Lentiviral hepatitis B pseudotype entry requires sodium taurocholate co-transporting polypeptide and additional hepatocyte-specific factors.

Hepatitis B virus (HBV) is one of the world's major unconquered infections, resulting in progressive liver disease, and current treatments rarely cure infection. A limitation to discovering new therapies is our limited knowledge of HBV entry and dissemination pathways that hinders the development of in vitro culture systems. To address this gap in our understanding we optimized the genesis of infectious lentiviral pseudoparticles (HBVpps). The recent discovery that the bile salt transporter sodium taurocholate co-transporting polypeptide (NTCP) acts as a receptor for HBV enabled us to assess the receptor dependency of HBVpp infection. HBVpps preferentially infect hepatoma cells expressing NTCP, whereas other non-liver cells engineered to express NTCP do not support infection, suggesting that additional hepatocyte-specific factors are required for HBVpp internalization. These results highlight the value of the HBVpp system to dissect the pathways of HBV entry and dissemination.

A peptide mimotope of hepatitis C virus E2 protein is immunogenic in mice and block human anti-HCV sera.

Conformational B-cell epitopes on the HCV E2 protein recognized by human antibodies were characterized by the use of a peptide mimotope named K1. K1 was identified by two HCV anti-E2 monoclonal antibodies (mAbs) following selection and purification of phage clones containing a 15-mer random peptide insert. Murine antisera to the mimotope K1 recognized the E2 protein. Five of eight human sera from patients who had cleared HCV recognized the K1 mimotope. Binding to E2 in four individuals with the capacity to block E2-CD81 interaction was inhibited by the mimotope K1. The results demonstrate that anti-E2 antibodies in sera from patients who have cleared HCV infection are directed against a conformational B-cell epitope on E2 that can be mimicked with linear synthetic peptides. These findings could have implications for vaccine design by employing linear mimotopes to direct B-cell responses against those specific E2 epitopes that may correlate with immunity.

Hepatitis B virus S gene mutants in a patient with chronic active hepatitis with circulating Anti-HBs antibodies.

An adult male farmer with chronic active hepatitis and cirrhosis despite previous circulating anti-HBs antibodies was studied. No markers of other hepatotropic viral infection were observed. HBV DNA was detected in serum by PCR and was characterized further by restriction fragment length polymorphism (RFLP) and sequencing of cloned PCR products derived from the S gene. The HBV DNA was ascribed to genotype F, and single-strand conformational polymorphism (SSCP) demonstrated the co-circulation of multiple quasispecies. Some of the variants exhibited changes located within the neutralizing "a" determinant, located between amino acids 124-147 of the S protein. Within this region, two clones showed either C124R or C124Y mutations. Other mutations were Q129R, C138R, C139R, and S140T (one clone each). Outside the "a" determinant several substitutions were documented. The high degree of the quasispecies variability was probably linked to the severity of the infection. Most members of the patient's family were infected with HBV, all with genotype F.

Recent advances in the development of peptide vaccines for hepatitis B.

The control of hepatitis B by vaccination is arguably one of medicine's greatest achievements in terms of protecting infants and adults at high risk of infection. Paradoxically, however, the existence of a large reservoir of chronically infected individuals will not diminish the risk of infection by those coming into close contact with such persons until universal infant immunisation is practised globally and vaccines are in place to ensure maximum efficacy in those with impaired immune responses, immunity is achieved with fewer doses, and immunisation as an adjunct to the antiviral treatment of chronic carriers is adopted. These imperatives have continued to stimulate research into vaccines based on chemically synthesised short peptides, and those systems best suited for their delivery. This review discusses the potential of synthetic peptide formulations as efficient inducers of both humoral and cellular immune responses against hepatitis B, and reviews recent advances in peptide delivery. Synthetic peptide and delivery systems technologies will, amongst others, be of paramount importance in the global fight for the eradication of hepatitis B in the 21st century.

Xenobiotics and breastfeeding.

Breastfeeding provides important benefits to mothers and infants and should be encouraged strongly as the optimal feeding choice for most infants. In assessing the effects of maternal medication on breastfeeding, clinicians must weigh the many benefits of breastfeeding for mothers and infants against the risk for exposing infants to a drug as it is present in breast milk. With regard to most medications, continued breastfeeding despite drug exposure is advantageous to mothers and infants.

