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We report muon spin rotation/relaxation (μSR) measurements of the heavy fermion superconductor UPt3 in external fields H ext∥ĉ. We find that the muon Knight shift is unchanged in the superconducting state, consistent with odd-parity pairing (such as p wave). The transverse field relaxation is observed to be strongly field dependent, decreasing with increasing field. Below Tc the increase is barely detectable in an applied field of 4 kG∥ĉ. On the basis of the high field measurements, we estimate the low temperature penetration depth to be λ(T→0)≥ (R18)11 000 Å.

We report muon spin rotation/relaxation (μSR) measurements of the heavy fermion superconductors UBe13 and UPt3. In both materials we find that the muon Knight shift is unchanged in the superconducting state, consistent with odd-parity pairing (such as p-wave). The magnetic field penetration depths in UPt3 and UBe13 are extremely long, greater than 10000 Å. We find no evidence of a magnetic transition in UBe13 below 10 K. © 1991.

OBJECTIVE: The lack of randomized controlled trials (RCTs) in pulmonary fibrosis in systemic sclerosis (SSc) has hampered an evidence-based approach to treatment. This RCT was undertaken to investigate the effects of intravenous (IV) cyclophosphamide (CYC) followed by azathioprine (AZA) treatment in pulmonary fibrosis in SSc. METHODS: Forty-five patients were randomized to receive low-dose prednisolone and 6 infusions (monthly) of CYC followed by oral AZA, or placebo. Primary outcome measures were change in percent predicted forced vital capacity (FVC) and change in single-breath diffusing capacity for carbon monoxide (DLCO). Secondary outcome measures included changes in appearance on high-resolution computed tomography and dyspnea scores. An intent-to-treat statistical analysis was performed. RESULTS: At baseline, there were no significant group differences in factors linked to outcome, including severity of pulmonary fibrosis and autoantibody status. Sixty-two percent of the patients completed the first year of treatment. Withdrawals included 9 patients (6 from the placebo group) with significant decline in lung function, 2 with treatment side effects (both from the active treatment group), and 6 with non-trial-related comorbidity. No hemorrhagic cystitis or bone marrow suppression was observed. Estimation of the relative treatment effect (active treatment versus placebo) adjusted for baseline FVC and treatment center revealed a favorable outcome for FVC of 4.19%; this between-group difference showed a trend toward statistical significance (P = 0.08). No improvements in DLCO or secondary outcome measures were identified. CONCLUSION: This trial did not demonstrate significant improvement in the primary or secondary end points in the active treatment group versus the group receiving placebo. However, for FVC there was a trend toward statistical significance between the 2 groups. This suggests that treatment of pulmonary fibrosis in SSc with low-dose prednisolone and IV CYC followed by AZA stabilizes lung function in a subset of patients with the disease. Therapy was well tolerated with no increase in serious adverse events. [ABSTRACT FROM AUTHOR]

Objective: To identify herd-level risk factors for bovine respiratory disease (BRD) in nursing beef calves., Design: Population-based cross-sectional survey., Sample: 2,600 US cow-calf producers in 3 Eastern and 3 Plains states., Procedures: The associations of herd characteristics with BRD detection in calves and cumulative BRD treatment incidence were determined., Results: 459 (177%) surveys were returned and met the inclusion criteria; 48% and 52% of these surveys were completed by producers in Plains and Eastern states, respectively. Mean (95% confidence interval) number of animals in herds in Plains and Eastern states were 102 (77 to 126) and 48 (40 to 56), respectively. Bovine respiratory disease had been detected in ≥ 1 calf in 21% of operations; ≥ 1 calf was treated for BRD and ≥ 1 calf died because of BRD in 89.2% and 46.4% of operations in which calf BRD was detected, respectively. Detection of BRD in calves was significantly associated with large herd size, detection of BRD in cows, and diarrhea in calves. Calving season length was associated with BRD in calves in Plains states but not Eastern states. Cumulative incidence of BRD treatment was negatively associated with large herd size and examination of cows to detect pregnancy and positively associated with calving during the winter, introduction of calves from an outside source, offering supplemental feed to calves, and use of an estrous cycle synchronization program for cows., Conclusions and Clinical Relevance: Results of this study indicated factors associated with calf BRD risk; modification of these factors could potentially decrease the incidence of BRD in nursing calves.

