Dielectrophoretic assembly of insulinoma cells and fluorescent nanosensors into three-dimensional pseudo-islet constructs.

Dielectrophoretic forces, generated by radio-frequency voltages applied to micromachined, transparent, indium tin oxide electrodes, have been used to condense suspensions of insulinoma cells (BETA-TC-6 and INS-1) into a 10 x 10 array of three-dimensional cell constructs. Some of these constructs, measuring approximately 150 microm in diameter, 120 microm in height and containing around 1000 cells, were of the same size and cell density as a typical islet of Langerhans. With the dielectrophoretic force maintained, these engineered cell constructs were able to withstand mechanical shock and fluid flow forces. Reproducibility of the process required knowledge of cellular dielectric properties, in terms of membrane capacitance and membrane conductance, which were obtained by electrorotation measurements. The ability to incorporate fluorescent nanosensors, as probes of cellular oxygen and pH levels, into these 'pseudo-islets' was also demonstrated. The footprint of the 10 x 10 array of cell constructs was compatible with that of a 1536 microtitre plate, and thus amenable to optical interrogation using automated plate reading equipment.

Genetic diversity in Indian common bean (Phaseolus vulgaris L.) using random amplified polymorphic DNA markers.

Genetic diversity of twenty-six common bean (Phaseolus vulgaris L.) accessions of diverse geographical origin was studied using Random Amplified Polymorphic DNA (RAPD) markers. Fifteen out of forty four primers screened showed polymorphism across present set of genotypes. A total of 124 amplicons were scored using these 15 primers. Ninety five percent of the amplified products showed polymorphism, indicating fair amount of variation at the DNA level among these accessions. Cluster analysis delineated the genotypes in to four groups.

Booster immunization with a hexavalent diphtheria, tetanus, acellular pertussis, hepatitis B, inactivated poliovirus vaccine and Haemophilus influenzae type b conjugate combination vaccine in the second year of life: safety, immunogenicity and persistence of antibody responses.

The immunogenicity and reactogenicity of booster vaccination with GSK Biologicals' hexavalent DTPa-HBV-IPV/Hib vaccine was assessed in toddlers aged 12-18 months previously primed with the same combination (N=341), or with DTPa-IPV/Hib and HBV administered separately (N=102; Trials 217744/059 and 217744/096). Antibody persistence at age 4-6 years was also assessed in children who had received a 4th consecutive dose of DTPa-HBV-IPV/Hib vaccine or separate DTPa-IPV/Hib and HBV vaccines in this study and in another study conducted under similar conditions in Germany. Prior to booster vaccination in the second year of life, antibody concentrations and seroprotection rates were similar irrespective of the primary vaccine used. One month after boosting with DTPa-HBV-IPV/Hib, substantial antibody increases were observed against all vaccine antigens indicative of previous immune priming. Seropositivity and booster response rates against all antigens were 97.4-100%. Reactogenicity following booster vaccination with DTPa-HBV-IPV/Hib was similar regardless of the primary regimen used. Three to four years after administration of the 4th DTPa-HBV-IPV/Hib dose, >90% vaccinees had persistent protective antibody concentrations against diphtheria, hepatitis B, Hib and the three poliovirus types. Anti-tetanus antibody concentrations were > or = 0.1 IU/ml in 76.4% subjects and seropositivity for pertussis antibodies ranged from 34.5% for PT to 98.9% for FHA. In conclusion, the combined hexavalent DTPa-HBV-IPV/Hib vaccine is immunogenic and safe when used for boosting in the second year of life, regardless of the primary vaccine used, and offers sustained protection during early childhood and beyond.

Assessment of nine candidate DTP-vaccines with reduced amount of antigen and/or without adjuvant as a fourth (booster-) dose in the second year of life.