The role of lactation specialists. A guide for physicians.

Breastfeeding support is a team event. The physician works with many health care professionals to provide complete care to the perinatal patient, including working with nurses trained in prenatal care, labor, delivery, and postpartum and newborn care, and working with midwives who provide prenatal care, labor, and delivery for the normal, uncomplicated patient. The pediatrician is part of this team and interacts with all of these players and with the office or clinic staff who provide follow-up, newborn, and child care.

A novel microencapsulated peptide vaccine against hepatitis B.

A 48 amino acid synthetic peptide (S121/48) representing residues 121-167 of the major envelope protein of hepatitis B virus (HBsAg) was successfully encapsulated into polylactide co-glycolide microspheres. A single immunization of the microspheres in BALB/c (H-2d) mice resulted in the production of high-titre anti-HBs antibodies (IgG1-type). The response was long lasting and was superior to that obtained using the same peptide adjuvanted with Freund's complete adjuvant. A T-cell memory response was detected 10 weeks after a booster immunization (approximately 35 weeks after initial immunization) as measured by in-vitro re-stimulation of splenocytes. This study illustrates the feasibility of a single dose vaccine for hepatitis B and is, to our knowledge, the first demonstration of a synthetic peptide immunogen inducing anti-native protein antibodies of comparable titre to those obtained with conventional vaccines for hepatitis B. The suitability of a synthetic peptide vaccine for hepatitis B is discussed.

An improved selection procedure for the screening of phage display peptide libraries.

Peptide sequences that bind to a wide range of ligands such as monoclonal antibodies, receptors and carbohydrates have been successfully identified after screening phage display peptide libraries. However, these procedures tend to select mainly medium to low affinity-binding clones. A modified screening procedure has been developed in order to improve the efficiency of this process such that high avidity/affinity binding clones are preferentially selected. Three different solid phase binding surfaces were evaluated for the attachment of antibody during the screening procedure and a stepwise decrease in the pH of the elution buffer introduced during the final round of biopanning. The monoclonal antibody MA 18/7 was used to screen a 15-mer peptide library. This antibody is well-characterised and its binding site has been mapped to residues 28-37 of the pre-S1 protein of the hepatitis B virus. The antibody was either biotinylated and attached to polystyrene plates via a streptavidin-biotin 'bridge', or bound directly to 1/4 in. polystyrene beads, or to 11 microm latex beads. A significant enrichment of binding clones was observed when the monoclonal antibody was attached directly to polystyrene or latex beads as compared to the biotinylated antibody. All mimotopes identified after biopanning with the antibody attached to the polystyrene beads possessed a central core motif, identical or similar to the sequence DPAF contained within the epitope binding site of MA 18/7 on the native pre-S molecule. However, this motif was only observed in 30% of clones isolated after biopanning using the 11 microm latex beads and in 2% of clones isolated after biopanning on the streptavidin-coated plates. Immunoblotting with the monoclonal antibody MA 18/7 confirmed binding to clones containing the DPAF sequence or a similar motif. A stepwise reduction in the pH of the elution buffer in the final round of biopanning resulted in the removal of clones that possessed low affinity binding motifs, thereby increasing the percentage of clones containing high affinity binding motifs in the final elution step at pH 2.0. Thus, the combined use of polystyrene beads and a stepwise decrease in the pH of the elution buffer in the final round of biopanning resulted in the elimination of non-binding clones and an increase in the efficiency in isolating high affinity binding clones.

Rotavirus cross-species pathogenicity: molecular characterization of a bovine rotavirus pathogenic for pigs.

