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1.
Clin Vaccine Immunol ; 18(10): 1728-36, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21852547

ABSTRACT

Lot 89SF has been the reference standard serum pool used in pneumococcal enzyme-linked immunosorbent assays (ELISAs) since 1990. In 2005, it was estimated that there remained between 2 and 5 years' supply of lot 89SF. Since lot 89SF was the reference standard used in the evaluation of the seven-valent pneumococcal conjugate vaccine Prevnar (PCV7), the link to clinical efficacy would be severed if stocks became completely depleted. Furthermore, demonstration of immune responses comparable to those elicited by PCV7 is a licensure approach used for new pneumococcal conjugate vaccines, so a replacement reference standard was required. A total of 278 volunteers were immunized with the 23-valent unconjugated polysaccharide vaccine Pneumovax II, and a unit of blood was obtained twice within 120 days following immunization. Plasma was prepared, pooled, and confirmed to be free from hepatitis B virus (HBV), hepatitis C virus (HCV), and HIV. The pooled serum was poured at 6 ml per vial into 15,333 vials and lyophilized. Immunological bridging of 007sp to 89SF was used to establish equivalent reference values for 13 pneumococcal capsular serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F) by five independent laboratories. Antibody concentrations in 007sp were established relative to the lot 89SF reference preparation using the WHO reference ELISA. Subsequently, 12 existing WHO calibration sera had concentrations reassigned for 13 pneumococcal serotypes using new serum 007sp as the reference, and these were compared to concentrations relative to the original reference serum. Agreement was excellent for the 12 WHO calibration sera. The 007sp preparation has replaced 89SF as the pneumococcal reference standard. Sufficient quantity of this new preparation is available such that, with judicious use, it should be available for at least 25 years.


Subject(s)
Antibodies, Bacterial/blood , Enzyme-Linked Immunosorbent Assay/standards , Streptococcus pneumoniae/immunology , Enzyme-Linked Immunosorbent Assay/methods , Human Experimentation , Humans , Pneumococcal Vaccines/administration & dosage , Reference Standards
3.
Vaccine ; 22(23-24): 3069-79, 2004 Aug 13.
Article in English | MEDLINE | ID: mdl-15297057

ABSTRACT

We developed a murine model for assessment of immunological memory and antibody-induced protection to nasopharyngeal (NP) challenges. BALB/c female mice (n = 10 mice per study parameter) were immunized with two priming doses of the licensed 7-valent pneumococcal (Pnc) conjugate vaccine and immune responses [antibody immunoglobulin G (IgG) levels, avidity and opsonophagocytic activity] were monitored for 26 weeks until IgG levels decreased to nearly baseline. A booster dose of either 2 microg conjugate or 5 microg polysaccharide vaccine was given at week 26. The ability of these two treatments to recall immune memory established by the conjugate vaccine was determined for types 4 and 14 for up to 63 days post-booster. The ability of challenge with pneumococcal type 14 to recall the immune response was also evaluated, as well as, the number of antibody secreting cells (ASC) specific to polysaccharide (Ps) 4, 6B, and 14. A higher dose of conjugate vaccine (2 microg) was necessary to elicit a significant increase in IgG levels after priming with one dose. Priming with lower doses (0.5 and 1.0 microg) only elicited modest increases in IgG levels. Recall of the immune response was found with either conjugate or Ps vaccines. NP challenge with type 14 at week 26 did not recall the immune response, although reduction in NP Pnc load was seen post-primary immunization at 5, 10 and 26 weeks. ASCs were detected in response to either conjugate or Ps booster doses. This model allows for the screening and determination of potential alternative vaccination regimens and the study of immunological markers of memory following Pnc vaccination.