BACKGROUND: The incidence of local reactions to diphtheria-, tetanus and acellular pertussis (DTaP-) vaccines in infants and toddlers increases with each subsequent dose, and entire thigh swellings (ETS) have been reported. Lowering the amount of antigen or of adjuvant may decrease the reactogenicity of DTaP while maintaining a protective immune response. OBJECTIVES: Following priming with three doses of a DTaP vaccine during infancy, the safety, reactogenicity and immunogenicity of nine different candidate DTaP-vaccines with reduced amounts of antigen and/or adjuvant given as fourth (booster) dose were evaluated. METHODS: Study participants were healthy infants aged 15-27 months at the time of booster vaccination. Each participant had received three doses of a DTaP vaccine (Infanrixtrade mark, GlaxoSmithKline, Rixensart, Belgium; "reference DTaP") at age 3, 4, and 5 months as part of a previous clinical trial. More than 20,000 children were eligible for participation in the current study protocol at the time. In a first phase at a University hospital-based vaccination study center, nine sequential cohorts of 63-119 study subjects received one of nine different candidate vaccines. Patients and study personal were blinded with regard to which vaccine was currently in use. Reactogenicity was solicited from parents using diary cards. Blood was drawn prior to and 4 weeks after vaccination and immediately centrifuged. The serum was stored at -20 degrees C until serology was performed by ELISA tests. As soon as the first candidate vaccine with adequate reactogenicity and immunogenicity profile was identified in the first study phase, a second study phase was initiated in parallel, to evaluate the safety and reactogenicity of the respective candidate vaccine in private practices in large cohorts (1613-2095 study subjects per group). RESULTS: In the first study phase, DTaP with no aluminum induced the highest frequency of ETS and fever. All other candidate vaccines caused lower rates of local and general reactions than the reference DTaP. As a general rule, vaccines with less antigen induced fewer reactions, although there was no strict dose-response effect and the difference, e.g. between a one-tenth and a one-fifth DTaP dose (DTaP 1/5; DTaP 1/10) was not clinically relevant. Separate injections of Td and aP caused fewer general reactions than the respective TdaP combination and local reactions were higher at the aP than at the Td injection site. Again, as a general rule, reduced amounts of antigen induced lower antibody concentrations, although all vaccines induced "protective" anti-tetanus and anti-diphtheria antibody responses. A total of 92-100% of children showed seroresponses to pertussis antigens even when vaccinated with reduced amounts of the respective pertussis antigen. Elimination of aluminum from DTaP vaccine induced higher anti-tetanus-antibody concentrations and so did a reduction of the amount of diphtheria antigen. Additional examples for antigen interaction were increased antibody concentrations, observed with injection of Td and aP into different limbs. In the second study phase, all three vaccines evaluated (one with a reduced amount of diphtheria antigen, TdaP; one with reduced amounts of all antigens, tdap; and one with a fifth dose of the reference vaccine (DTaP 1/5)) were safe and had an acceptable reactogenicity profile in a total of 4871 study subjects. CONCLUSIONS: Local reactions due to DTaP booster doses in the second year of life can be reduced by reducing the amount of antigen in the respective vaccine while an adequate immunogenicity is maintained. Aluminum-free vaccines induced ETS and fever most commonly. Any changes in vaccine composition should lead to a full evaluation of the new product.

Cell mediated and antibody immune response to inactivated hepatitis A vaccine.

The humoral and cellular immune response to inactivated hepatitis A vaccine was investigated dynamically in a time elapse study over 1 year. Fourty-five healthy volunteers, seronegative for anti-HAV, were vaccinated with 1440 enzyme-linked immunosorbent assay units (EU) of formalin-inactivated hepatitis A virus following a 0--6-month schedule. Serum anti-HAV levels and HAV-specific proliferation of peripheral blood mononuclear cells were measured at several time points over a 26- and 28-week period after the first and second injection, respectively. Distinct B and T cell responses were determined within 14 days after primary vaccination. The booster vaccination-induced immediate peak levels for the humoral (anti-HAV GMC=5376mIU/ml) as well as the cellular (median Deltacpm=14173cpm) response.

Comparison of the immunogenicity and reactogenicity of two commercially available hexavalent vaccines administered as a primary vaccination course at 2, 4 and 6 months of age.

