double-blind, placebo-controlled study was conducted to compare two vaccines using different adjuvants with regard to their ability to stimulate antibody production against the alpha- and beta-toxins and the exopolysaccharide of Staphylococcus aureus. The vaccines contained identical antigens, consisting of inactivated whole bacteria of two strains of S. aureus in addition to alpha- and beta-toxoid. One vaccine contained mineral oil, while the other used a water-soluble acrylic acid polymer resin (Carbopol) as adjuvant. Saline served as the placebo. One hundred and forty ewes were vaccinated twice before lambing, by subcutaneous injection with vaccine or placebo in the region of the supramammary lymph node, and were observed and sampled over a period of 6 months. The vaccine containing mineral oil as adjuvant induced significantly greater immune responses to the alpha- and beta-toxins than did the vaccine containing Carbopol. The latter vaccine induced higher levels of antibodies to exopolysaccharide. The degree of local adverse reactions did not differ between the two groups. The results indicate differences between the oil-adjuvanted and Carbopol-adjuvanted vaccines with regard to their ability to stimulate antibody production against S. aureus protein antigens in sheep.

Imaging the surface of Staphylococcus aureus by atomic force microscopy.

The surfaces of four strains of Staphylococcus aureus, which differed in their expression of capsular polysaccharides, were examined using atomic force microscopy. The images show that it is possible to get information about surface characteristics of S. aureus using atomic force microscopy (AFM) following simple preparation. Strains Smith Diffuse (serotype 2), Reynolds (serotype 5), Wood-46 (capsule negative) and JL243 (capsule negative) were grown on medium known to promote the expression of capsular polysaccharides. The bacteria were air-dried prior to being imaged using tapping-mode AFM. Differences in the appearance of the bacterial surfaces were evident between the strains. The two capsule-negative strains exhibited a smooth regular surface, as opposed to the mucoid appearance of the two strains having polysaccharide capsules. Moreover, comparison of images of the heavily encapsulated serotype 2 strain and the serotype 5 strain indicates that a type 2 capsule can be distinguished from a type 5 microcapsule.

Staphylococcus aureus capsular polysaccharide type 5 conjugate and whole cell vaccines stimulate antibody responses in cattle.

Dairy heifers were immunized subcutaneously with one of four different vaccines which contained preparations of Staphylococcus aureus capsular polysaccharide type 5 (CP5) and a mineral oil adjuvant, or received a placebo containing saline and adjuvant. The vaccine containing a CP5-human serum albumin conjugate (CP5-HSA) and the vaccine with formaldehyde inactivated whole cells expressing CP5, both elicited strong anti-CP5 antibody responses. After two injections three weeks apart and a third injection 10 months later, the mean level and duration of the anti-CP5 antibody response was significantly higher in the whole cell group. No differences were found between the two groups with regard to the relative proportion of IgG subclasses, and the antibody responses to the polysaccharide were composed of both the IgG1 and IgG2. Vaccines containing only free CP5 or CP5 mixed with HSA produced weak and transient humoral immune responses. Only animals vaccinated with the whole cell vaccine or the conjugate vaccine showed responses to CP5 in a lymphocyte proliferation assay conducted one year after the third vaccination. This study indicates that CP5 expressed on the surface of formaldehyde inactivated whole cells, emulsified in an oil adjuvant, gives a strong and long lasting immune response in cattle. The use of conjugation technology, although effective, might not be necessary in order to achieve an immune response against S. aureus CP5 in cattle.

Dendritic cell activation and cytokine production induced by group B Neisseria meningitidis: interleukin-12 production depends on lipopolysaccharide expression in intact bacteria.