Subject(s)
Immunologic Memory/immunology , Pneumococcal Vaccines/immunology , Animals , Antibodies, Bacterial/analysis , Antibodies, Bacterial/biosynthesis , Antibody Affinity/physiology , Antibody-Producing Cells/immunology , Dose-Response Relationship, Immunologic , Enzyme-Linked Immunosorbent Assay , Female , Immunization, Secondary , Immunoglobulin G/analysis , Immunoglobulin G/biosynthesis , Mice , Mice, Inbred BALB C , Models, Immunological , Nasopharynx/microbiology , Opsonin Proteins/pharmacology , Vaccines, Conjugate/immunology
4.
Clin Diagn Lab Immunol ; 8(6): 1115-9, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11687449

ABSTRACT

We evaluated the functional activities of antibodies, serum bactericidal activity (SBA), and immunoglobulin G (IgG) antibody avidity indices, using sodium thiocyanate (NaSCN) elution, elicited after vaccination with fractional doses of the Haemophilus influenzae type b conjugate (polyribosylribitol phosphate [PRP] conjugated to tetanus toxoid [PRP-T]) vaccine. A cohort of 600 infants from the Dominican Republic were randomized to receive one of three regimens of the PRP-T vaccine at ages 2, 4, and 6 months: full doses (10 microg of PRP antigen), one-half doses (5.0 microg), and one-third doses (3.3 microg) (J. Fernandez et al., Am. J. Trop. Med. Hyg. 62:485-490, 2000). Sixty serum samples, collected at age 7 months, with > or =2.0 microg of anti-PRP IgG per ml were randomly selected for avidity determinations. Geometric mean IgG concentrations were 13, 14, and 17 microg/ml for infants who received the full-dose (n = 19), one-half-dose (n = 19), and one-third-dose (n = 22) regimens, respectively. SBA geometric mean titers (1/dilution) were 85.0, 82.0, and 76.1 in sera from infants receiving the full-, one-half-, and one-third-dose regimens, respectively. Avidity indices (mean +/- standard error weighted average of NaSCN molar concentration x serum dilution factor) were 71.9 +/- 9.4, 123.6 +/- 26.8, and 150.9 +/- 24.9 for the full-, one-half-, and one-third-dose regimens, respectively. Upon comparison, the only significant difference (P = 0.024) found was a greater avidity index for sera from infants receiving the one-third-dose regimen than for sera from infants receiving the the full-dose regimen. We conclude that fractional doses elicit similar functional antibody activities in infants with > or = 2 microg of anti-PRP IgG per ml, corresponding to 89, 90, and 97% of infants receiving three doses of either the full concentration or one-half or one-third of the labeled concentration, respectively. This approach offers an alternative strategy for the prevention of H. influenzae type b disease in countries with limited resources.


Subject(s)
Antibodies, Bacterial/blood , Diphtheria Toxoid/administration & dosage , Diphtheria Toxoid/immunology , Haemophilus Infections/prevention & control , Haemophilus Vaccines/administration & dosage , Haemophilus Vaccines/immunology , Haemophilus influenzae/immunology , Cohort Studies , Developing Countries , Diphtheria Toxoid/economics , Dominican Republic , Haemophilus Infections/immunology , Haemophilus Vaccines/economics , Health Care Costs , Humans , Immunoglobulin G/blood , Infant
5.
Vaccine ; 20(3-4): 545-53, 2001 Nov 12.
Article in English | MEDLINE | ID: mdl-11672921

ABSTRACT

In a double-blinded, randomized trial, human immunodeficiency virus (HIV)-infected adults with > or = 200 CD4 cells/microl received placebo (PL), 7-valent conjugate, or 23-valent pneumococcal polysaccharide (PS) vaccine in one of the following two-dose combinations given 8 weeks apart: conjugate-conjugate, conjugate-polysaccharide, placebo-polysaccharide, placebo-placebo. A total of 67 persons completed the study. Neither significant increases in HIV viral load nor severe adverse reactions occurred in any group. After controlling for confounders, when compared with persons receiving placebo-polysaccharide, persons receiving conjugate-conjugate and conjugate-polysaccharide had higher antibody concentrations (serotypes 4, 6B, 9V and serotype 23F, respectively) and opsonophagocytic titers (functional antibody assay, serotypes 9V, 23F and serotypes 4, 6B, 9V, respectively) after the second dose (P<0.05). The second dose with either conjugate or polysaccharide following the first conjugate dose, however, produced no further increase in immune responses.