Objective: To compare immune responses following modified-live virus (MLV) vaccination at weaning after intranasal or SC administration of an MLV vaccine to beef calves at 2 or 70 days of age., Animals: 184 calves., Procedures: Calves were allocated to 1 of 5 groups. The IN2 (n = 37) and IN70 (37) groups received an MLV vaccine containing bovine herpesvirus 1 (BHV1), bovine viral diarrhea virus (BVDV) types 1 and 2, bovine respiratory syncytial virus (BRSV), and parainfluenza 3 virus intranasally and a Mannheimia haemolytica and Pasteurella multocida bacterin SC at median ages of 2 and 70 days, respectively. The SC2 (n = 36) and SC70 (37) groups received a 7-way MLV vaccine containing BHV1, BVDV1, BVDV2, BRSV, parainfluenza 3 virus, M haemolytica, and P multocida SC at median ages of 2 and 70 days, respectively; the control group (37) remained unvaccinated until weaning. All calves received the 7-way MLV vaccine SC at median ages of 217 (weaning) and 231 days. Serum neutralizing antibody (SNA) titers against BHV1, BVDV1, and BRSV and intranasal IgA concentrations were determined at median ages of 2, 70, 140, 217, and 262 days. Cell-mediated immunity (CMI) against BHV1, BRSV, BVDV1, and P multocida was determined for 16 calves/group., Results: At median ages of 140 and 217 days, BVDV1 SNA titers were significantly higher for the SC70 group than those for the other groups. Intranasal IgA concentrations and CMI increased over time for all groups. Vaccination at weaning increased SNA titers and CMI in all groups., Conclusions and Clinical Relevance: SC administration of an MLV vaccine to 70-day-old calves significantly increased BVDV1 antibody titers before weaning.

In 2008, an unusual strain of methicillin-sensitive Staphylococcus aureus (MSSA68111), producing both Panton-Valentine leukocidin (PVL) and toxic shock syndrome toxin-1 (TSST-1), was isolated from a fatal case of necrotizing pneumonia. Because PVL/TSST-1 co-production in S. aureus is rare, we characterized the molecular organization of these toxin genes in strain 68111. MSSA68111 carries the PVL genes within a novel temperate prophage we call ФPVLv68111 that is most similar, though not identical, to phage ФPVL--a phage type that is relatively rare worldwide. The TSST-1 gene (tst) in MSSA68111 is carried on a unique staphylococcal pathogenicity island (SaPI) we call SaPI68111. Features of SaPI68111 suggest it likely arose through multiple major recombination events with other known SaPIs. Both ФPVLv68111 and SaPI68111 are fully mobilizable and therefore transmissible to other strains. Taken together, these findings suggest that hypervirulent S. aureus have the potential to emerge worldwide.

A previously healthy male with subacute onset right leg weakness was suspected to have an astrocytoma as imaging showed a lesion. Subsequent biopsy showed the presence of foamy macrophages containing periodic acid-Schiff staining granules, suggesting Whipple disease as a possible diagnosis.

Diabetic retinopathy can result in vision loss and involves progressive neurovascular degeneration of the retina. This study tested the hypothesis that diabetes decreases the retinal expression of presynaptic proteins involved in synaptic function. The protein and mRNA contents for synapsin I, synaptophysin, vesicle-associated membrane protein 2, synaptosomal-associated protein of 25 kDa and postsynaptic density protein of 95 kDa were measured by immunohistochemistry, immunoblotting and real-time quantitative polymerase chain reaction in whole retinas and retinal synaptosomes from streptozotocin-diabetic and control Sprague-Dawley rats. There was less presynaptic protein immunoreactivity after 1 and 3 months of diabetes than in controls. Discrete synaptophysin-immunoreactive puncta were significantly smaller and fewer in sections from 1- and 3-month diabetic rat retinas than in those from controls. The content of presynaptic proteins was significantly less in whole retinas of 1- and 3-month diabetic rats, and in synaptosomes from 1-month diabetic rats, than in controls. Whole retinas had significantly less mRNA for these genes after 3 months but not 1 month of diabetes, as compared to controls (with the exception of postsynaptic density protein of 95 kDa). In contrast, there was significantly less mRNA for synaptic proteins in synaptosomes of 1-month diabetic rats than in controls, suggesting a localized depletion at synapses. Protein and mRNA for beta-actin and neuron-specific enolase were unchanged by diabetes. The ratio of phosphorylated to total synapsin I was also reduced in whole retina and isolated synaptosomes from 1-month diabetic rats, as compared to controls. These data suggest that diabetes has a profound impact on presynaptic protein expression in the retina, and may provide a mechanism for the well-established defects in vision and the electrophysiological response of the retina in diabetes.