Interactions between dendritic cells (DCs) and microbial pathogens are fundamental to the generation of innate and adaptive immune responses. Upon stimulation with bacteria or bacterial components such as lipopolysaccharide (LPS), immature DCs undergo a maturation process that involves expression of costimulatory molecules, HLA molecules, and cytokines and chemokines, thus providing critical signals for lymphocyte development and differentiation. In this study, we investigated the response of in vitro-generated human DCs to a serogroup B strain of Neisseria meningitidis compared to an isogenic mutant lpxA strain totally deficient in LPS and purified LPS from the same strain. We show that the parent strain, lpxA mutant, and meningococcal LPS all induce DC maturation as measured by increased surface expression of costimulatory molecules and HLA class I and II molecules. Both the parent and lpxA strains induced production of tumor necrosis factor alpha (TNF-alpha), interleukin-1alpha (IL-1alpha), and IL-6 in DCs, although the parent was the more potent stimulus. In contrast, high-level IL-12 production was only seen with the parent strain. Compared to intact bacteria, purified LPS was a very poor inducer of IL-1alpha, IL-6, and TNF-alpha production and induced no detectable IL-12. Addition of exogenous LPS to the lpxA strain only partially restored cytokine production and did not restore IL-12 production. These data show that non-LPS components of N. meningitidis induce DC maturation, but that LPS in the context of the intact bacterium is required for high-level cytokine production, especially that of IL-12. These findings may be useful in assessing components of N. meningitidis as potential vaccine candidates.

Gram-negative bacteria induce proinflammatory cytokine production by monocytes in the absence of lipopolysaccharide (LPS).

Tumour necrosis factor-alpha (TNF-alpha), IL-1alpha and IL-6 production by human monocytes in response to a clinical strain of the Gram-negative encapsulated bacteria Neisseria meningitidis and an isogenic lpxA- strain deficient in LPS was investigated. Wild-type N. meningitidis at concentrations between 105 and 108 organisms/ml and purified LPS induced proinflammatory cytokine production. High levels of these cytokines were also produced in response to the lpxA- strain at 107 and 108 organisms/ml. The specific LPS antagonist bactericidal/permeability-increasing protein (rBPI21) inhibited cytokine production induced by LPS and wild-type bacteria at 105 organisms/ml but not at higher concentrations, and not by LPS-deficient bacteria at any concentration. These data show that proinflammatory cytokine production by monocytes in response to N. meningitidis does not require the presence of LPS. Therapeutic strategies designed to block LPS alone may not therefore be sufficient for interrupting the inflammatory response in severe meningococcal disease.

Inactivated meningococci and pertussis bacteria are immunogenic and act as mucosal adjuvants for a nasal inactivated influenza virus vaccine.

Whole killed meningococci (Nm) and pertussis bacteria (Bp) were tested for mucosal immunogenicity and as mucosal adjuvants for an inactivated influenza virus vaccine given intranasally to unanaesthetized mice. Virus was given alone, or simply mixed with one of the bacterial preparations, in four doses at weekly intervals. The virus alone induced low but significant increases of influenza-specific IgG antibodies in serum measured by ELISA, whereas IgA responses in serum and saliva were insignificant compared to non-immunized controls. With Bp or Nm admixed, serum IgG and IgA and salivary IgA responses to the influenza virus were substantially augmented (P<0.005). However, this adjuvant effect of the bacterial preparations was not significant for responses in the intestine as measured by antibodies in faeces. Antibody responses to Bp itself, but not to Nm, were inhibited by the admixture of the virus vaccine. Moreover, the pertussis preparation induced salivary antibodies which cross-reacted with Nm. Whole-cell bacteria with inherent strong mucosal immunogenicity may also possess mucosal adjuvanticity for admixed particulate antigens which are weakly immunogenic by the nasal route.

Surveillance of antimicrobial resistance in Denmark.

Recent data of the Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP) show that, in Denmark, resistance levels among Salmonella enterica are modest and that resistance in Escherichia coli isolates causing disease in anim

A mouse model utilising human transferrin to study protection against Neisseria meningitidis serogroup B induced by outer membrane vesicle vaccination.

We have previously developed a mouse model based on transient bacteraemia in normal B10.M mice to evaluate the protective efficacy of outer membrane vesicle vaccines against serogroup B meningococci. To obtain a course of infection similar to that observed in man, we have in this work modified the mouse model by administration of human holo-transferrin upon bacterial challenge. Co-challenge with holo-transferrin induced increasing bacteraemia and subsequent death in normal non-immune mice, but not in vaccinated animals. The model system is dependent on challenge with meningococci expressing the transferrin receptor which is obtained by culturing the bacteria under iron restriction. The modified model system for protection against meningococcal infection presented here makes it possible to measure outer membrane vesicle vaccine induced protection by using bacteraemia as well as survival as parameters.

