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David S Stephens

Publications and source records attributed to David S Stephens.

At least 37 records · Page 2Linked to original sources
PubMed2004

Neisseria meningitidis serogroup W-135 carriage among US travelers to the 2001 Hajj.

In 2000, a large international outbreak of meningococcal disease caused by Neisseria meningitidis serogroup W-135 was identified among pilgrims returning from the Hajj in Saudi Arabia. To assess ongoing risk, we evaluated N. meningitidis carriage among US travelers to the 2001 Hajj. Of 25 N. meningitidis isolates obtained, 15 (60%) were nongroupable and 8 (32%) were serogroup W-135 when tested by standard slide-agglutination techniques. Two additional nongroupable isolates were characterized as serogroup W-135 when tested by polymerase chain reaction. Nine of 10 serogroup W-135 isolates were indistinguishable from the Hajj-2000 clone. None of the departing, but 9 (1.3%) of the returning, pilgrims carried serogroup W-135 (P=.01); all carriers reported previous vaccination. Carriage of N. meningitidis serogroup W-135 increased significantly in pilgrims returning from the Hajj. Although the risk of disease to pilgrims appears to be low, the risk of spread to others of this pathogenic strain remains a concern.

Adult↗
PubMed2004

Immune responses to Bacillus anthracis protective antigen in patients with bioterrorism-related cutaneous or inhalation anthrax.

Anti-protective antigen (PA) immunoglobulin (Ig) G, toxin neutralization, and PA-specific IgG memory B cell responses were studied in patients with bioterrorism-related cutaneous or inhalation anthrax and in a patient with laboratory-acquired cutaneous anthrax. Responses were determined for >1 year after the onset of symptoms. Eleven days after the onset of symptoms (15 days after likely exposure), anti-PA IgG was detected in 16 of 17 patients with confirmed or suspected clinical anthrax who were tested. Anti-PA IgG remained detectable 8-16 months after the onset of symptoms in all 6 survivors of inhalation anthrax and in 7 of 11 survivors of cutaneous anthrax who were tested. Anti-PA IgG levels and serum toxin neutralizing activity were strongly associated (R2=0.83). PA-specific IgG memory B cells were detectable in all 6 survivors of inhalation anthrax but in only 2 of 7 patients with cutaneous anthrax who were tested. Anti-PA IgG is an important diagnostic marker of anthrax, a predictor of serum anti-toxin activity, and a marker of immunological memory against anthrax.

Anthrax↗
PubMed2004

The Neisseria meningitidis serogroup A capsular polysaccharide O-3 and O-4 acetyltransferase.

Neisseria meningitidis serogroup A capsular polysaccharide (CPS) is composed of a homopolymer of O-acetylated, alpha1-->6-linked ManNAc 1-phosphate that is distinct from the capsule structures of the other meningococcal disease-causing serogroups, B, C, Y, and W-135. The serogroup A capsule biosynthetic genetic cassette consists of four open reading frames, mynA-D (sacA-D), that are specific to serogroup A, but the functions of these genes have not been well characterized. mynC was found to encode an inner membrane-associated acetyltransferase that is responsible for the O-acetylation of the CPS of serogroup A. The wild-type CPS as revealed by 1H NMR had 60-70% O-acetylated ManNAc residues that contained acetyl groups at O-3, with some species acetylated at O-4 and at both O-3 and O-4. A non-polar mynC mutant generated by introducing an aphA-3 kanamycin resistance cassette produced CPS with no O-acetylation. A serogroup A capsule-specific monoclonal antibody was shown to recognize the wild-type O-acetylated CPS, but not the CPS of the mynC mutant, which lacked O-acetylation. MynC was C-terminally His-tagged and overexpressed in Escherichia coli to obtain the predicted approximately 26-kDa protein. The acetyltransferase activity of purified MynC was demonstrated in vitro using [14C]acetyl-CoA. MynC O-acetylated the O-acetylated CPS of the mynC mutant and further acetylated the wild-type CPS of serogroup A meningococci, but not the CPS of serogroup B or C meningococci. Genetic complementation of the mynC mutant confirmed the function of MynC as the serogroup A CPS O-3 and O-4 acetyltransferase. MynC represents a new subclass of O-acetyltransferases that utilize acetyl-CoA to decorate the D-mannosamine capsule of N. meningitidis serogroup A.

Acetyltransferases↗
PubMed2004

The MisR/MisS two-component regulatory system influences inner core structure and immunotype of lipooligosaccharide in Neisseria meningitidis.