Rotaviruses which cause disease in heterologous animal species have been reported but the molecular basis of cross-species infectivity and disease is not established. We report the molecular characterization of a cloned rotavirus, PP-1, which was originally obtained from cattle and which had been biologically characterized in vivo in two target animal species, gnotobiotic pigs and calves. In pigs, PP-1 caused severe clinical disease but in experimental calves it replicated subclinically. PP-1 was characterized as a G3 reassortant with a porcine VP4 and NSP4 but a bovine NSP1. The PP-1 VP4 had 96 to 97% deduced amino acid identity to P[7] porcine rotaviruses and P[7] specificity was confirmed with VP4-specific monoclonal antibodies. Sequence analysis of the PP-1 NSP1 showed 94 to 99.6% deduced amino acid identity to bovine rotaviruses but the NSP4 protein had 94 to 98% identity to the NSP4 genotype B porcine rotaviruses. G-typing PCR initially classified PP-1 as a G10 rotavirus but sequence analysis revealed 92 to 96% identity of the PP-1 VP7 with porcine, simian, and human G3 rotaviruses. These results, combined with the in vivo properties of PP-1 in the two target species, supported the concept that species-specific VP4 and NSP4, but not NSP1, are required to induce rotavirus disease, at least in calves and pigs. The results illustrate experimentally that rotaviruses circulating in one animal species can pose a risk to another by the emergence of a pathogenic reassortant rotavirus under appropriate conditions.

Naked DNA when co-administered intranasally with heat-labile enterotoxin of Escherichia coli primes effectively for systemic B- and T-cell responses to the encoded antigen.

In this study a novel prime-boost immunisation strategy was evaluated. Priming of BALB/c mice by the intranasal route with plasmid DNA encoding beta-galactosidase (LacZ) with or without heat-labile enterotoxin (LT) of Escherichia coli as a mucosal adjuvant, resulted in the induction of weak serum antibody and proliferative T-cell responses. However, following an intraperitoneal booster injection with the beta-galactosidase protein (beta-gal), strong antibody and proliferative T-cell responses were induced in all the mice. These responses were highest in mice primed intranasally with a mixture of LacZ+LT as compared to those mice primed with DNA (LacZ) or protein (beta-gal) alone. Moreover, LacZ+LT primed mice produced high avidity antibodies and the subclasses of serum antibodies were IgG1 and IgG2a, suggesting a mixed Th1/Th2-type response. Priming of mice with either protein (beta-gal) or DNA (LacZ) alone, produced predominantly IgG1 antibodies, suggesting a Th2-type response. These findings suggest that the use of a heterologous DNA-prime, protein-boost immunisation scheme combining different routes of administration, might be an advantageous strategy for the induction of accelerated immune responses.

Long-term effect of dust control on blood lead concentrations.

BACKGROUND: Dust control is recommended to prevent children's exposure to residential lead hazards, but the long-term effect of dust control on children's exposure to environmental lead is unknown. OBJECTIVE: To determine the effect of dust control on children's exposure to lead, as measured by blood lead concentration at 48 months of age. DESIGN: A randomized, controlled trial. Setting. Rochester, New York. PARTICIPANTS: A total of 275 urban children were randomized at 6 months of age; 189 (69%) were available for the 48-month follow-up blood test. Intervention. Children and their families were randomly assigned to an intervention group that received cleaning equipment and up to 8 visits by a trained lead hazard control advisor or to a control group. The intervention was terminated when the children were 24 months of age. OUTCOME MEASURES: Geometric mean blood lead concentration and prevalence of elevated blood lead concentration (ie, >/=10 microg/dL, >/=15 microg/dL, and >/=20 microg/dL), by group assignment. RESULTS: For children with 48-month blood tests, baseline geometric mean blood lead concentrations were 2.8 microg/dL (95% confidence interval [CI]: 2.6, 3.0); there were no significant differences in baseline characteristics or lead exposure by group assignment. At 48 months of age, the geometric mean blood lead was 5.9 microg/dL (95% CI: 5.3, 6.7) for the intervention group and 6.1 microg/dL (95% CI: 5.5,6.9) for the control group. The percentage of children with a 48-month blood lead >/=10 microg/dL, >/=15 microg/dL, and >/=20 microg/dL was 19% versus 19%, 2% versus 9%, and 1% versus 2% in the intervention and control groups, respectively. CONCLUSIONS: We conclude that dust control, as performed by families and in the absence of lead hazard controls to reduce ongoing contamination from lead-based paint, was not effective in preventing children's exposure to residential lead hazards.

48-mer synthetic peptide analogue of the hepatitis B virus "a" determinant induces an anti-HBs antibody response after a single injection.