Crossbred (Bos taurus) yearling beef bulls were assessed for breeding soundness and physical traits prior to multi-sire natural mating at pasture. Bulls (n = 60) were assigned to six groups of nine or 10 bulls and two bull-groups were rotated on 14-day intervals during a 63-day mating season in each breeding herd (n = 3) of 191-196 cows. The remaining bulls (n = 14) were maintained under similar environmental conditions without mating exposure. Bulls were observed during mating and assessed for breeding soundness and changes following mating. Bulls used for breeding (UFB) lost 77 kg of body weight and declined from body condition scores of 6 to 4.5, whereas bulls not used for breeding (NUB) maintained body condition scores of 6 and gained 27 kg. The UFB bulls incurred a 75% total injury rate with 63% incidence of lameness and 12% incidence of reproductive injuries, resulting in a 22% attrition rate. Only 45% were physically sound at the end of mating. Scrotal circumference declined in UFB bulls (-4.58%) and increased in NUB bulls (2.49%). From the 98% BSE-satisfactory rate (UFB) prior to breeding, only 61% were BSE-satisfactory post-breeding. The NUB bulls declined from 57 to 36% satisfactory. The BSE classification was influenced by significant increases in abnormal spermatozoa (primary and secondary), which was significantly associated with injuries incurred during mating. Group and breed differences in injury rates and BSE-status following mating were evident. Environmental conditions and mating activity influenced bull seminal quality and physical condition. Pregnancy rates in all three breeding herds (91-96%) were similar, with insignificant differences between bull-groups; the effects of physical and reproductive changes on individual bull fertility were immeasurable.

Purpose: To evaluate topical acidulated phosphate fluoride (APF) and low fluence argon laser (Ar) treatment effects on in vitro caries formation in primary tooth enamel., Methods: 20 extracted or exfoliated primary teeth with sound buccal and lingual surfaces underwent soft tissue debridement and a fluoride-free prophylaxis. Treatment groups were: (1) Control [n=5]; (2) Ar [231mW, 10 seconds, 11.5J/cm2; n=5], (3) 1.23% APF for 4 minutes before Ar [n=5]; (4) Ar before APF [n=5]. Buccal and lingual enamel surfaces were treated, and then rinsed in deionized,distilled water (24 hours). An acid-resistant coating was applied leaving buccal and lingual sound enamel windows exposed. In vitro enamel caries was created (2.2 mM calcium, 2.2 mM phosphate, 5.0 mM fluoride, pH 3.90, 10 days). Following longitudinal sectioning, two lesions per primary tooth (10 lesions total) with each group were evaluated for lesion depth (polarized light, water imbibition) and compared (ANOVA, Duncan's Multiple Range)., Results: Argon laser irradiation alone provided a 41% reduction in lesion depth (176 +/- 21 microm, P< 0.05) when compared with that for the no treatment controls (297 +/- 31 microm). The combination of argon laser irradiation with APF treatment resulted in lesion depth decreases of slightly over 50% (140 +/- 23 microm for APF before ArTx; 124 +/- 17 microm for ArTx before APF, P< 0.05) compared with control lesion depths, and 20 to 30% over that for argon laser treatment alone (P< 0.05). There was no statistical difference in lesion depth regardless of whether fluoride treatment occurred before or after laser irradiation (P> 0.05).