Lipooligosaccharide (LOS) of Neisseria meningitidis is the major inflammatory mediator that contributes to meningococcal pathogenesis. Variable attachments to the HepII residue of the LOS inner core together with the alpha-chain heterogeneity result in immunologically distinct LOS structures, which may be selected for during human infection. Lpt-3, a phosphoethanolamine (PEA) transferase, and LgtG, a glucosyltransferase, mediate the substitution of PEA or glucose at the O-3 position of HepII in L3 or L2 LOS immunotypes, respectively. Inactivation of a two-component response regulator, encoded by NMB0595, in N. meningitidis strain NMB resulted in the loss of all PEA decorations on the LOS inner core expressed by the NMB0595 mutant. When compared with the parental strain NMB that predominantly expresses L2 immunotype LOS and other minor LOS structures, the NMB0595 mutant expresses a pure population of a novel LOS structure completely substituted at the HepII O-3 position with glucose, but lacking other PEA decorations on the inner core. Quantitative real time PCR experiments showed increased transcription of lgtG in the NMB0595 mutant, and no significant change in lpt-3 transcription. Inactivation of lgtG resulted in LOS inner cores without glucose, but these structures, even though the lpt-3 transcription was unaffected, also lacked the O-3-linked PEA. Consistently, a double mutation of lgtG and misR in strain NMB yielded a LOS structure without PEA or Glc substitution of HepII. These data indicated a new pathway for the regulation of LOS inner core structure in N. meningitidis through an environmental sensing two-component regulatory system, named misR(NMB0595)/misS(NMB0594) for regulator and sensor of the meningococcal inner core structure.

Blotting, Southern↗
PubMed2004

Neisseria meningitidis lipooligosaccharide structure-dependent activation of the macrophage CD14/Toll-like receptor 4 pathway.

Meningococcal lipopoly(oligo)saccharide (LOS) is a major inflammatory mediator of fulminant meningococcal sepsis and meningitis. Highly purified wild-type meningococcal LOS and LOS from genetically defined mutants of Neisseria meningitidis that contained specific mutations in LOS biosynthesis pathways were used to confirm that meningococcal LOS activation of macrophages was CD14/Toll-like receptor 4 (TLR4)-MD-2 dependent and to elucidate the LOS structural requirement for TLR4 activation. Expression of TLR4 but not TLR2 was required, and antibodies to both TLR4 and CD14 blocked meningococcal LOS activation of macrophages. Meningococcal LOS alpha or beta chain oligosaccharide structure did not influence CD14/TLR4-MD-2 activation. However, meningococcal lipid A, expressed by meningococci with defects in 3-deoxy-D-manno-octulosonic acid (KDO) biosynthesis or transfer, resulted in an approximately 10-fold (P < 0.0001) reduction in biologic activity compared to KDO2-containing meningococcal LOS. Removal of KDO2 from LOS by acid hydrolysis also dramatically attenuated cellular responses. Competitive inhibition assays showed similar binding of glycosylated and unglycosylated lipid A to CD14/TLR4-MD-2. A decrease in the number of lipid A phosphate head groups or penta-acylated meningococcal LOS modestly attenuated biologic activity. Meningococcal endotoxin is a potent agonist of the macrophage CD14/TLR4-MD-2 receptor, helping explain the fulminant presentation of meningococcal sepsis and meningitis. KDO2 linked to meningococcal lipid A was structurally required for maximal activation of the human macrophage TLR4 pathway and indicates an important role for KDO-lipid A in endotoxin biologic activity.

Animals↗
PubMed2004

Biofilm formation by Neisseria meningitidis.

Biofilm formation by the human pathogen Neisseria meningitidis was analyzed. Biofilm-forming meningococcal strains were identified and quantitated by crystal violet staining. Laser scanning confocal microscopy of the meningococcal biofilm revealed variable layers up to 90 microm in thickness. A total of 39 meningococcal isolates were studied; 23 were nasopharyngeal-carriage isolates, and 16 were invasive-disease isolates. Thirty percent of carriage isolates and 12.5% of invasive-disease isolates formed biofilms proficiently on a polystyrene surface. Generally, the strains that formed biofilms showed high-level cell surface hydrophobicity, characteristic of strains lacking a capsule. The inhibitory role of capsule in biofilm formation was further confirmed by comparing the biofilm-forming capabilities of a serogroup B wild-type strain of a disease-associated isolate to those of its capsule-deficient mutant (ctrA). Some strains of meningococci form biofilms, and this process is likely important in menigococcal colonization.

Bacterial Capsules↗
PubMed2004

The binding of acetaminophen, lidocaine, and valproic acid to human milk.