An extended (48 amino acid) synthetic peptide analogue of the hepatitus B virus (HBV) S protein (HBsAg) 'a' determinant has been produced by using 9-fluorenylmethoxycarbonyl (fmoc) chemistry and a low substitution polystyrene resin as the solid phase support. This peptide (S121/48) elicited a sustained anti-peptide antibody response in BALB/c (H-2(d)) mice when immunised with Freund's complete adjuvant (FCA). Cross-reactive, anti-HBs antibodies were induced, directed against a significant proportion of the conformationally restrained epitope repertoire on the native HBsAg particles. Similar responses were obtained by injection of guinea pigs, a species known both to be exquisitely sensitive to HBsAg and to produce a wide range of B cell responses to HBsAg antigens. Taken together, these data show for the first time, that a synthetic peptide mimicking conformational epitopes can be produced by chemical synthesis and can be used to induce significant titres of anti-HBs antibodies after a single injection. This immunogen has considerable potential for incorporation into novel delivery systems, e.g., microspheres, thus offering the potential of a controlled release, single dose hepatitis B vaccine.

A randomized, controlled trial of a eutectic mixture of local anesthetic cream (lidocaine and prilocaine) versus penile nerve block for pain relief during circumcision.

OBJECTIVE: We set out to compare a eutectic mixture of local anesthetic cream (lidocaine and prilocaine) to dorsal penile nerve block with lidocaine for anesthesia during circumcision. STUDY DESIGN: In a double-blind study, term newborns were randomized to local anesthetic cream and sodium chloride solution dorsal penile nerve block (n = 31) or to placebo cream and lidocaine dorsal penile nerve block (n = 29). Pain was assessed by determination of heart rate, respiratory rate, and behavioral distress scoring. Group differences were evaluated with repeat-measures analyses of variance. RESULTS: Distress scores and heart rates were significantly higher in the eutectic mixture group than in the lidocaine group. Respiratory rates were higher in the eutectic mixture group but did not reach statistical significance. CONCLUSIONS: Distress scores and heart rates were significantly higher in infants treated with the anesthetic mixture than in infants treated with lidocaine. Dorsal penile nerve block with lidocaine is a more efficacious means of providing anesthesia for neonatal circumcision than the mixture of local anesthetics.

Physiologic stability of newborns during cup- and bottle-feeding.

BACKGROUND: To prevent breastfeeding problems, cup-feeding has been recommended as a method of providing medically necessary supplemental feedings to breastfed infants. OBJECTIVES: To compare amounts ingested, administration time, and infant physiologic stability during cup-, bottle-, and breastfeeding. DESIGN/METHODS: A total of 98 term, healthy newborns were randomized to either cup-feeding (n = 51) or bottle-feeding (n = 47). The heart (HR), respiratory (RR), and oxygen (O(2)) saturation rates were monitored on these infants and 25 breastfed newborns during 1 feeding. Differences in amounts ingested and administration times were evaluated with t tests and physiologic data with repeat measures analysis of variance. RESULTS: There were no significant differences in administration time, amounts ingested or overall HR, RR, and (O(2)) saturation rates, between cup and bottle groups. Breastfed infants had longer administration times and lower overall HR, RR, and higher O(2) saturation as compared with cup- and bottle-fed infants. CONCLUSIONS: Administration times, amounts ingested, and infant physiologic stability do not differ with cup- and bottle-feeding. Breastfeeding takes longer than cup- or bottle-feeding, but infants experience less physiologic variability. These data support cup-feeding as an alternative to bottle-feeding for supplying supplements to breastfed infants.

Mapping the specificity of an anti-feline immunodeficiency virus monoclonal antibody using a filamentous phage displayed peptide library.

Recent developments in peptide technology enable the use of random peptide libraries for identifying linear amino acid sequences (mimotopes) which can mimic conformational epitopes without necessarily exhibiting amino acid sequence homology with the native linear sequence. In this study a 15-mer random peptide library displayed on the surface of a filamentous phage has been used to characterise the conformational epitopes recognised by a monoclonal antibody raised against the envelope protein gp120 of feline immunodeficiency virus (FIV). Three mimotopes were identified that reacted with the selecting antibody in an immunoblot assay. Sequence analysis revealed that, whereas the three mimotopes had several amino acids in common, there was no significant homology with the primary amino acid sequence of gp120 although some amino acids were shared between the variable region (V3) and the three mimotopes. Petide mimtopes of complex retroviral glycoproteins may have potential uses as novel vaccines and for the serological diagnosis of FIV.