Subject(s)
Antibodies, Bacterial/blood , HIV Infections/immunology , Pneumococcal Vaccines/immunology , Adult , CD4 Lymphocyte Count , Enzyme-Linked Immunosorbent Assay , HIV Infections/virology , Humans , Phagocytosis , Pneumococcal Vaccines/adverse effects , Vaccines, Conjugate/immunology , Viral Load
6.
J Infect Dis ; 182(5): 1553-6, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11023481

ABSTRACT

Concentrations of serum anti-Haemophilus influenzae type b (anti-Hib) capsular polysaccharide (CPS) >/=0.15 and >/=1.0 microgram/mL are widely used as surrogates for protection against invasive Hib disease. However, the relationship between serum anti-Hib CPS following immunization and protection against colonization is not known, making it difficult to evaluate new Hib vaccines or combination vaccines. In the Dominican Republic, nasopharyngeal swabs were collected from 546 9-month-old infants who had received Hib conjugate vaccine at ages 2, 4, and 6 months and from 600 unvaccinated infants of the same age. The prevalence of Hib colonization was lower among vaccinated infants than among unvaccinated infants (0.9% vs. 2.3%). Among vaccinated infants, protection against colonization was significantly correlated with anti-Hib CPS concentrations >/=5 microgram/mL 1 month following the third dose of vaccine. These results suggest that the concentration of serum anti-Hib CPS needed for protection against colonization is greater than that needed for protection for invasive disease.


Subject(s)
Antibodies, Bacterial/blood , Bacterial Capsules/immunology , Haemophilus Infections/prevention & control , Haemophilus Vaccines/immunology , Haemophilus influenzae/immunology , Immunoglobulin G/blood , Polysaccharides, Bacterial/immunology , Humans , Infant , Vaccination
7.
J Infect Dis ; 181(3): 1162-6, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10720547

ABSTRACT

Children with sickle cell disease were immunized with either 2 doses of 7-valent pneumococcal conjugate vaccine followed by 1 dose of 23-valent pneumococcal polysaccharide vaccine or a single dose of 23-valent vaccine. Functional antibodies to 7 vaccine serotypes were measured by a flow cytometric opsonophagocytic assay (OPA) and compared with IgG anticapsular polysaccharide antibody concentrations measured by ELISA. Moderate correlations were found between OPA and ELISA antibody titers for all 7 serotypes (r values, 0.41-0.70; P<.001 for all serotypes). After immunization with 23-valent vaccine, geometric mean antibody titers by OPA were significantly higher in the combined schedule group for 5 of 7 vaccine serotypes but were significantly higher for only 2 of 7 serotypes as measured by ELISA. The ability of OPA to show a greater differential response to the 2 immunization schedules used in this study suggests that it may be useful in the evaluation of immunization regimens involving pneumococcal conjugate vaccines.


Subject(s)
Anemia, Sickle Cell/immunology , Antibodies, Bacterial/blood , Bacterial Vaccines/immunology , Immunoglobulin G/blood , Phagocytosis , Streptococcus pneumoniae/immunology , Adolescent , Child , Enzyme-Linked Immunosorbent Assay , Humans , Immunization , Pneumococcal Vaccines , Vaccines, Conjugate/immunology
8.
Am J Trop Med Hyg ; 62(4): 485-90, 2000 Apr.
Article in English | MEDLINE | ID: mdl-11220764

ABSTRACT

To assess the immunogenicity of more economical regimens of Haemophilus influenzae type b (Hib) conjugate vaccine, a randomized trial of fractional doses of polyribosylribitol phosphate-tetanus toxoid (PRP-T) Hib vaccine was undertaken in the Dominican Republic. Six hundred children were assigned to one of six regimens with PRP-T vaccine: full-dose, half-dose, and one-third-dose of Hib vaccine given separately or combined with diphtheria, tetanus, and pertussis (DTP) vaccine at ages 2, 4, and 6 months. Regimens that elicited antibody levels > 1.0 microg/mL in >70% of children and < or = 0.15 microg/mL in > 90% of children were considered acceptable. At 1 month post Dose 3, all regimens met the criteria for acceptable response. Among those who received Hib as a separate injection, geometric mean concentrations of anti-PRP bodies (GMCs) at age 1 month post Dose 3 were 11.2, 11.9, and 16.3 in the full, half, and one-third dose groups, respectively. Among those who received Hib and DTP combined, the GMCs were 6.4, 5.2, and 5.7 in the full-, half-, and one-third-dose groups respectively.