The binding of acetaminophen, lidocaine, and valproic acid to pooled normal mature human milk was studied in vitro by using equilibrium dialysis. Scatchard analysis revealed high-affinity, low-capacity binding for acetaminophen (Ka [affinity constant of association], 1.47 x 10(4) L/mol; Bo [concentration of binding sites], 9.01 x 10(-4) mol/L) and some minimal, nonspecific binding. Binding ranged up to 85%. For lidocaine, low-affinity, high-capacity binding was noted (Ka, 1.42 x 10(2) L/mol; Bo, 1.69 x 10(-2) mol/L). Binding ranged up to 72%. For valproic acid, only minimal, nonspecific binding was noted at low drug concentrations with binding ranging up to 64%. The binding of these drugs to milk might enhance their excretion and subsequent ingestion by infants who are breast-fed. In addition, the low pH of the milk (6.24) may cause "ion trapping" of acetaminophen (pKa, 9.5) and lidocaine (pKa, 7.9).

Acetaminophen↗
PubMed2004

Meningococcal conjugate vaccines.

Neisseria meningitidis is a leading cause of bacterial meningitis and sepsis in the US, Europe and in many other parts of the world, including parts of sub-Saharan Africa (known as the African 'meningitis belt'). There are > 500000 cases of meningococcal disease annually with an estimated death toll of 135000 worldwide. Approximately 10 - 15 % of survivors experience significant morbidity in the form of neurological sequelae, including hearing loss, speech disorders, loss of limbs, mental retardation and paralysis. Disease is usually caused by N. meningitidis serogroups A, B, C, Y or W-135. Prevention of meningococcal disease includes isolation, chemoprophylaxis and vaccination with available polysaccharide vaccines. However, the polysaccharide meningococcal vaccines (i.e., A and C; A, C and W-135; or A, C, Y and W-135) initially developed in the 1970s are generally poorly immunogenic in children or require repeated doses and do not produce long-lasting immunity. Conjugate vaccine technology has been very successfully used in childhood vaccines for the prevention of other bacterial meningitis pathogens, including vaccines against Haemophilus influenzae serotype b (Hib) and more recently, the seven- and nine-valent conjugate pneumococcal vaccines. Newly released meningococcal conjugate vaccines against N. meningitidis serogroup C have been highly efficacious in young children and adolescents, with minimal side effects. Conjugate vaccines targeting other important meningococcal serogroups (e.g., N. meningitidis serogroup A, responsible for the large pandemic outbreaks and the majority of disease in sub-Saharan Africa and serogroups Y and W-135) are under development and together with the serogroup C conjugates, have the potential to significantly impact worldwide sporadic and epidemic meningococcal disease. The search for an effective serogroup B meningococcal vaccine remains elusive. This manuscript reviews the conjugate meningococcal vaccines and their potential for meningococcal disease prevention.

Humans↗
PubMed2003

Genetic basis for nongroupable Neisseria meningitidis.

Nongroupable Neisseria meningitidis may constitute one-third or more of meningococcal isolates recovered from the nasopharynx of human carriers. The genetic basis for nongroupability was determined in isolates obtained from a population-based study in which 60 (30.9%) of 194 meningococcal isolates from asymptomatic carriers were not groupable. Forty-two percent of nongroupable isolates were related to serogroup Y ET-508/ST-23 clonal complex strains, the most common groupable carrier isolate from the study population. Nongroupable isolates were all rapidly killed by 10% normal human serum. The capsule loci of 6 of the ET-508/ST-23 complex strains and of 25 other genetically diverse nongroupable meningococci were studied in detail. Serogroup A or novel capsule biosynthesis genes were not found. Nongroupable isolates were genetically serogroup Y, B, or C isolates that did not express capsule but were related to groupable isolates found in the population (class I); capsule deficient because of insertion element-associated deletions of capsule biosynthesis genes (class II); or isolates that lacked all capsule genes and formed a distinct genetic cluster not associated with meningococcal disease (class III).

Adolescent↗
PubMed2003

Pathology and pathogenesis of bioterrorism-related inhalational anthrax.