Purpose: The purpose of this in vitro study was to evaluate the effect of low-fluence argon laser (AL) irradiation and acidulated phosphate fluoride (APF) gel treatment on enamel microhardness., Methods: Twelve mandibular permanent molars were selected for this study. The teeth were sectioned generating 4 flat enamel surfaces per tooth. The flattened enamel tooth surfaces were randomly assigned to 1 of 4 treatment groups: (1) no treatment (control); (2) the enamel surface was exposed to a 4-minute, 1.23% APF gel treatment; (3) the enamel surface was exposed to AL irradiation of 11.5 J/cm2 (0.23 1-W, 5-mm beam size, 10 seconds); and (4) the enamel surface was exposed to the same AL irradiation followed by an APF gel treatment. Using a Buehler Micromet II Digital Microhardness Tester, Knoop hardness was determined using a 1,000-gram load and a dwell time of 12 seconds. Five hardness values were recorded for each enamel surface. Data were analyzed using ANOVA and Fisher's least significant difference post-hoc test., Results: Mean surface hardness values (+/-SD) were 298+/-37 Knoop hardness (HK) for the no treatment (control), 270+/-70 HK for the APF-only group, 316+/-5 HK for the AL-only group, and 317+/-25 HK for the AL-before-APF group. The AL-only and AL-before-APF groups had significantly higher (P<.05) surface hardness values vs the APF-only group., Conclusions: Enamel surface microhardness is higher when exposed to low AL irradiation only or AL before APF vs a no treatment (control) enamel surface.

Influenza vaccines have been produced for decades in eggs by a classical technology. A range of new vaccine technologies and approaches has been applied to improving these vaccines. Foremost among these are the use of mammalian cells to produce influenza viruses, the use of new adjuvants, and a live attenuated vaccine. Mammalian cell-derived vaccines, produced in nontumorigenic cell lines, have been developed and shown to be effective production systems as alternatives to eggs. This manuscript discusses the advantages and challenges of mammalian cell production, as well as alternative technologies for influenza vaccines.

This paper summarizes major technologies, with emphasis on applications to preventive vaccines for infectious diseases. A limited number of examples of each technology are provided.

Application of a non-cavitating ultrasonic standing wave to suspended microparticles brings the particles into close approximation and has been used previously to enhance the performance of several diagnostic agglutination tests. The sensitivity of rotavirus detection by ultrasound enhanced latex agglutination was compared with conventional test-card agglutination. Application of ultrasound gave a 32-fold improvement in the sensitivity of detection of rotavirus antigen in buffer compared with the test card method. A novel turbidimetric approach was used to measure agglutination occurring following the test-card procedure (in place of visual examination) and following exposure of commercial rotavirus latex reagents to a 4.5 MHz ultrasonic field (in place of microscopy). The sensitivity enhancement over the conventional method achievable through ultrasonic exposure was comparable whether agglutination measurements were made visually or turbidimetrically and demonstrates the potential for turbidimetry in combination with the ultrasonic method. Turbidimetry offers an alternative to visual assessment that may be more easily incorporated into automated systems.

Microbial antigen in clinical specimens can be detected rapidly by commercial test-card latex agglutination, but poor sensitivity is a potential difficulty. Antigen detection by immuno-agglutination of coated latex micro-particles can be enhanced in comparison with the conventional test-card method in both rate and sensitivity by the application of a non-cavitating ultrasonic standing wave. Antibody-coated micro-particles suspended in the acoustic field are subjected to physical forces that promote the formation of agglutinates by increasing particle-particle contact. This report reviews the application of ultrasound to immuno-agglutination testing with several commercial antibody-coated diagnostic micro-particles. This technique is more sensitive than commercial card-based agglutination tests by a factor of up to 500 for fungal cell-wall antigen, 64 for bacterial polysaccharide and 16 for viral antigen (in buffer). The detection sensitivity of meningococcal capsular polysaccharide in patient serum or CSF has been increased to a stage where serotyping by ultrasound-enhanced agglutination is comparable to that achievable with the PCR, but is available more rapidly. Serum antigen concentration as measured by ultrasonic agglutination has prognostic value. Increasing the sensitivity of antigen detection by increasing the acoustic forces that act on suspended particles is considered. Employing turbidimetry to measure agglutination as part of an integrated ultrasonic system would enable the turnover of large numbers of specimens. Ultrasound-enhanced latex agglutination offers a rapid, economical alternative to molecular diagnostic methods and may be useful in situations where microbiological and molecular methods are impracticable.

The highly conserved NspA protein has been found in the outer membrane of every Neisseria meningitidis strain tested so far. Two monoclonal antibodies (MAbs) directed against this protein were used to demonstrate that biologically important epitopes of the NspA protein are exposed at the surface of serologically distinct meningococcal strains. Analysis of sera collected from mice that survived a deadly meningococcal challenge following immunization with recombinant NspA protein (rNspA) revealed the presence of cross-reactive antibodies which efficiently attached to and killed the four serogroup B strains tested. These data are additional proof that the NspA protein is exposed at the surface of intact meningococcal cells, which is an important characteristic for a vaccine candidate.