Subject(s)
Antibodies, Bacterial/biosynthesis , Haemophilus Vaccines/administration & dosage , Haemophilus Vaccines/immunology , Tetanus Toxoid/administration & dosage , Tetanus Toxoid/immunology , Antibodies, Bacterial/blood , Dose-Response Relationship, Immunologic , Enzyme-Linked Immunosorbent Assay , Humans , Infant , Single-Blind Method , Vaccines, Conjugate/administration & dosage , Vaccines, Conjugate/immunology
9.
Clin Infect Dis ; 29(2): 281-8, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10476727

ABSTRACT

The pneumococcal polysaccharide vaccine is recommended as a means of preventing invasive disease in the elderly. We compared responses to the 23-valent polysaccharide vaccine in 46 previously unvaccinated, healthy, institutionalized elderly persons (mean age, 85.5 years) with those in 12 healthy younger adults (mean age, 37 years) by measuring prevaccination and postvaccination serum IgG antibody concentrations (by ELISA), functional antibody activity (by opsonophagocytosis), IgG antibody avidity, and passive protection in mice. Postvaccination IgG antibody concentrations for two serotypes (6B and 19F) of the five studied (4, 6B, 14, 19F, and 23F) were significantly lower in elderly than in younger adults; however, opsonophagocytic activity was significantly reduced for all serotypes in the elderly. Sera with reduced opsonophagocytic activity (titer, <64) correlated with low IgG antibody avidity and protected mice poorly against pneumococcal challenge. In elderly persons receiving polysaccharide vaccination, there was a significant reduction in the functionality of postvaccination antibodies, and this appeared to increase with advanced age.


Subject(s)
Aging/immunology , Antibodies, Bacterial/immunology , Bacterial Vaccines/immunology , Streptococcus pneumoniae/immunology , Adult , Aged , Aged, 80 and over , Animals , Antibody Affinity , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunization, Passive , Immunoglobulin G/blood , Immunoglobulin G/immunology , Male , Mice , Pneumococcal Vaccines , Vaccination
10.
Clin Diagn Lab Immunol ; 6(4): 581-6, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10391867

ABSTRACT

Opsonophagocytosis is the primary mechanism for clearance of pneumococci from the host, and the measurement of opsonophagocytic antibodies appears to correlate with vaccine-induced protection. We developed a semiautomated flow cytometric opsonophagocytosis assay using HL-60 granulocytes as effector cells and nonviable 5, 6-carboxyfluorescein, succinimidyl ester-labeled Streptococcus pneumoniae (serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F) as bacterial targets. The flow cytometric opsonophagocytosis assay was highly reproducible (for 87% of repetitive assays the titers were within 1 dilution of the median titer) and serotype specific, with >/=97% inhibition of opsonophagocytic titer by addition of homologous serotype-specific polysaccharide. In general, opsonophagocytic titers were not significantly inhibited by the presence of either heterologous pneumococcal polysaccharide or penicillin in the serum. The flow cytometric assay could reproducibly measure functional antibody activity in prevaccination (n = 28) and postvaccination (n = 36) serum specimens from healthy adult volunteers vaccinated with the 23-valent pneumococcal polysaccharide vaccine. When compared with a standardized manual viable opsonophagocytic assay, a high correlation (r = 0.89; P

Subject(s)
Antibodies, Bacterial/blood , Bacterial Vaccines/immunology , Flow Cytometry/methods , Vaccination , Adult , Cross Reactions/immunology , Humans , Opsonin Proteins/metabolism , Phagocytosis/physiology , Pneumococcal Vaccines , Reproducibility of Results , Sensitivity and Specificity
11.
J Infect Dis ; 180(1): 133-40, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10353871

ABSTRACT

An infant mouse assay system for assessment of protective concentrations of human serum pneumococcal anticapsular antibodies is described. Passive immunization of anticapsular antibodies was evaluated for protection of infant mice challenged with Streptococcus pneumoniae serotypes 1, 4, 5, 6B, 18C, and 23A, with bacteremia as an end point. Protection was defined as no detectable bacteremia in 70% of mice 48 h after challenge. Type-specific anticapsular concentrations required for protection varied with serotype (0.4 microg/mL). Across serotypes, there was no significant correlation between human IgG concentration in mouse serum and protection from bacteremia or between IgG concentration and opsonophagocytic titer. Significant correlation (r=.84, P<.001) was observed between opsonophagocytic titer of human IgG antibody in mouse sera and protection from bacteremia. Thus, protective concentrations of anticapsular antibodies against bacteremia are serotype dependent. Opsonophagocytosis is a better predictor of in vivo protective capacity of pneumococcal anticapsular antibodies than are ELISA IgG antibody concentrations.


