Biomedical subjects

C R Howard

Publications and source records attributed to C R Howard.

At least 19 recordsLinked to original sources

PubMed2003

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.

Amino Acid Substitution↗

PubMed2001

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.

Amino Acid Sequence↗

PubMed2001

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.

Amino Acid Sequence↗

PubMed2001

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.

Amino Acid Sequence↗

PubMed2001

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.

Breast Feeding↗

PubMed2001

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.

Allied Health Personnel↗

PubMed2001

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.

Hepatitis B↗

PubMed2000

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.

Adjuvants, Immunologic↗

PubMed2000

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.

Animals↗

PubMed2000

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.

Child, Preschool↗

PubMed1999

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.

Animals↗

PubMed1999

DNA is as effective as protein at inducing antibody in fish.

Antiviral vaccines are needed for fish. 50 microg plasmid DNA in saline by the intramuscular route and 10 microg beta-gal protein in a commercial oil adjuvant by the peritoneal route induced serum antibody of the same titre and avidity in goldfish. The DNA expressed beta-gal under control of the immediate early promoter/enhancer gene of human cytomegalovirus. Commercial bacterin vaccines are administered to fish by the intraperitoneal route with oil and this route for DNA induced only 2-fold less antibody than DNA by the intramuscular route. Bacterin vaccines and antiviral plasmid DNA could therefore be co-injected into the peritoneum of fish in an oil adjuvant as a single dose.

Animals↗

PubMed1999

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.

Amino Acid Sequence↗

PubMed1999

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.

Administration, Cutaneous↗

PubMed1999

Why screen newborns for cocaine: service patterns and social outcomes at age one year.

OBJECTIVE: To compare baseline characteristics, service provision, and child placement for infants exposed to cocaine in utero based on postnatal screening results. METHODS: We studied a retrospective cohort of 40 consecutive drug-exposed, but seemingly healthy term infants who underwent urine drug screening in the newborn nursery of a community hospital. Using clinical and service agency data, two cocaine-exposed cohorts were compared (a) screen-positive at birth (n = 22) versus (b) screen-negative at birth (n = 18). RESULTS: Both cocaine-exposed groups had similar infant birth weights, levels of paternal involvement, maternal ages, gravidity, parity, and lengths of gestation. Mothers in both groups had similar histories of prostitution, poor home environment, drug use, and prenatal drug rehabilitation. Mothers of screen-positive infants were more likely than mothers of screen-negative infants to have other children in foster care (27% vs. 6%, p = .07), to have experienced previous interventions by child protective services (CPS) (55% vs. 17%, p < .01), to have had no prenatal care (32% vs. 6%, p = .09), and fewer prenatal visits (4.7 vs. 8.6, p = .02). Compared to screen-negative infants, more screen-positive infants were referred to a high-risk infant tracking program (91% vs. 6%), referred to CPS (100% vs. 33%), placed outside the mother's home (50% vs. 22%), and had their mothers referred to drug rehabilitation (36% vs. 11%), (p < .01 for each). By 1 year of age, support services differed little between exposed cohorts. However, 6 of 22 screen-positive infants were in foster care and 3 were placed for adoption, while only 1 of the 18 screen-negative infants was in foster care and only 1 had been placed for adoption. There were no services available in this community to provide coordinated or comprehensive services or drug treatment specific to the needs of drug using mothers and drug exposed infants. CONCLUSIONS: Despite similarities between cocaine-exposed infants cared for in a normal newborn setting (with and without positive urine drug screens at birth), differences in referral services were noted. More striking than these differences was that services for families with drug-exposed infants are inadequate to even meet the needs of those families in our setting deemed to be at highest risk. Neonatal drug screening needs to be paired with effective services.

Cocaine-Related Disorders↗

PubMed1999

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.

Amino Acid Sequence↗

PubMed1999

The safety and longevity of DNA vaccines for fish.

A plasmid that contained the cytomegalovirus (CMV)-promoter-driven lacZ reporter gene (pCMV-lacZ) remained in the epaxial muscle of five of eight goldfish as covalently closed circles, the most functional form of plasmid, for at least 70 days at 22 degrees. It was not present in the gills or elsewhere by polymerase chain reaction and was not integrated. Its expressed protein, Escherichia coli beta-galactosidase (beta-gal), which was in the injected myofibres, was detected in all the fish at 4-21 days and in about half the fish from 28 days until the end of the experiment at 70 days. The numbers of cells that secreted antibody to beta-gal in the kidney peaked at 14 days. Serum antibody and proliferating kidney cells to beta-gal were in all fish from 14 days with a plateau of the responses from 21 days onwards. The plasmid did not induce autoimmune-like antibodies to itself or to single- or double-stranded salmon testis DNA. Plasmids can therefore induce long-term foreign protein expression whilst inducing humoral and cell-mediated immunity without autoimmunity or integration in goldfish.

Animals↗

PubMed1999

The effects of early pacifier use on breastfeeding duration.

OBJECTIVE: To evaluate the effects of pacifier use and the timing of pacifier introduction on breastfeeding duration, problems, and frequency. METHODS: A cohort of 265 breastfeeding mother-infant dyads was followed prospectively. Maternal interviews were conducted at delivery, 2, 6, 12, and 24 weeks, and thereafter every 90 days until breastfeeding ended. Information was obtained regarding pacifier use, infant feeding, use of supplemental foods and breastfeeding frequency, duration, and problems. The effect of pacifier introduction by 6 weeks of age on breastfeeding duration was evaluated with Kaplan-Meier and Cox proportional hazards models. The effect of the timing of pacifier introduction (</=2 weeks and </=6 weeks) on breastfeeding duration at 2 and 3 months was evaluated using logistic regression modeling. RESULTS: A total of 181 mothers (68%) introduced a pacifier before 6 weeks. In adjusted analyses, pacifier introduction by 6 weeks was associated with a significantly increased risk for shortened duration of full (hazard ratio, 1.53; 95% confidence interval: 1.15, 2.05) and overall (hazard ratio, 1.61; 95% confidence interval: 1.19,2.19) breastfeeding. Women who introduced pacifiers tended to breastfeed their infants fewer times per day, with significant differences noted at 2 (8.1 +/- 2.6 vs 9.0 +/- 2.3) and 12 weeks' (6.3 +/- 2.0 vs 7.4 +/- 1.6) postpartum. At 12 weeks postpartum, women who introduced pacifiers also were more likely to report that breastfeeding was inconvenient and that they had insufficient milk supplies. Pacifier use begun either before 2 weeks or before 6 weeks' postpartum was not significantly associated with breastfeeding duration at 2 and 3 months. CONCLUSIONS: Pacifier use was independently associated with significant declines in the duration of full and overall breastfeeding. Breastfeeding duration in the first 3 months' postpartum, however, was unaffected by pacifier use. Women who introduced pacifiers tended to breastfeed their infants less frequently and experienced breastfeeding problems consistent with infrequent feeding. Findings from this study suggest that the decreases in breastfeeding duration associated with pacifier use may be a consequence of less frequent breastfeeding among women who introduce pacifiers to their infants.

Breast Feeding↗