Infants (N = 459) were randomly assigned to receive either Infanrix hexa or Hexavac vaccines at 2, 4 and 6 months of age as a primary vaccination schedule. The immunogenicity of the hepatitis B component was statistically significantly higher for Infanrix hexa compared to Hexavac in terms of both seroprotection (98.6% versus 94.7%, p = 0.0302) and GMCs (905.6 versus 226.4, p < 0.0001). Significantly (p < or =0.0001) higher antibody levels against diphtheria and the 3 polio components were also induced by Infanrix hexa. The responses to tetanus, Hib and pertussis components were similar. The incidences of clinically relevant solicited symptoms, unsolicited symptoms or serious adverse events were low in both groups.

Electrokinetic measurements of membrane capacitance and conductance for pancreatic beta-cells.

Membrane capacitance and membrane conductance values are reported for insulin secreting cells (primary -cells and INS-1 insulinoma cells), determined using the methods of dielectrophoresis and electrorotation. The membrane capacitance value of 12.57 (+/-1.46) mFm(-2), obtained for -cells, and the values from 9.96 (+/-1.89) mFm(-2) to 10.65 (+/-2.1) mFm(-2), obtained for INS-1 cells, fall within the range expected for mammalian cells. The electrorotation results for the INS-1 cells lead to a value of 36 (+/-22) Sm(-2) for the membrane conductance associated with ion channels, if values in the range 2-3 nS are assumed for the membrane surface conductance. This membrane conductance value falls within the range reported for INS cells obtained using the whole-cell patch-clamp technique. However, the total 'effective' membrane conductance value of 601 (+/-182) Sm(-2) obtained for the INS-1 cells by dielectrophoresis is significantly larger (by a factor of around three) than the values obtained by electrorotation. This could result from an increased membrane surface conductance, or increased passive conduction of ions through membrane pores, induced by the larger electric field stresses experienced by cells in the dielectrophoresis experiments.

Safety, reactogenicity and immunogenicity of a combined hexavalent tetanus, diphtheria, acellular pertussis, hepatitis B, inactivated poliovirus vaccine and Haemophilus influenzae type b conjugate vaccine, for primary immunization of infants.

Safety, reactogenicity and immunogenicity of GSK Biologicals' hexavalent DTPa-HBV-IPV/Hib vaccine (Infanrix)hexa) was assessed when used for primary vaccination at 3, 4 and 5 months of age (N = 2163), compared to the separate administration of DTPa-IPV/Hib and HBV vaccines (N = 720). A similar safety and reactogenicity profile was demonstrated for both vaccine regimens, as well as a good immune response for all antigen components. By offering protection against six diseases in a series of single injections, the hexavalent DTPa-HBV-IPV/Hib vaccine was shown to be a safe, well tolerated and immunogenic alternative to primary immunization with licensed separately administered vaccines.

Pandemic preparedness: lessons learnt from H2N2 and H9N2 candidate vaccines.

Vaccination against influenza is considered to be one of the key interventions in case of a pandemic. Unfortunately, shortages in vaccine supplies will occur because of the substantial increase in vaccine demands worldwide and the limited available supply resources. The recommended use of monovalent--instead of current trivalent--vaccines containing 15 micro g hemagglutinin (HA) per dose can theoretically triple vaccine volumes but is unlikely to meet the demand. Furthermore, previous experiences demonstrated that one dose of 15 micro g HA will not be sufficient to elicit protective antibody levels in unprimed individuals. Modified formulation approaches were investigated, that would be suitable to provide significantly higher volumes of potent vaccine within a given period of time. Low doses of HA combined with aluminum (Al) adjuvants and the use of whole virus instead of split or subunit antigens can lead to substantial increases in process yield. In addition, production of whole virus vaccines will reduce manufacturing complexity. In a dose-finding study in healthy adults and elderly, immune responses after administration of Al-adjuvanted low-dose formulations were compared to a standard split virus vaccine (Fluarix, GlaxoSmithKline Biologicals, Rixensart, Belgium). All vaccines were safe and well tolerated. Antigen concentrations as low as 1.9 micro g HA/strain per dose of adjuvant-containing experimental vaccines induced protective antibody levels in primed populations. Reactogenicity profiles of Al-adjuvanted low-dose vaccines were investigated in a feasibility trial. Neither the use of Al-adjuvant nor of whole virus had a significant effect on general reactions. Studies in unprimed populations with H2N2 and H9N2 candidate vaccines showed different results, with a potential need for a two-dose schedule. Indeed, hemagglutination inhibition titers did not reach protective levels after a single vaccine dose but could be met following administration of a second dose. The same is true for Al-adjuvanted whole virus formulations with an up to eightfold-reduced antigen content. It may be concluded that the use of Al-adjuvanted whole virus vaccines with low HA content can raise protective antibody levels after two vaccine doses, which may, in turn, result in significant increases of vaccine supplies in the case of a pandemic.