The purpose of this study is to prospectively evaluate a strategy in which prophylaxis with amphotericin B lipid complex at 3 different dosages was targeted to liver transplant recipients at high risk for the development of invasive fungal infection (IFI). High risk was defined as a postoperative requirement for prolonged (>/=5 days) intensive care unit (ICU) treatment. Consecutive high-risk patients were administered prophylaxis with amphotericin B lipid complex from day 5 after orthotopic liver transplantation (OLT) until ICU discharge or death. The first 10 eligible patients were administered 5 mg/kg/d, the next 10 patients were administered 2.5 mg/kg/d, and a final 10 patients were administered 1 mg/kg/d. Drug safety and efficacy were assessed before each dosage reduction. During the study period, 130 adult patients underwent 137 OLTs. Thirty patients fulfilled the entry criteria and were administered prophylaxis with amphotericin B lipid complex. No patient developed proven IFI during prophylaxis. Cultures from normally sterile sites (blood and abdominal drain fluid) always showed negative results. All fungal isolates were sensitive in vitro to amphotericin B. There was no significant difference in colonization scores among the groups of patients administered different dosages of amphotericin B lipid complex. No death, serious adverse reaction, or nephrotoxicity was attributed to amphotericin B lipid complex. We conclude that prophylaxis with amphotericin B lipid complex targeted to patients requiring prolonged ICU treatment after OLT appears to be well tolerated and may prevent IFI. Our current policy is to use amphotericin B lipid complex, 1 mg/kg/d, as antifungal prophylaxis in this high-risk group.

The distribution of Neisseria meningitidis serogroup B and C polysaccharide antigen in blood and the prognostic significance of antigen concentration was examined by ultrasound-enhanced immunoagglutination of coated microparticles. Specimens (169 sera/plasma from 145 patients with confirmed meningococcal disease) were tested retrospectively. The ultrasonic immunoassay detected serum antigen in 136 samples from 112 patients. Titration of antigen-positive specimens allowed estimation of blood antigen concentration. The modal blood antigen titre was 1/16, corresponding to an estimated polysaccharide concentration of 0.85 microg/ml. The lowest mean blood antigen concentration found ultrasonically was 0.05 microg/ml; compared to the 1.98 microg/ml found by conventional latex agglutination, this represents an approximately 30-fold improvement in sensitivity. Three grades of outcome were correlated with the presenting antigen titre in 83 patients: (i) <2 weeks hospitalisation, (ii) > or =2 weeks hospitalisation and (iii) mortality. High polysaccharide concentrations correlated with mortality. Nine of 15 patients with a serum antigen titre of 1/64 or greater (> or =3.4 microg/ml polysaccharide) died, whereas no patient with titres equal to or less than 1/4 (< or = 0.21 microg/ml) died, including those patients in whom antigen was undetectable by ultrasonic immunoassay. Increasing antigen concentration significantly correlated with severity of outcome (P<0.001). Ultrasound-enhanced agglutination provides a rapid prognostic indicator by sensitive measurement of serum antigen level.

Purpose: The purpose of this in vitro study was to compare caries resistance of sound human enamel following argon laser (AL) irradiation, as well as, combinations of topical fluoride foams and AL irradiation., Methods and Materials: Thirty extracted human teeth were sectioned into four buccal windows and assigned to one of the following treatment groups: (1) no treatment/control; (2) low fluence (11.5 J/cm2) AL irradiation for 10 seconds; (3) 1.23% APF foam for 4 minutes followed by low fluence AL irradiation; (4) 2.0% NaF foam for 4 minutes followed by low fluence AL irradiation. Caries-like lesions were created by submerging the teeth in ten Cate solution (pH 4.5). Following a 96-hour exposure period, 100 microns longitudinal sections were prepared for polarized light evaluation. Visilog 5.1.1. image analysis software was used to obtain quantitative lesion depths. The Scheffe F-test was used to compare the lesion depths for each of the four treatment groups., Results: Lesion depths were: 16.1 +/- 6 microns for control; 13.7 +/- 4 microns for AL irradiation alone; 12.1 +/- 4.3 microns for 1.23% APF foam before AL irradiation; and 11.4 +/- 5.9 microns for 2.0% NaF foam before AL irradiation. Significant difference (p < 0.05) was found between the control group and the 2.0% NaF foam before AL irradiation group. AL irradiation alone reduced lesion depth by 15% compared with the control lesion. When AL irradiation was combined with 1.23% APF foam treatment, lesion depth decreased by 25% compared with control lesions, and by 29% when combined with 2.0% NaF foam., Conclusion: Combining AL irradiation and 2.0% NaF foam treatment significantly enhances the resistance of sound enamel to an in vitro cariogenic challenge.

