Biomedical subjects

D H Walker

Publications and source records attributed to D H Walker.

At least 37 records · Page 2Linked to original sources

PubMed2001

Identification of protective components of two major outer membrane proteins of spotted fever group Rickettsiae.

Fragments representing the genes of the two major outer membrane proteins of spotted fever group rickettsiae (rOmpA and rOmpB) were tested as DNA vaccines. Immunizations with each of three fragments (rompA4999-6710, rompB1550-2738, and rompB2459-4123) conferred a degree of protection on vaccinated mice against virulent rickettsial challenge. Protection was achieved when DNA immunizations were followed by booster immunizations with the homologous recombinant protein. Proliferation and gamma-interferon secretion were detected after in vitro stimulation of lymphocytes from immunized animals with whole Rickettsia conorii antigen. The data validate particular segments of rOmpA and rOmpB as potent immunogens and hence as sources of immunostimulatory elements with specificity for T lymphocytes, which are the key effectors of protective immunity against rickettsial infections.

Animals↗

PubMed2001

Rickettsial infection in murine models activates an early anti-rickettsial effect mediated by NK cells and associated with production of gamma interferon.

Natural killer (NK) cell activity was significantly increased on days 2-6 of infection in the Rickettsia conorii-infected C3H/HeN mice and on day 2 in the Rickettsia typhi-infected C57BL/6 mice. Depletion of NK cell activity utilizing anti-NK1.1 monoclonal antibody enhanced the susceptibility of normally resistant C57BL/6 mice to infection with R. typhi, and depletion of NK cell activity with antibody to asialo GM1 enhanced the susceptibility of C3H/HeN mice to infection with R. conorii. Serum gamma interferon was increased in R. conorii-infected C3H/HeN mice compared with NK cell-depleted, infected mice during the early course of infection. Additionally, the NK cell activating cytokine IL-12 was elevated in the sera of infected mice during the time period representing enhanced NK cell activity compared with uninfected mice. Thus, it appears that NK cells contribute to the early anti-rickettsial immune response, likely via a mechanism involving gamma interferon.

Animals↗

PubMed2001

Rickettsial phospholipase A2 as a pathogenic mechanism in a model of cell injury by typhus and spotted fever group rickettsiae.

Phospholipase A2 activity by typhus group rickettsiae causes hemolysis in vitro. Rickettsial phospholipase A2 has been proposed to mediate entry into the host cell, escape from the phagosome, and cause injury to host cells by both typhus and spotted fever group rickettsiae. In a rickettsial contact-associated cytotoxicity model, the interaction of Rickettsia prowazekii or R. conorii with Vero cells caused temperature-dependent release of 51Cr from the cells. Treatment of rickettsiae, but not the cells, with a phospholipase A2 inhibitor (bromophenacyl bromide) or with antibody to king cobra venom inhibited cell injury. Rickettsial treatment with bromophenacyl bromide inhibited the release of free fatty acids from the host cell. Neither the inhibitor nor antivenom impaired rickettsial active transport of L-lysine. Thus, host cell injury was mediated by a rickettsial phospholipase A2-dependent mechanism.

Acetophenones↗

PubMed2001

Hemostatic/fibrinolytic protein changes in C3H/HeN mice infected with Rickettsia conorii--a model for Rocky Mountain spotted fever.

Changes in plasma hemostatic and fibrinolytic proteins were determined during courses of a murine model of fatal and non-fatal Rocky Mountain spotted fever. C3H/HeN mice were infected with Rickettsia conorii and coagulation and histopathologic studies were performed at prescribed periods of time. A significant decrease in plasma factor VIII activity and rise in plasma factor V procoagulant activity correlated with a fatal infection. Factor VII levels were unchanged; factor XI levels dropped early in the course in the lethally infected animals, but returned to normal. Factor XII, high molecular weight kininogen, and prekallikrein levels were unchanged by the sublethal infection. Prekallikrein levels fell during the lethal infection. Antithrombin concentrations were decreased significantly in all animals, but plasma plasminogen levels did not change in either group of animals. Nonocclusive thrombi were microscopically observed rarely and only in animals surviving a sublethal infection. A fall in tissue plasminogen activator activity and a rise in plasminogen activator inhibitor activity highly correlated with a lethal outcome. Lethal infection with R. conorii is associated with primary endothelial cell injury resulting in decreased tissue plasminogen activator and increased plasminogen activator inhibitor.