Disposition of recombinant human interleukin-10 in subjects with various degrees of renal function.

The pharmacokinetics of intravenously administered recombinant human interleukin-10 (rHuIL-10) were evaluated in 18 subjects with creatinine clearances (Clcr) between 2.7 and 116.7 mL/min/1.73 m2. Serum samples for rHuIL-10 were obtained over a 48-hour period after a single 25 micrograms/kg i.v. bolus infusion. AUC, total body clearance (Clp), and steady-state volume of distribution (Vdss) were derived by compartmental methods. Analysis of serum concentrations showed statistically significant group differences for log-transformed AUC and original scale Clp (p < 0.01). The AUC and effective half-life increased, while the mean Clp of rHuIL-10 decreased as renal function declined. A linear relationship between AUC and Clcr as well as Clp and Clcr demonstrates that the disposition of rHuIL-10 is altered in subjects with renal insufficiency. No serious adverse events were noted.

Endothelial adhesion molecule expression and its inhibition by recombinant bactericidal/permeability-increasing protein are influenced by the capsulation and lipooligosaccharide structure of Neisseria meningitidis.

Vascular endothelial injury is responsible for many of the clinical manifestations of severe meningococcal disease. Binding and migration of activated host inflammatory cells is a central process in vascular damage. The expression and function of adhesion molecules regulate interactions between leukocytes and endothelial cells. Little is known about how meningococci directly influence these receptors. In this study we have explored the effect of Neisseria meningitidis on endothelial adhesion molecule expression and found this organism to be a potent inducer of the adhesion molecules CD62E, ICAM-1, and VCAM-1. Exposure of endothelium to a serogroup B strain of Neisseria meningitidis, B1940, and a range of isogenic mutants revealed that lipooligosaccharide (LOS) structure and capsulation influence the expression of adhesion molecules. Following only a brief exposure (15 min) to the bacteria, there were large differences in the capacity of the different mutants to induce vascular cell adhesion molecules, with the unencapsulated and truncated LOS strains being most potent (P < 0.05). Furthermore, the pattern of cell adhesion molecule expression was different with purified endotoxin from that with intact bacteria. Meningococci were more potent stimuli of CD62E expression than was endotoxin, whereas endotoxin was at least as effective as meningococci in inducing ICAM-1 and VCAM-1. The effect of bactericidal/permeability increasing protein (rBPI(21)), an antibacterial molecule with antiendotoxin properties, was also dependent on LOS structure. The strains which possessed a truncated or nonsialylated LOS, whether capsulated or not, were more sensitive to the inhibitory effects of rBPI(21). These findings could have important implications for the use of antiendotoxin therapy in meningococcal disease.

Towards a nasal vaccine against meningococcal disease, and prospects for its use as a mucosal adjuvant.

A Norwegian outer membrane vesicle (OMV) vaccine against group B meningococcal disease proved to be strongly immunogenic when administered intranasally in mice. The OMV preparation, made from Neisseria meningitidis and intended for parenteral use, was therefore given without adjuvant to human volunteers (n = 12) in the form of nose drops or nasal spray. Such immunizations, which were carried out at weekly intervals during a three-week period, were able to induce systemic antibodies with bactericidal activity in more than half of the individuals. In addition, all vaccinees developed marked increases in OMV-specific IgA antibodies in nasal secretions. The potential of the OMV particles as carriers for other less immunogenic antigens were elucidated in mice with use of whole inactivated influenza virus. Even though influenza virus alone did induce some systemic and salivary antibody responses after being administered intranasally, these responses were greatly augmented when the virus was presented together with OMVs. Thus, it is possible that a nasal OMV vaccine may induce protection against invasive meningococcal disease, and also that it might be used as a vehicle for nasal vaccines against other diseases.

Intranasal administration of a meningococcal outer membrane vesicle vaccine induces persistent local mucosal antibodies and serum antibodies with strong bactericidal activity in humans.