Objective: This clinical pilot study was conducted to investigate the effectiveness of argon laser irradiation to reduce demineralization or loss of tooth structure in vivo., Summary Background Data: In vitro research previously demonstrated the ability of argon laser irradiation to reduce demineralization or loss of tooth structure., Methods: Using the Ogaard model of producing demineralization, the experimental teeth were irradiated with argon laser of 250 mW (producing approximately 12 J/cm2) prior to banding. Polarized light evaluation of the sectioned, extracted teeth was used to determine the amount of demineralization., Results: Results showed a 29.1% reduction in demineralization in the experimental teeth as compared to the bilateral control teeth., Conclusion: Low-power argon laser irradiation significantly reduced demineralization clinically.

Purpose: This in-vitro study evaluated shear bond strength and analyzed, via scanning electron microscopy, enamel prepared for pit and fissure sealant placement. Various surface pretreatment methods were conducted prior to short-term (72-hour) and long-term (120-day) analysis., Method: Six treatment groups of 14 teeth, having 28 bonding surfaces (buccal and lingual) were treated. Cylinders of Delton pit and fissure sealant were placed on the prepared buccal and lingual surfaces and light-cured for 60 seconds. One-half of each group's specimens were stored in distilled water for 72 hours and the other half were stored in water distilled for 120 days followed by thermocycling. All specimens were subjected to shear bond strength analysis as determined on an Instron testing machine., Results: All acid treated groups were equivalent and greater than air abrasion alone after 72 hours of water storage. Scanning electron microscopy of air-abraded and combination treated enamel surfaces revealed a more detailed retentive pattern in the combination treatment than in either treatment alone., Conclusion: Based on in-vitro shear bond strength values, air abrasion with 50 micron alumina is an effective pre-etch treatment for sealant placement and in concert with phosphoric acid treatment significantly enhanced the long-term bond of a sealant to enamel. The clinical relevance of this has not been established.

Breakthroughs in molecular biology, biochemistry, process development, immunology and related fields have provided the means for improving current vaccines and developing new ones. The increasing availability of these vaccines is providing the unprecedented opportunity to prevent serious infectious disease in different age groups and to significantly reduce associated morbidity and mortality. However, since the great majority of licensed and experimental vaccines in advanced clinical studies are injected, each new vaccine creates the need for additional needlesticks which could become so numerous as to discourage full compliance with immunization schedules. Therefore, the development of combination vaccines is a major way to assure compliance with immunization needs. The leading types of licensed and experimental vaccines which have been or may be made into combination vaccines will be described. In addition, key challenges common to the development of all combination vaccines will be discussed; these include pharmaceutical, immunological, clinical, regulatory, manufacturing, public health and marketing issues. In order to assure timely development and availability of such combination products, these issues must be integrated successfully early in a development program.

Technologies for making active vaccines fall into 3 general groups: live, subunit (killed or inactivated) and genetic. Each of these groups is further divisible into multiple categories, which include recombinant-derived antigens as well as native microorganisms and their components. In addition, there are new enabling technologies such as delivery systems and vectors which can be applied to these approaches. Most disease targets, whether infectious or noninfectious in origin, can be approached by the application of several different vaccine technologies, as can be tested during the discovery phase of research. The criteria for choosing early in a development program which of the vaccine technologies are likely to ultimately be most fruitful for a given application include: knowledge of the pathogenesis of the given infection/disease; technical feasibility; immunobiology and associated mechanisms; preclinical efficacy profile; anticipated clinical safety; regulatory; manufacturing; and marketing. All of these criteria should be considered together in making selections for an R&D program. This paper is reviewing the major vaccine technologies and relevant examples of how these criteria are used to make decisions in vaccine development.