A new accelerated vaccination schedule for rapid protection against hepatitis A and B.

BACKGROUND: Increasing travel stresses the requirement for rapid protection against infections such as hepatitis A and B. METHODS: This randomised, multicentre study investigated an accelerated vaccination schedule using a combined hepatitis A and B vaccine (Twinrix, Smithkline Beecham Biologicals) compared with simultaneous administration of the two corresponding monovalent vaccines. The combined vaccine was administered on days 0, 7 and 21, whereas the comparison group received hepatitis A vaccine on day 0 and hepatitis B vaccine on days 0, 7 and 21. All subjects received booster vaccination at month 12. RESULTS: At month 1, 100% of subjects in the combined group and 99% of the controls were seropositive for anti-HAV antibodies. The corresponding seroprotection rates for anti-HBs antibodies were 82.0 and 83.9%, respectively. Examination of the 95% confidence intervals (CIs) for the treatment differences showed the two vaccines to be equivalent in terms of immunogenicity 1 week after the initial vaccination course. Just prior to the booster, the seropositivity rate for anti-HAV was 96.2% in the combined group and 95% in the control group. For anti-HBs, this was 94 and 91.6%, respectively. All subjects were seropositive for anti-HAV and seroprotected against hepatitis B at month 13. The anti-HAV GMCs were 9571mIU/ml with the combined vaccine and 5206mIU/ml in control subjects. The anti-HBs titre was 26002 and 29,196mIU/ml, respectively. Both groups had a similar reactogenicity profile. CONCLUSIONS: The accelerated schedule of the combined vaccine provides a good immune response against hepatitis A and B antigens and is suitable for last minute immunisation.

Development and application of a self-referencing glucose microsensor for the measurement of glucose consumption by pancreatic beta-cells.

Glucose gradients generated by an artificial source and beta-cells were measured using an enzyme-based glucose microsensor, 8-microm tip diameter, as a self-referencing electrode. The technique is based on a difference measurement between two locations in a gradient and thus allows us to obtain real-time flux values with minimal impact of sensor drift or noise. Flux values were derived by incorporation of the measured differential current into Fick's first equation. In an artificial glucose gradient, a flux detection limit of 8.2 +/- 0.4 pmol.cm(-2).s(-1) (mean +/- SEM, n = 7) with a sensor sensitivity of 7.0 +/- 0.4 pA/ mM (mean +/- SEM, n = 16) was demonstrated. Under biological conditions, the glucose sensor showed no oxygen dependence with 5 mM glucose in the bulk medium. The addition of catalase to the bulk medium was shown to ameliorate surface-dependent flux distortion close to specimens, suggesting an underlying local accumulation of hydrogen peroxide. Glucose flux from beta-cell clusters, measured in the presence of 5 mM glucose, was 61.7 +/- 9.5 fmol.nL(-1).s(-1) (mean +/- SEM, n = 9) and could be pharmacologically modulated. Glucose consumption in response to FCCP (1 microM) transiently increased, subsequently decreasing to below basal by 93 +/- 16 and 56 +/- 6%, respectively (mean +/- SEM, n = 5). Consumption was decreased after the application of 10 microM rotenone by 74 +/- 5% (mean +/- SEM, n = 4). These results demonstrate that an enzyme-based amperometric microsensor can be applied in the self-referencing mode. Further, in obtaining glucose flux measurements from small clusters of cells, these are the first recordings of the real-time dynamic of glucose movements in a biological microenvironment.