Animals↗

PubMed2000

Rickettsia felis rickettsiosis in Yucatán.

Three patients with fever, exanthem, headache, and central-nervous-system involvement were diagnosed with Rickettsia fells infection by specific PCR of blood or skin and seroconversion to surrogate Rickettsia antigens. Although R. felis's relationship to other Rickettsia species is known and the pathogenic potential of this clade is well documented, R. felis's role as a pathogen has not been fully understood.

Adult↗

PubMed2000

A conserved, transcriptionally active p28 multigene locus of Ehrlichia canis.

Antigenic diversity of Ehrlichia chaffeensis and Ehrlichia canis may involve independent or differential expression of the P28 outer membrane proteins genes, enabling persistent infections of the natural hosts. In this study, we analyzed the transcriptional activity of a five gene locus in E. canis encoding homologous, but non-identical, p28 genes. The p28 multigene locus contained three previously identified complete gene sequences and one partial gene sequence. A new p28 gene was identified and sequenced, and the complete sequence of a second partial p28 gene was determined. The new p28 gene joined two previously separate loci, forming the single p28 multigene locus. The amino acid homology of the E. canis P28 proteins ranged from 51 to 74%. The nucleic acid sequence of regions compared within the locus spanning four p28 genes from two geographically distinct E. canis isolates was completely conserved. Analysis of the five p28 genes demonstrated that all were transcriptionally active in in-vitro cultures of E. canis incubated at the vertebrate host (37 degrees C) and ambient tick temperatures (27 degrees C). Polycistronic copies of multiple p28 genes were not detected by RT-PCR, but monocistronic p28 mRNA transcripts were detected by Northern blotting from E. canis infected DH82 cells, indicating that the genes are transcribed as monocistronic messages.

Amino Acid Sequence↗

PubMed2000

Characterization of the complete transcriptionally active ehrlichia chaffeensis 28 kDa outer membrane protein multigene family.

The 28kDa outer membrane proteins (P28) of Ehrlichia chaffeensis are encoded by a multigene family. The purpose of this study was to determine all the p28 gene sequences and their transcriptional activities. There were 21 members of the p28 multigene family located in a 23kb DNA fragment in the genome of E. chaffeensis. The p28 genes each contained 816-903 nucleotides with intergenic spaces of 10-605 nucleotides. All the genes were complete and were predicted to have a signal sequence. The molecular masses of the mature proteins were predicted to be 28-32kDa. The amino acid sequence identity of the P28 proteins was 20-83%. Ten p28 genes were investigated for transcriptional activity by using RT-PCR amplification of mRNA. Six of 10 tested p28 genes were actively transcribed in cell-culture grown E. chaffeensis. RT-PCR also indicated that each of the p28 genes was monocistronic. These results suggest that the p28 genes are active genes and encode polymorphic forms of the P28 proteins. The P28s were divergent among isolates of E. chaffeensis also. The large repertoire of the p28 genes in a single ehrlichial organism and antigenic diversity of the P28 among the isolates of E. chaffeensis suggest that P28s may be involved in immune avoidance.

Amino Acid Sequence↗

PubMed2000

The 120 kDa outer membrane protein of Ehrlichia chaffeensis: preferential expression on dense-core cells and gene expression in Escherichia coli associated with attachment and entry.