Detailed instructions for performance and interpretation of ultrasound-enhanced latex agglutination tests for the rapid identification of bacteria causing meningitis are described. This recently developed technique, which enhances the sensitivity of most latex immunoagglutination assays, has been studied mainly in the context of detection of antigens of meningitis-causing bacteria. The test concentrates on the Wellcogen bacterial antigen kit (Murex Diagnostics Ltd) that contains five latex suspensions specific for Haemophilus influenzae type b, Neisseria meningitidis ACYW135, N. meningitidis B/Escherichia coli K1, Streptococcus group B and Streptococcus pneumoniae. Light photomicrographs of positive agglutination are shown. Particular attention is paid to the appearance of the latex in negative control samples following exposure to ultrasound. Guidance is given on interpretation and assessment in clinical samples.

A large body of evidence exists that implicates a number of microbial agents in the pathogenesis of coronary heart disease (CHD). This, if proven, may have far-reaching implications for the prevention and treatment of CHD and other atherosclerotic disease. The histopathology of atherosclerosis and its natural history suggest infectious causation at many points along the progression of disease, particularly with regard to CHD, and a number of pathogens have been the focus of study. Viral agents implicated include Coxsackie B4 virus, for which tenuous sero-epidemiological associations exist, and the Herpesviridae. The animal herpesvirus causing Marek's disease in chickens causes atherosclerotic lesions in these animals. Herpes simplex virus I and II have been found in aortic smooth muscle and produce changes in vitro in smooth muscle that are similar to those seen at the beginning of atherosclerosis and which may also explain some of the features of atherosclerotic complications. Cytomegalovirus is implicated more strongly sero-epidemiologically by in-vivo detection in atherosclerotic lesions and by its links with post-cardiac transplant vasculopathya syndrome similar to atherosclerosis. Bacteria have also been shown to have links with CHD. Chlamydia pneumoniae and Helicobacter pylori have both been associated sero-epidemiologically with CHD, and these findings have been consolidated by recent work showing their presence in atherosclerotic lesions in adults. Bacterial infections in general lead to many changes in lipid, thrombic and other acute-phase protein metabolism, and some of these changes occur with both C. pneumoniae and H. pylori infections. The ubiquity and similar epidemiological features to CHD of all these microbial pathogens make the resolution of the causative issue impossible by retrospective means. All that can be shown at present are a variety of weak and strong links, the significance of which can only be determined by large and perhaps lifetime prospective studies.

A comparison was made of antibody responses generated to live varicella (Oka/Merck) vaccine (Varivax) produced during three different manufacturing campaigns to evaluate the quality of the antibody responses and demonstrate consistency of the manufacturing process. Anti-varicella zoster virus (VZV) antibody titers were measured by an enhanced neutralization assay and VZV glycoprotein-based enzyme-linked immunosorbent assay (gpELISA). For sera taken from children who had received one dose of vaccine an excellent linear concordance in titers was observed between the two assays. Sera from adults who had received two doses demonstrated continuing increased neutralization at high gpELISA titers. The immunogenicity measured by the two assays demonstrates that the overall performance of the vaccine was very similar over the three production series.

There have been two major focal points for the development of recombinant viral subunit vaccines. Expression strategies for the candidate antigens have focused increasingly on producing more natural antigenic structures, for example, virus-like particles. Also, new adjuvants have been used in order to increase immunogenicity.

The live attenuated varicella vaccine, which is available for the prevention of chickenpox, was produced by a classic technology that also has been used for polio, measles, mumps, and rubella vaccines. There are many newer technologies that have been applied to the research and development of other vaccines. Each of these other approaches offers potential advantages and disadvantages relative to the current varicella vaccine.

Laboratory and clinical studies have been directed toward development of a vaccine against rotavirus gastroenteritis in infants. First, bovine rotavirus strain WC3, which did not induce neutralizing antibodies to predominant human rotavirus (HRV) serotypes, was determined to be safe and immunogenic; however, it was not protective in all efficacy trials. HRVs adapted to cell culture retained some virulence for infants, but when further attenuated by cold adaptation, they were poorly immunogenic. Reassortant rotaviruses were designed to express HRV surface proteins VP7 (G) or VP4 (P) while retaining a bovine WC3 genome background. Reassortants containing either HRV surface protein and as few as four bovine rotavirus genes were safe in infants. A monovalent WC3 reassortant of serotype G1 specificity was 64%-100% protective in placebo-controlled trials. A quadrivalent WC3 reassortant vaccine with components of HRV G1, G2, G3, and P[8] specificity induced 67% protection against all rotavirus disease in a multicenter efficacy trial.