A hepatitis B virus variant found in the sera of immunised children induces a conformational change in the HBsAg "a" determinant.

The emergence of variants in the outer envelope proteins of hepatitis B virus (HBV) are found among individuals vaccinated against HBV and asymptomatic carriers of the infection. For example, children in The Gambia vaccinated against hepatitis B may show serological evidence of breakthrough infections, particularly if anti-HBs antibodies induced by the vaccine are low in titre. A single-point mutation at nucleotide 421 of the S gene is associated with such breakthrough infections. In the present study, the antigenicity of variant HBV S protein expressed as HBsAg particles in a vaccinia virus expression system has been characterised using a panel of monoclonal antibodies directed against linear and conformational determinations of the S protein. A cellular ELISA procedure using expressed antigen in Vero cells revealed differences in reactivity using four of the six antibodies that had been raised against the adw subtype of HBV and recognise conformational epitopes in the a determinant. In two instances, an enhanced reactivity for the variant antigen was found, confirming that point mutations in the a determinant of the S protein between residues 139 and 147 may result in significant changes in conformation. These findings also demonstrate that there are distinct antigenic differences between the vaccine strains of HBsAg/ adw subtype and the predominant HBsAg subtype circulating in West Africa. The implications of this work are that serodiagnosis of HBV infections may be unreliable in populations where there is a possibility of variant HBV infections emerging in the face of increasing herd immunity to HBV as a result of vaccination, particularly using monoclonal antibody-based diagnostic tests. Such variants may play a role in the maintenance of HBV infections in endemic regions.

Antibody responses in humans to an inactivated hantavirus vaccine (Hantavax).

Hantaviruses cause haemorrhagic fever with renal syndrome (HFRS) and result in severe human morbidity and mortality. Safe and effective vaccines are needed urgently in order to reduce the incidence of human illness. Hitherto studies of hantavirus vaccine efficiency have been limited to individuals at low risk of infection. In this study the immune response to an inactivated hantavirus vaccine was measured in 64 human volunteers at high risk of infection by virtue of residence and occupation. 30 d after vaccination, 79% of subjects developed a significant hantavirus antibody titre as measured by immunofluorescence (IFA) and 62% by enzyme linked immunosorbent assay (ELISA). Seroconversion rates increased to 97% one month after the booster dose. Neutralising antibody titres paralleled this trend with 13% of vaccine recipients producing neutralising antibody one month after the first dose and 75% of vaccine recipients responding one month after boosting. Antibody titres had declined by one year, however, with only 37% and 43% of sera positive by IFA and ELISA, respectively. Re-vaccination at this time produced a vigorous anamnestic response with 94% and 100% of vaccine recipients yielding positive antibody titres. Only 50% of the sampled population, however, produced neutralising antibodies following the booster dose one year later. The vaccine was well tolerated and there were no apparent differences in the responses of males and females. However, further improvement of this vaccine is necessary in order to induce a more longlasting humoral immune response.

Drugs and breastfeeding.

Breastfeeding provides important benefits to mother and infant and should be strongly encouraged as the optimal infant feeding choice for most infants. In assessing the impact of maternal medication on breastfeeding, the clinician must always weigh the many benefits of breastfeeding for the mother and infant against the risk of exposing the infant to the drug as it appears in breast milk. With regard to the vast majority of medications, continued breastfeeding is advantageous to both mother and infant.

Given the benefits of breastfeeding, are there any contraindications?

There are many benefits to the infant to be breastfed and to the mother to breastfeed. There are, however, a few conditions where the tremendous benefit may be outweighed by maternal infection, disease, drugs, or contaminants as these are rare. HIV is the only infection that is an absolute contraindication in developed countries. Galactosemia is the only infant disease and there are a few medications that are contraindicated.