The immunodominant 120 kDa protein (p120) of Ehrlichia chaffeensis was demonstrated to be exposed on the surface of purified whole ehrlichial cells examined by immunoelectron microscopy with a rabbit antibody against a portion of the domain containing tandem repeat units. In the intracellular location, the 120 kDa protein was detected by immunoelectron microscopy in the outer membrane of the cell wall of dense-core forms of the ehrlichiae in infected canine macrophage-like cells and as a component of the intramorular fibrillary matrix. No 120 kDa protein was detected in the cell wall of ehrlichial reticulate cells. Recombinant Escherichia coli with a plasmid containing the entire 120 kDa protein gene, but no bacteria with non-recombinant plasmid, attached to the surface of HeLa cells as visualized by electron microscopy. Some of the recombinant 120 kDa protein expressing E. coli invaded the HeLa cells as determined by gentamicin protection assays and by intravacuolar localization ultrastructurally.

Animals↗

PubMed2000

Establishment of a novel endothelial target mouse model of a typhus group rickettsiosis: evidence for critical roles for gamma interferon and CD8 T lymphocytes.

A mouse model of typhus rickettsiosis that reproduces the hematogenous dissemination to the critical target organs, including brain, lungs, heart, and kidneys, primary endothelial and, to a lesser degree, macrophage intracellular rickettsial infection, and typical vascular-based lesions of louse-borne typhus and murine typhus was established. Intravenous inoculation of C3H/HeN mice with Rickettsia typhi caused disease with a duration of the incubation period and mortality rate that were dependent on the infective dose of rickettsiae. Lethal infection was associated with high concentrations of R. typhi in the lungs and brain, despite a brisker humoral immune response to the rickettsiae than in the sublethal infection. Gamma interferon and CD8 T lymphocytes were demonstrated to be crucial to clearance of the rickettsiae and recovery from infection in experiments in which specific monoclonal antibodies were administered to deplete these components. Death of animals depleted of gamma interferon or CD8 T lymphocytes was associated with overwhelming rickettsial infection demonstrated by titers of infectious rickettsiae and by immunohistochemistry. An effective antirickettsial immune response was associated with elevated serum concentrations of IL-12 on Day 5 and increased secretion of IL-12 by concanavalin-A-stimulated spleen cells on Day 5. Evidence for transient suppression of the immune response consisted of marked reduction in the secretion of IL-2 and IL-12 by concanavalin-A-stimulated spleen cells on Days 10 and 15. This model offers excellent opportunities for study of attenuation and pathogenetic mechanisms of typhus rickettsiae, which are established biologic weapons of potential use in bioterrorism.

Animals↗

PubMed2000

Laparoscopic surgery in children with ventriculoperitoneal shunts.

BACKGROUND/PURPOSE: Children with ventriculoperitoneal shunts may require laparoscopic surgery. The authors aimed to determine if this group of children are at greater risk for complications or technical problems. METHODS: Children with ventriculoperitoneal shunts who underwent laparoscopic surgery between 1995 and 1998 were reviewed. In addition, the subset of children undergoing laparoscopic fundoplication were compared with the group of children without shunts who- had the same operation during the same period. RESULTS: Ten children with ventriculoperitoneal shunts underwent laparoscopic surgery. Three had complications, none of which were caused by the shunt. None had any evidence of shunt dysfunction related to the laparoscopic procedure. The 6 children with shunts who underwent laparoscopic fundoplication were compared with 17 similar children without shunts who underwent the same operation during the same period. There were no differences between the groups with respect to operating time, conversion to an open approach, or complications. CONCLUSION: Laparoscopic surgery can be performed safely and effectively in children with ventriculoperitoneal shunts.

Adolescent↗

PubMed2000

The rickettsial outer-membrane protein A and B genes of Rickettsia australis, the most divergent rickettsia of the spotted fever group.