Genital infections with human papillomavirus (HPV) are increasingly recognized as a significant source of human disease; HPV is now implicated in up to 90% of cervical carcinomas. Neutralizing antibodies against papillomaviruses recognize conformational epitopes formed when viral capsid proteins assemble into virions or virus-like particles. Immunization with plasmid DNA encoding the major viral capsid protein L1 was studied as a means of inducing neutralizing antibodies and protection against virus challenge. In a cottontail rabbit papillomavirus (CRPV) model, immunization with plasmid DNA encoding L1 elicited conformationally specific neutralizing antibodies and provided immunity against papilloma formation upon challenge with CRPV. Immunization with DNA encoding the capsid protein may provide a means of protecting humans against HPV and would simplify the production of multivalent vaccines by combining plasmids that encode the viral capsid proteins of different strains. This may be of importance given the multiplicity of HPV types capable of causing disease.

Bovine rotavirus strain WC3 (P7[5], G6) administered at the 12th passage level was well tolerated clinically in infants and efficiently induced serum virus neutralizing antibody (VNA) with bovine rotavirus G6 specificity. The protective efficacy of WC3 vaccine against all rotavirus disease was inconsistent, varying in four separate trials from 76% to 0%; some selective protection against severe disease was seen in all trials. WC3 reassortants containing the gene for an individual human rotavirus VP7 (G) or VP4 (P) surface antigen were also well tolerated, but preferentially induced VNA to the WC3 parent. Efficacy trials of human G1 VP7 reassortant WI79-9 (P7[5], G1) consistently led to > 60% protection against all rotavirus disease. A quadrivalent WC3 reassortant vaccine was developed to contain four separate monovalent reassortants expressing human rotaviruses surface proteins G1, G2, G3, and P1A [8] respectively. In a multicenter trial including 439 infants, this vaccine induced 67.1% protection against all rotavirus disease (defined as positive for rotavirus antigen by ELISA only [p = < 0.001]) and 72.6% protection when the standard for rotavirus diagnosis was a positive test of stool for both rotavirus antigen by ELISA and rotavirus RNA by electropherotype analysis (p = < 0.001). In this trial, episodes of the most severe rotavirus disease (clinical severity score > 16.0 eight cases) occurred only in placebo recipients.

Papillomaviruses infect epithelia of the skin and mucous membranes and cause benign or malignant tumours in animals and in humans. The viruses are highly species-specific, and cell culture systems for propagating human papillomaviruses (HPVs) do not exist. However, there are several animal papillomavirus models. In the cottontail rabbit papillomavirus (CRPV) system, we demonstrated that recombinant CRPV virus-like particles (VLPs) consisting of the capsid proteins L1 or L1+L2 can be produced in the yeast Saccharomyces cerevisiae. Three immunizations with L1 VLPs formulated on aluminum adjuvant at 1-100 micrograms dose-1 efficiently protected rabbits from challenge with CRPV. Sera of immunized rabbits were shown to contain high-titered serum antibodies to CRPV L1 VLPs and to neutralize CRPV in vitro. Our results suggest that recombinant yeast-derived VLPs could be the basis for a candidate HPV vaccine.

An enhanced neutralization assay was developed to permit the sensitive, specific, and reproducible measurement of antibodies to varicella-zoster virus (VZV). Optimal neutralization was achieved using a combination of guinea pig complement (C') and rabbit anti-human IgG. This provided 625-, 160- and 13- to 64-fold increases in dilution endpoints of human post-zoster serum, varicella-zoster immune globulin and representative sera from recipients of live attenuated varicella vaccine, respectively, above those measured in the absence of C' and anti-IgG. The specificity of the assay was shown by the absorption of serum neutralization capacity with VZV-specific antigen and the lack of concordance between antibody titers to VZV with those to either herpes simplex virus type-2 or cytomegalovirus. The antibody status of recipients of live attenuated varicella vaccine was established from the amount of neutralizing activity produced at a single optimal serum dilution.