Oxygen consumption oscillates in single clonal pancreatic beta-cells (HIT).

Based on population studies, we have hypothesized that changes in metabolism in pancreatic beta-cells precede changes in Ca2+. It is well known from single-cell Ca2+ studies that variable oscillatory patterns in Ca2+ occur in response to glucose stimulation. The present studies, using the clonal beta-cell line HIT-T-15, were undertaken to evaluate the relationship between glucose concentration, insulin secretion, and O2 consumption and to determine the Ca2+ dependency of glucose-induced changes in O2 consumption. In population studies, an excellent correlation was found between respiration and insulin secretion, with half-maximal values at approximately 1 mmol/l glucose for both respiration and secretion. In the absence of Ca2+, glucose stimulated O2 consumption but not insulin secretion. In single clonal beta-cells, a self-referencing O2 electrode was used to assess O2 consumption. Large-amplitude oscillations were found to occur in response to stimulation by glucose and were blocked by uncoupling respiration with carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP). They were also blocked and respiration totally inhibited by antimycin A, an inhibitor of complex III of the respiratory chain. Half of the cells sampled (approximately 100 total) exhibited increased oscillatory O2 consumption in response to glucose. Oscillations in O2 occurred in response to glucose even in the absence of Ca2+, and their amplitude increased further on restoration of a normal extracellular Ca2+ level. These studies indicated that oscillatory O2 consumption was not dependent on Ca2+ but that the amplitude of the O2 oscillations increased in the presence of Ca2+, possibly reflecting the additional work involved in insulin secretion and Ca2+ pumping. These studies demonstrated, for the first time, a direct correlation between O2 consumption and insulin secretion, the oscillatory nature of O2 consumption in single cells, and the feasibility of using a highly sensitive noninvasive on-line self-referencing O2 electrode to monitor single beta-cell respiration.

Primary vaccination of infants with diphtheria-tetanus-acellular pertussis-hepatitis B virus- inactivated polio virus and Haemophilus influenzae type b vaccines given as either separate or mixed injections.

OBJECTIVE: The aim of this open, multicenter, randomized trial was to evaluate the immunogenicity and reactogenicity of a candidate combined diphtheria-tetanus-acellular pertussis-hepatitis B virus-inactivated polio virus (DTaP-HBV-IPV) vaccine when given as either a mixed or as separate concomitant injections with Haemophilus influenzae type b (Hib) vaccine. STUDY DESIGN: A total of 359 subjects were randomized to receive either DTaP-HBV-IPV/Hib (mixed administration - 180 subjects) or DTaP-HBV-IPV + Hib (separate administration in opposite limbs - 179 subjects) at 2, 3, and 4 months of age. RESULTS: After vaccination, seroprotective antibody concentrations against diphtheria, tetanus, hepatitis B, and polio viruses and a high (> or = 97%) pertussis vaccine response were seen in almost all study participants. All subjects except one in the mixed administration group had postvaccination Hib anti-PRP antibody concentrations > or = 0.15 microg/mL. Of subjects in the mixed and separate group, 77.2% (geometric mean antibody concentration, 2. 62 microg/mL) and 88.6% (geometric mean antibody concentration, 4.45 microg/mL) had Hib anti-PRP concentrations > or = 1 microg/mL, respectively. The addition of the Hib component to the 5-component vaccine did not increase the incidence of local or general reactions. CONCLUSION: Both administrations of the candidate vaccine were found to be safe, immunogenic, and well tolerated. Although anti-PRP geometric mean antibody concentrations and the percent of subjects achieving the 1 microg/mL seroprotective level were lower after the mixed administration, they were in the range seen with monovalent Hib vaccines or with other DTaP-based/Hib combinations licensed in some European countries. Therefore both administrations have the potential to simplify childhood immunization.

Can monovalent hepatitis A and B vaccines be replaced by a combined hepatitis A/B vaccine during the primary immunization course?