A nasal vaccine, consisting of outer membrane vesicles (OMVs) from group B Neisseria meningitidis, was given to 12 volunteers in the form of nose drops or nasal spray four times at weekly intervals, with a fifth dose 5 months later. Each nasal dose consisted of 250 microg of protein, equivalent to 10 times the intramuscular dose that was administered twice with a 6-week interval to 11 other volunteers. All individuals given the nasal vaccine developed immunoglobulin A (IgA) antibody responses to OMVs in nasal secretions, and eight developed salivary IgA antibodies which persisted for at least 5 months. Intramuscular immunizations did not lead to antibody responses in the secretions. Modest increases in serum IgG antibodies were obtained in 5 volunteers who had been immunized intranasally, while 10 individuals responded strongly to the intramuscular vaccine. Both the serum and secretory antibody responses reached a maximum after two to three doses of the nasal vaccine, with no significant booster effect of the fifth dose. The pattern of serum antibody specificities against the different OMV components after intranasal immunizations was largely similar to that obtained with the intramuscular vaccine. Five and eight vaccinees in the nasal group developed persistent increases in serum bactericidal titers to the homologous meningococcal vaccine strain expressing low and high levels, respectively, of the outer membrane protein Opc. Our results indicate that meningococcal OMVs possess the structures necessary to initiate systemic as well as local mucosal immune responses when presented as a nasal vaccine. Although the serum antibody levels were less conspicuous than those after intramuscular vaccinations, the demonstration of substantial bactericidal activity indicates that a nonproliferating nasal vaccine might induce antibodies of high functional quality.

Outer membrane vesicle vaccines made from short-chain lipopolysaccharide mutants of serogroup B Neisseria meningitidis: effect of the carbohydrate chain length on the immune response.

Outer membrane vesicle (OMV) vaccines were made from Neisseria meningitidis strain 44/76 and its two short-chain lipopolysaccharide (LPS) mutants, Mu-1 and Mu-4. Only the 44/76 vaccine contained LPS with the host antigen lacto-N-neotetraose. The protein composition of the vaccines was similar. The LPS carbohydrate chain length proved to influence drastic changes in the LPS immunogenicity as well as the outer membrane proteins (OMPs) ability to elicit functional antibodies in mice. Only LPS in the Mu-1 and Mu-4 vaccines were immunogenic, and the 44/76 vaccine differed also by not inducing antibodies to the class 4 OMP. The Mu-1 vaccine, with a LPS carbohydrate chain comprising only two residues of 2-keto-3-deoxy-octonic acid, induced lower bactericidal activity and less antibodies to the class 1 OMP, compared to the two other vaccines. This indicates that LPS of a certain carbohydrate chain length is required for adequate exposure of the class 1 OMP epitopes essential for inducing bactericidal antibodies.

Accuracy of technicians versus pharmacists in checking syringes prepared for a dialysis program.

The accuracy of pharmacy technicians versus pharmacists in checking drug doses prepared in syringes for a dialysis program was studied. Three pharmacy technicians from the pharmacy of a regional kidney disease program in Minnesota participated in the study after completing a training program and after common preparation errors had been identified by pharmacists. From November 1995 to April 1996, the technicians used labels printed from a database of pharmacist-verified orders to prepare and label i.v. syringes. Four medications were used-epoetin alfa, calcitriol, heparin prepared from beef lung, and heparin prepared from porcine intestinal mucosa. Each syringe was checked by one of nine pharmacists for accuracy of dose and medication, and all errors were recorded. The technicians checked syringes prepared by other technicians and also recorded errors. Accuracy rates (percentages of syringes correctly evaluated) for pharmacists and technicians were compared. A total of 10,608 syringes were checked. Accuracy rates for pharmacists and pharmacy technicians were 99.86% and 99.83%, respectively. Accuracy rates in checking syringes did not differ significantly between pharmacists and technicians in this study setting.

The eye and its diseases in Ancient Egypt.

Based on a study of mummies, skeletons, burial rites, medical instruments, medicaments, literature and objets d'art from Ancient Egypt before the Hellenistic Period, the understanding of the eye, its diseases and their treatment at that time is described. Magic spells, religious rites and medical treatments, especially with eye ointments, were probably used often complementary to one another. We must be very cautious about our conclusions in regard to the effectiveness of the treatments. Eye diseases have been depicted only exceptionally in Egyptian art, except for blindness and 'symbolic blindness'.