Subject(s)
Antibodies, Bacterial/blood , Bacterial Capsules/immunology , Biological Assay/methods , Immunization, Passive , Streptococcus pneumoniae/immunology , Animals , Animals, Newborn , Bacteremia , Humans , Mice , Opsonin Proteins , Phagocytosis , Streptococcus pneumoniae/pathogenicity
12.
Infect Immun ; 67(5): 2327-33, 1999 May.
Article in English | MEDLINE | ID: mdl-10225891

ABSTRACT

Streptococcus pneumoniae undergoes spontaneous phase variation between a transparent and an opaque colony phenotype, the latter being more virulent in a murine model of sepsis. Opaque pneumococci have previously been shown to express lower amounts of C polysaccharide (cell wall teichoic acid) and in this study were shown to have a higher content of capsular polysaccharide by immunoelectron microscopy. This report then examined the relationship between expression of these two cell surface carbohydrate structures and their relative contribution to the increased virulence of opaque variants. Comparison of genetically related strains showed that the differential content of capsular polysaccharide did not affect the amount of teichoic acid as measured by a capture enzyme-linked immunosorbent assay (ELISA). In contrast, when the teichoic acid structure was altered by replacing choline in the growth medium with structural analogs, the quantity of capsular polysaccharide as measured by a capture ELISA was decreased, demonstrating a linkage in the expression of the two surface carbohydrate structures. A standardized assay was used to assess the relative contribution of cell surface carbohydrates to opsonophagocytosis. The opaque variants required 1.2- to 30-fold more immune human serum to achieve 50% opsonophagocytic killing than did related transparent variants (types 6B and 9V). The opsonophagocytic titer was proportional to the quantity of capsular polysaccharide rather than teichoic acid. The major factor in binding of the opsonin, C-reactive protein (CRP), was also the amount of capsular polysaccharide rather than the teichoic acid ligand. Only for the transparent variant (type 6B), which bound more CRP, was there enhanced opsonophagocytic killing in the presence of this serum protein. Increased expression of capsular polysaccharide, therefore, appeared to be the major factor in the decreased opsonophagocytic killing of opaque pneumococci.


Subject(s)
Carbohydrates/immunology , Streptococcus pneumoniae/immunology , Animals , Antibodies, Bacterial , C-Reactive Protein/metabolism , Cell Membrane/immunology , Genetic Variation , HL-60 Cells , Humans , Mice , Microscopy, Immunoelectron , Opsonin Proteins , Phagocytosis , Phenotype , Polysaccharides, Bacterial/immunology , Streptococcus pneumoniae/pathogenicity , Streptococcus pneumoniae/ultrastructure , Teichoic Acids/immunology , Virulence/immunology
13.
J Trauma ; 44(5): 760-5; discussion 765-6, 1998 May.
Article in English | MEDLINE | ID: mdl-9603075

ABSTRACT

OBJECTIVES: Pneumococcal polysaccharide vaccine is given after emergency splenectomy for trauma to lessen the risk of overwhelming postsplenectomy sepsis. This study was undertaken to determine optimal timing of vaccine administration as determined by serum type-specific polysaccharide antibody concentration titer and functional activity of the resulting antibodies. METHODS: Fifty-nine consecutive patients undergoing splenectomy after trauma were randomized to receive pneumococcal vaccine postoperatively at 1, 7, or 14 days. Immunoglobulin G serum antibody concentrations against serogroup 4 and serotypes 6B, 19F, and 23F were measured before vaccination and 4 weeks postvaccination. Antibody concentrations were determined by enzyme-linked immunosorbent assay, and functional antibody by opsonophagocytosis. Results were compared with a normal adult control group (n = 12). RESULTS: Postvaccination enzyme-linked immunosorbent assay immunoglobulin G antibody concentrations for all serogroups and serotypes studied were not significantly different in splenectomized patients and control subjects. Postvaccination functional antibody activity was significantly reduced in early vaccination groups (serotype 6B excepted). However, with the exception of 19F, all titers for the 14-day group approached those of the control subjects (p > 0.05). Fold-increases of opsonophagocytic titers for serogroup 4 and serotypes 6B and 19F showed progressive increases with delay in vaccination. Except for serotype 23F, the number of postsplenectomy patients with opsonophagocytic titers <64 significantly decreased with a delay in vaccination (14 days). CONCLUSIONS: Postvaccination immunoglobulin G serum antibody concentrations were not significantly different from normal control subjects regardless of the time of vaccination (1, 7, or 14 days). Although concentrations approach normal, functional antibody activity was significantly lower. Better functional antibody responses against the serogroup and serotypes studied seemed to occur with delayed (14-day) vaccination.