A combined hepatitis A/B vaccine (Twinrix Adult) has been licensed in Germany since 1997. We investigated possible differences in immunogenicity and safety when changing over from vaccinations with monovalent vaccines made by different manufacturers to vaccinations with the combined hepatitis A/B vaccine in an open, randomized, multicenter trial. We therefore compared four different schemes changing over from concomitant vaccinations with monovalent vaccines against hepatitis A and B (Havrix 1440+Engerix-B or Vaqta+Gen H-B-Vax) to combined vaccination against hepatitis A+B with three injections of the combined hepatitis A/B vaccine (0, 1, and 6 month schedule). Local and general symptoms were mostly mild in all five groups. With complete three-dose course using the combined vaccine or an early changeover from monovalent vaccines to the combined vaccine, higher overall anti-HBs seroprotection rates and geometric mean concentrations (GMCs) against hepatitis B could be achieved as early as after 2 months as compared to those groups switching later to the combined vaccine. This study demonstrated for the first time that switching from monovalent hepatitis A and B vaccinations to the combined hepatitis A and B vaccination has no negative influence on the tolerability and improves the immunogenicity.

Self-referencing, non-invasive, ion selective electrode for single cell detection of trans-plasma membrane calcium flux.

Biological systems have very different internal ion compositions in comparison with their surrounding media. The difference is maintained by transport mechanisms across the plasma membrane and by internal stores. On the plasma membrane, we can classify these mechanisms into three types, pumps, porters, and channels. Channels have been extensively studied, particularly since the advent of the patch clamp technique, which opened new windows into ion channel selectivity and dynamics. Pumps, particularly the plasma membrane Ca(2+)-ATPase, and porters are more illusive. The technique described in this paper, the self-referencing, ion-selective (or Seris) probe, has the ability to monitor the behavior of membrane transport mechanisms, such as the pumps and porters, in near to real-time by non-invasively measuring local extracellular ion gradients with high sensitivity and square micron spatial resolution. The principles behind the self-referencing technique are described with an overview of systems utilizing ion, electrochemical and voltage sensors. Each of these sensors employs the simple expedient of increasing the system resolution by self-referencing and, thereby, removing the drift component inherent to all electrodes. The approach is described in detail, as is the manner in which differential voltage measurements can be converted into a flux value. For the calcium selective probes, we can resolve flux values in the low to sub pmol.cm(-2)s(-1) range. Complications in the use of the liquid ion exchange cocktail are discussed. Applications of the calcium selective probe are given, drawing on examples from the plant sciences, developmental biology, muscle physiology, and the neurosciences.

The self-referencing oxygen-selective microelectrode: detection of transmembrane oxygen flux from single cells.

A self-referencing, polarographic, oxygen-selective microelectrode was developed for measuring oxygen fluxes from single cells. This technique is based on the translational movement of the microelectrode at a known frequency through an oxygen gradient, between known points. The differential current of the electrode was converted into a directional measurement of flux using the Fick equation. Operational characteristics of the technique were determined using artificial gradients. Calculated oxygen flux values matched theoretical values derived from static measurements. A test preparation, an isolated neuron, yielded an oxygen flux of 11.46+/-1.43 pmol cm-2 s-1 (mean +/- s.e.m.), a value in agreement with those available in the literature for single cells. Microinjection of metabolic substrates or a metabolic uncoupler increased oxygen flux, whereas microinjection of KCN decreased oxygen flux. In the filamentous alga Spirogyra greveilina, the probe could easily differentiate a 16.6% difference in oxygen flux with respect to the position of the spiral chloroplast (13.3+/-0.4 pmol cm-2 s-1 at the chloroplast and 11.4+/-0.4 pmol cm-2 s-1 between chloroplasts), despite the fact that these positions averaged only 10.6+/-1.8 microm apart (means +/- s.e.m.). A light response experiment showed real-time changes in measured oxygen flux correlated with changes in lighting. Taken together, these results show that the self-referencing oxygen microelectrode technique can be used to detect local oxygen fluxes with a high level of sensitivity and spatial resolution in real time. The oxygen fluxes detected reliably correlated with the metabolic state of the cell.

Workforce issues in pediatric dentistry: recommendations of a recent conference.