During October and November 2001, public health authorities investigated 11 patients with inhalational anthrax related to a bioterrorism attack in the United States. Formalin-fixed samples from 8 patients were available for pathological and immunohistochemical (IHC) study using monoclonal antibodies against the Bacillus anthracis cell wall and capsule. Prominent serosanguinous pleural effusions and hemorrhagic mediastinitis were found in 5 patients who died. Pulmonary infiltrates seen on chest radiographs corresponded to intraalveolar edema and hyaline membranes. IHC assays demonstrated abundant intra- and extracellular bacilli, bacillary fragments, and granular antigen-staining in mediastinal lymph nodes, surrounding soft tissues, and pleura. IHC staining in lung, liver, spleen, and intestine was present primarily inside blood vessels and sinusoids. Gram's staining of tissues was not consistently positive. In 3 surviving patients, IHC of pleural samples demonstrated abundant granular antigen-staining and rare bacilli while transbronchial biopsies showed granular antigen-staining in interstitial cells. In surviving patients, bacilli were not observed with gram's stains. Pathological and IHC studies of patients who died of bioterrorism-related inhalational anthrax confirmed the route of infection. IHC was indispensable for diagnosis of surviving anthrax cases. The presence of B. anthracis antigens in the pleurae could explain the prominent and persistent hemorrhagic pleural effusions.

Adult↗
PubMed2003

Genetic basis for biosynthesis of the (alpha 1-->4)-linked N-acetyl-D-glucosamine 1-phosphate capsule of Neisseria meningitidis serogroup X.

The genetic basis for biosynthesis of the (alpha1-->4)-linked N-acetyl-D-glucosamine 1-phosphate capsule of Neisseria meningitidis serogroup X was defined. The biosynthesis gene cassette was a approximately 4.2-kb region located between ctrA of the capsule transport operon and galE, which encodes the UDP-glucose-4-epimerase. This location was identical to the locations of the biosynthesis cassettes in other meningococcal serogroups. Three open reading frames unique to meningococcus serogroup X were identified. Deletion-insertion mutation and colony immunoblotting confirmed that these three genes were essential for serogroup X capsule expression, and the genes were designated xcbA, xcbB, and xcbC (serogroup X capsule biosynthesis). Reverse transcriptase PCR indicated that the xcbABC genes form an operon and are cotranscribed divergently from ctrA. XcbA exhibited 52% amino acid similarity to SacB, the putative capsule polymerase of meningococcus serogroup A, suggesting that it plays a role as the serogroup X capsule polymerase. An IS1016 element was found within the intergenic region separating ctrA and xcbA in multiple strains, and this element did not interfere with capsule expression.

Acetylglucosamine↗
PubMed2003

Development and evaluation of an improved mouse model of meningococcal colonization.

Studies of meningococcal pathogenesis have been severely restricted due to the absence of an adequate animal model. Given the significance of iron in meningococcal pathogenesis, we developed a model of Neisseria meningitidis colonization in outbred adult mice that included daily administration of iron dextran. While receiving iron, the animals were inoculated intranasally with the initial doses of bacterial suspension. Meningococci were recovered from the animals by nasopharyngeal washes. Approximately half of the animals inoculated with 10(7) CFU remained colonized 13 days after the initial bacterial inoculation. The model was further evaluated with genetically defined isogenic serogroup B mutant strains, and the colonization capabilities of the mutants were compared to that of the wild-type parent. A mutant that produces truncated lipooligosaccharide (KDO(2)-lipid A) and a mutant defective in capsule transport were dramatically impaired in colonization. A mutant defective in pilus transport (pilQ) showed moderately impaired colonization. The immunological aspect of the model was also evaluated by challenging mice after immunization with homologous whole-cell meningococci. The immunized mice were protected from colonization of the homologous strain. In this model, long-term meningococcal colonization was maintained, allowing us to study the effects of specific genetic mutation on colonization. In addition, this model allows investigation of the role of active immune response against meningococci.

Animals↗
PubMed2002

Genotype-specific carriage of Neisseria meningitidis in Georgia counties with hyper- and hyposporadic rates of meningococcal disease.

Carriage of Neisseria meningitidis in a Georgia county with hypersporadic incidence of meningococcal disease ("hypersporadic county") and in a county with no cases of meningococcal disease was determined by a cross-sectional pharyngeal culture study of high school students. Among 2730 students from whom culture samples were obtained, meningococcal carriage was 7.7% (140/1818) in the hypersporadic county and 6.1% (56/912) in the comparison county. Carriage rates by serogroup and genetic type (i.e., electrophoretic type [ET]) did not differ significantly between counties, but apartment or mobile home residency was a risk factor for carriage in the hypersporadic county. Although most cases of meningococcal disease in the hypersporadic county were caused by members of the serogroup C ET-37 clonal group, no ET-37 meningococcal isolates were recovered from carriers in this county. However, 38% of all meningococcal isolates recovered from carriers in both counties were members of the serogroup Y ET-508 clonal group, an emerging cause of meningococcal disease in Georgia and throughout the United States during 1996-2001. Shifts in carriage and transmission of meningococcal strains with different pathogenic potential are important determinants of meningococcal disease incidence.

Adolescent↗