Subject(s)
Bacterial Vaccines/administration & dosage , Bacterial Vaccines/immunology , Immunoglobulin G/blood , Splenectomy , Streptococcus pneumoniae/immunology , Adolescent , Adult , Aged , Drug Administration Schedule , Enzyme-Linked Immunosorbent Assay , Female , Humans , Male , Middle Aged , Opsonin Proteins/immunology , Phagocytosis , Pneumococcal Vaccines , Postoperative Period , Reference Values , Wounds and Injuries/surgery
14.
Clin Diagn Lab Immunol ; 4(4): 415-22, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9220157

ABSTRACT

Host protection against pneumococcal disease i primarily mediated by phagocytosis. We developed and standardized an opsonophagocytic assay using HL-60 cells (human promyelocytic leukemia cells). Fifty-five serum samples were analyzed for the presence of functional antibody against seven pneumococcal serogroups or serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) by using differentiated HL-60 cells (granulocytes) and peripheral blood leukocytes (PBLs). Six of the 55 serum samples were from unvaccinated adult volunteers, 31 serum samples were from adults who received one dose of the 14-valent or the 23-valent polysaccharide vaccine, and 18 serum samples were from 16-month-old infants who received four doses of an investigational 7-valent polysaccharide-protein conjugate vaccine. The results of an opsonophagocytic assay with HL-60 cells correlated highly with those of an assay with PBLs as effector cells (median r for seven serotypes = 0.87: P < 0.01). Opsonophagocytic titers were compared with the immunoglobulin G antibody concentrations determined by enzyme-linked immunosorbent assay (ELISA). The r values for serogroups or serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F were 0.61, 0.60, 0.67 0.90, 0.61, 0.39, and 0.57, respectively, when HL-60 cells were used as effector cells and 0.56, 0.47, 0.61, 0.90, 0.71, 0.31, and 0.62, respectively, when PBLs were used. The assay requires small amounts of serum (40 microliters per serotype), making this test suitable for assaying infant sera. Culturable cells aid in assay standardization and likely reduce donor-to-donor variability. This standardized assay, in combination with the standardized ELISA, can be used to evaluate current and developing pneumococcal vaccines, in which functional opsonophagocytic antibody activity may correlate with protection against pneumococcal disease.


Subject(s)
Antibodies, Bacterial , Opsonin Proteins/immunology , Pneumococcal Infections/diagnosis , Pneumococcal Infections/immunology , Adult , Animals , Animals, Newborn , Binding, Competitive/immunology , Complement System Proteins/immunology , Enzyme-Linked Immunosorbent Assay , Female , HL-60 Cells/chemistry , HL-60 Cells/immunology , HL-60 Cells/microbiology , Humans , Immunoglobulin G , Immunoglobulins, Intravenous/administration & dosage , Infant , Male , Mice , Mice, Inbred BALB C , Middle Aged , Pneumococcal Infections/therapy , Rabbits , Receptors, Cell Surface/immunology
15.
Can J Microbiol ; 43(4): 315-21, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9115089

ABSTRACT

The hyaluronate lyase (hyaluronidase) gene from Propionibacterium acnes was cloned and sequenced. The gene was isolated on an EcoRI-generated 3-kb piece of DNA. Expression was less in Escherichia coli than in P. acnes; in E. coli, active enzyme was only cell associated and not secreted. The gene is 2256-pb long and codes for a protein of 82 kDa. Amino terminal sequencing of the protein of the partially purified gene indicated the presence of a 32-amino-acid leader sequence. The leader sequence showed a membrane-spanning domain and all of the features usually associated with the leader for a secreted protein. The amino acid sequence is predicted to share homology with the hyaluronidase enzymes from Streptococcus pneumoniae, Streptococcus agalactiae, and Staphylococcus aureus. A potential hyaluronate-binding domain was identified and antibody against this domain was inhibitory to the enzyme.