A recent conference of the California Society of Pediatric Dentists discussed issues surrounding the shortage of pediatric dentists in the state. Several contributing factors were identified and recommendations made regarding ways of working with the dental schools to help increase the number of practitioners. This article frames the problem regarding servicing the state's children and discusses some recommended actions.

The influence of major histocompatibility complex class II genes and T-cell Vbeta repertoire on response to immunization with HBsAg.

Nonresponsiveness to HBsAg vaccination is observed in 5-10% of vaccine recipients and is possibly caused by a defect in the T helper cell compartment. The immune response to HBsAg is influenced by genes of the major histocompatibility complex. We have investigated MHC class I and class II antigens in 53 adult responders and 73 nonresponders. Results obtained in this first study were tested in a second study with 56 responders and 62 nonresponders from an infant vaccination trial. In addition, the peripheral Vbeta-chain T-cell receptor repertoire was investigated using monoclonal antibodies and flow-cytometry in 26 adult responders and 38 nonresponders. As previously reported, nonresponsiveness to HBsAg vaccination was associated with DRB1*3 and DRB1*7. In addition, DRB1*13 was significantly increased among vaccine responders (35.2% vs 5.4%;p < 0.0001) suggesting an immune response promoting effect for this allele whereas the closely related allele DRB1*14 was associated with nonresponse in the infant study. There was no evidence for a hole in the T cell receptor Vbeta repertoire. In conclusion, in agreement with results obtained in mice there appears to be a hierarchy of DRB1* genes in the HBsAg immune response. The possible differential association of DRB1*13 and DRB1*14 may allow the identification of differences between responsiveness and nonresponsiveness to a few amino acid differences in the beta1-domain of the class II heterodimer.

Booster immunization of low- and non-responders after a standard three dose hepatitis B vaccine schedule--results of a post-marketing surveillance.

Seventy-nine low-responders and 83 non-responders after a previous three-dose hepatitis B (HB) vaccine course at 0.1, and 6 months were enrolled to receive additional 20 micrograms recombinant HB vaccine doses every 2 months until all had anti-HBs levels > or = 100 mIU ml-1. After the first booster, 65.4% had anti-HBs levels > or = 100 mIU ml-1, 17.9% were low-responders (10-99 mIU ml-1), and 16.7% remained non-responders (< 10 mIU ml-1). All complying non-responders developed anti-HBs levels > or = 100 mIU ml-1 after the third booster at the latest, whereas all low-responders reached this level after the second booster. Although body mass index affected the response to the first hepatitis B booster, when full compliance to regular revaccination was ensured, all non- and low-responders eventually reached sufficient anti-HBs levels.

O2 availability modulates transmembrane Ca2+ flux via second-messenger pathways in anoxia-tolerant hepatocytes.

Transmembrane Ca(2+)-flux was studied from single isolated turtle hepatocytes by using a noninvasive Ca(2+)-selective self-referencing microelectrode. Cells in Ca(2+)-reduced culture medium demonstrated a vanadate- and lanthanum-inhibitable Ca(2+)-efflux of 4 x 10(-17) mol Ca2+. microns-2. s-1 continuously over 170 h. This flux diminished with 50 nM phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator, and was reinstated on PKC deactivation with sphingosine. Progressive hypoxia resulted in a reversible suppression of Ca2+ efflux to 90% of normoxic controls with an apparent Michaelis constant for oxygen of 145 microM. PKC activation was critical in this suppression, as anaerobic administration of sphingosine caused a Ca2+ influx and cell rupture. Hypoxia was also associated with an altered pattern of adenosine-mediated control over Ca2+ efflux. Adenosine (100 microM) elevated Ca2+ efflux twofold in normoxia, but neither adenosine nor the A1-purinoreceptor antagonist 8-phenyltheophylline altered the observed anaerobic suppression. Aerobic administration of 2-10 mM KCN failed to reproduce the anaerobic suppression; however, in conjunction with 10 mM iodoacetate, complete metabolic blockade caused a Ca2+ influx and cell rupture. These observations suggest modulatory control by oxygen over transmembrane Ca2+ efflux involving second-messenger systems in the hypoxic transition.