Identification of a locus involved in meningococcal lipopolysaccharide biosynthesis by deletion mutagenesis.

A novel method for insertion/deletion mutagenesis in meningococci was devised. This consisted of ligating a digest of total chromosomal DNA to a 1.1 kb restriction fragment containing an erythromycin-resistance marker (ermC), and subsequent transformation of the ligation mixture into the homologous meningococcal strain H44/76. Southern blotting of a number of the resulting erythromycin-resistant transformants demonstrated that all carried the ermC gene inserted at different positions in the chromosome. Mutants with a specific phenotype were identified by screening with the anti-lipopolysaccharide (LPS) monoclonal antibody MN4A8B2, which is specific for immunotype L3. In this way, two independent L3-negative mutant strains were isolated. In transformation experiments with chromosomal DNA from these mutants, erythromycin-resistance and lack of MN4A8B2 reactivity were always linked, showing that the insertion/deletion was in a locus involved in LPS biosynthesis. On SDS-PAGE, the mutant LPS displayed an electrophoretic mobility intermediate between that produced by the previously isolated galE and rfaF mutant strains. Chemical analysis of the mutant LPS revealed that the structure was probably lipid A-(KDO)2-(Hep)2. Chromosomal DNA flanking the ermC insertion in these two mutant strains was cloned, and used as probe for the isolation of the corresponding region of the wild-type strain. From hybridization and polymerase chain reaction (PCR) analysis, it could be concluded that both mutations map to the same locus. The affected gene probably encodes the glycosyltransferase necessary for adding N-acetylglucosamine to heptose.

Short-chain lipopolysaccharide mutants of serogroup B Neisseria meningitidis of potential value for production of outer membrane vesicle vaccines.

Four lipopolysaccharide (LPS) mutants (Mu-1 to Mu-4) were isolated after exposing Neisseria meningitidis strain 44/76 to pyocins from Pseudomonas aeruginosa. Parent strain LPS contained one major SDS-PAGE band expressing the immunotype determinants of L3, L3,7 and L3,7,9 and a minor band of higher mobility expressing the immunotype determinants of L8, L8a, L1,8,10 and L11. Each mutant LPS appeared as one SDS-PAGE band of higher mobility than the bands of the parent strain. None of these LPSs expressed the immunotype determinants of the parent strain, except Mu-4 LPS which reacted with the L11-specific MAb 4C4. Strain 44/76 LPS was found to contain galactose (Gal), glucose (Glc), heptose (Hep), glucosamine (GlcN), and 2-keto-3-deoxy-octulosonic acid (Kdo) in the molar ratios of 1.9:1.3:1.7:3.5:2.1. The corresponding ratios of the mutants were: Mu-4, 0:1.7:1.7:2.8:2.0; Mu-3, 0:0:1.7:2.4:1.6; Mu-2, 0:0:2.1:1.8:2.0, Mu-1, 0:0:1.8:1.9. Thus, all mutant LPSs lacked Gal and possessed less GlcN as compared to strain 44/76 LPS. Consequently, these mutants do not express the lacto-N-neo-tetraose (Gal1-4GlcN1-3Gal1-4Glc) commonly found as a part of meningococcal LPS and also on structures of human erythrocytes. These LPS mutants will be considered for use in production of OMV vaccines without host-like antigens, which might favour induction of antibodies to more conserved epitopes of meningococcal LPS.

Distribution of tetracycline resistance determinants among gram-negative bacteria isolated from polluted and unpolluted marine sediments.

Tetracycline-resistant gram-negative bacteria were isolated from four different marine sediments in Scandinavia and analyzed with DNA probes for the determinant classes A to E. Colony hybridizations of 429 isolates revealed that class E is the dominating resistance determinant in these marine sediments. Comparison of fecally polluted and unpolluted sediments showed few determinant classes in unpolluted sediment and a complex composition of several determinant classes in polluted sediment. Total DNA extraction and analysis with DNA probes for determinant classes A to E resulted in no hybridization signal, because of the low number of gram-negative tetracycline-resistant bacteria. Identification of class E isolates revealed that this determinant is present not only in Aeromonas hydrophila, Escherichia coli, and Vibrio salmonicida but also in additional strains.