Subject(s)
Genes, Bacterial , Polysaccharide-Lyases/genetics , Propionibacterium acnes/genetics , Amino Acid Sequence , Cloning, Molecular , Molecular Sequence Data
16.
J Bacteriol ; 176(18): 5771-9, 1994 Sep.
Article in English | MEDLINE | ID: mdl-8083169

ABSTRACT

The pca branch of the beta-ketoadipate pathway in Pseudomonas putida is responsible for the complete degradation of p-hydroxybenzoate through ortho cleavage of the initial pathway metabolite, protocatechuate. The pcaR regulatory locus has been found to be required for both induction of all of the genes within the pca regulon (pcaBDC, pcaIJ, and pcaF) and the chemotactic response of the bacteria to aromatic compounds. Insertional inactivation mutagenesis, using Tn5 and mini-Tn5 transposons, was used to locate, clone, and sequence this pcaR regulatory gene. The pcaR gene product, when overexpressed in Escherichia coli, possessed a specific affinity for the pcaIJ promoter region and demonstrated that the entire PcaR protein was required for this function. The deduced amino acid sequence of the PcaR regulatory peptide bears little resemblance to its counterpart in the other branch of the pathway, CatR, but exhibits significant homology to its regulatory antecedent, PobR, which regulates the initial breakdown of p-hydroxybenzoate into protocatechuate. Comparisons of the pcaIJ and pcaR promoter regions revealed conservation of a 15-bp sequence centered around the -10 region in both sequences. This, together with previously defined deletional studies with the pcaIJ promoter region, suggests that PcaR exerts its regulatory effect through protein-DNA interactions within this region, which would be unusually close to the transcriptional start site of pcaIJ for a positive regulator.


Subject(s)
Bacterial Proteins/genetics , DNA-Binding Proteins/genetics , Genes, Regulator/genetics , Parabens/metabolism , Pseudomonas putida/genetics , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Base Sequence , Cloning, Molecular , DNA, Bacterial/analysis , DNA, Bacterial/metabolism , Gene Expression , Genes, Bacterial/genetics , Molecular Sequence Data , Molecular Weight , Mutagenesis, Insertional , Promoter Regions, Genetic , Restriction Mapping , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Transcription, Genetic/genetics
17.
J Clin Microbiol ; 28(1): 27-31, 1990 Jan.
Article in English | MEDLINE | ID: mdl-2298877

ABSTRACT

Aerobic and anaerobic bacteria isolated from human axillae were tested for their capacity to adhere to buccal epithelial cells, immortalized human epithelial (HEp-2) cells, and undifferentiated and differentiated human epithelial cells. In general, both aerobic and anaerobic diphtheroids adhered better to differentiated human epithelial cells than to HEp-2 and undifferentiated human epithelial cells (P less than 0.05). Mannose, galactose, fucose, N-acetyl-D-glucosamine, and fibronectin were also assayed for their capacity to inhibit the adherence of diphtheroids to human epithelial cells. A great deal of variability was observed in the capacity of the latter compounds to inhibit the attachment of aerobic diphtheroids to undifferentiated and differentiated epithelial cells. Overall, mannose appeared to be best at inhibiting the adherence of the aerobic diphtheroids to undifferentiated human epithelial cells. Galactose, fucose, N-acetyl-D-glucosamine, and fibronectin showed a greater capacity to inhibit attachment of aerobic diphtheroids to differentiated than to undifferentiated human epithelial cells. The inhibition of adherence to differentiated human epithelial cells varied with the microorganism and the compound tested; however, the highest and most consistent inhibition of adherence (76.1 to 88.6%) was observed with a 5% solution of N-acetyl-D-glucosamine. The in vitro adherence and adherence inhibition assays presented here demonstrate that a number of adhesins and receptors are involved in the adherence of skin bacteria to human epithelial cells and receptors on human epithelial cells are apparently altered during differentiation.


Subject(s)
Bacterial Adhesion , Skin/microbiology , Bacteria, Aerobic/isolation & purification , Bacteria, Anaerobic/isolation & purification , Cell Differentiation , Cell Line , Corynebacterium/isolation & purification , Epithelial Cells , Epithelium/microbiology , Female , Humans , In Vitro Techniques , Male , Mouth Mucosa/cytology , Mouth Mucosa/microbiology , Skin/cytology
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