The genes for rickettsial outer-membrane protein A (rOmpA), a distinguishing feature of spotted fever group (SFG) rickettsiae, and rOmpB, a genus-specific protein, were identified and sequenced in Rickettsia australis. The amino acid sequences of domains I, III and IV of the R. australis rOmpA share close homology with those of rOmpA of other SFG rickettsiae, but the repeat region (domain II) is dramatically different from that of other known SFG rOmpA. R. australis rOmpB is more similar to rOmpB of other SFG rickettsiae than to that of typhus group rickettsiae.

Amino Acid Substitution↗

PubMed2000

Glycosylation of homologous immunodominant proteins of Ehrlichia chaffeensis and Ehrlichia canis.

The glycoprotein genes of Ehrlichia chaffeensis (1,644 bp) and Ehrlichia canis (2,064 bp) encode proteins of 548 to 688 amino acids with predicted molecular masses of only 61 and 73 kDa but with electrophoretic mobilities of 120 kDa (P120) and 140 kDa (P140), respectively. The 120-kDa protein gene of E. chaffeensis contains four identical 240-bp tandem repeat units, and the 140-kDa protein gene of E. canis has 14 nearly identical, tandemly arranged 108-bp repeat units. Conserved serine-rich motifs identified in the repeat units of P120 and P140 were also found in the repeat units of the human granulocytotropic ehrlichiosis agent 130-kDa protein and of the fimbria-associated adhesin protein Fap1 of Streptococcus parasanguis. Nearly the entire (99%) E. chaffeensis P120 gene (1,616 bp), the 14-repeat region (78%) of the E. canis P140 gene (1,620 bp), and a 2-repeat region from the E. chaffeensis P120 gene (520 bp) were expressed in Escherichia coli. The recombinant proteins exhibited molecular masses ranging from 1.6 to 2 times larger than those predicted by the amino acid sequences. Antibodies against the recombinant proteins reacted with E. chaffeensis P120 and E. canis P140, respectively. Carbohydrate was detected on the E. chaffeensis and E. canis recombinant proteins, including the two-repeat polypeptide region of E. chaffeensis P120. A carbohydrate compositional analysis identified glucose, galactose, and xylose on the recombinant proteins. The presence of only one site for N-linked (Asn-Xaa-Ser/Thr) glycosylation, a lack of effect of N-glycosidase F, the presence of 70 and 126 Ser/Thr glycosylation sites in the repeat regions of P120 and P140, respectively, and a high molar ratio of carbohydrate to protein suggest that the glycans may be O linked.

Amino Acid Sequence↗

PubMed2000

Mechanisms of intracellular killing of Rickettsia conorii in infected human endothelial cells, hepatocytes, and macrophages.

The mechanism of killing of obligately intracellular Rickettsia conorii within human target cells, mainly endothelium and, to a lesser extent, macrophages and hepatocytes, has not been determined. It has been a controversial issue as to whether or not human cells produce nitric oxide. AKN-1 cells (human hepatocytes) stimulated by gamma interferon, tumor necrosis factor alpha, interleukin 1beta, and RANTES (regulated by activation, normal T-cell-expressed and -secreted chemokine) killed intracellular rickettsiae by a nitric oxide-dependent mechanism. Human umbilical vein endothelial cells (HUVECs), when stimulated with the same concentrations of cytokines and RANTES, differed in their capacity to kill rickettsiae by a nitric oxide-dependent mechanism and in the quantity of nitric oxide synthesized. Hydrogen peroxide-dependent intracellular killing of R. conorii was demonstrated in HUVECs, THP-1 cells (human macrophages), and human peripheral blood monocytes activated with the cytokines. Rickettsial killing in the human macrophage cell line was also mediated by a limitation of the availability of tryptophan in association with the expression of the tryptophan-degrading enzyme indoleamine-2,3-dioxygenase. The rates of survival of all of the cell types investigated under the conditions of activation and infection in these experiments indicated that death of the host cells was not the explanation for the control of rickettsial infection. This finding represents the first demonstration that activated human hepatocytes and, in some cases, endothelium can kill intracellular pathogens via nitric oxide and that RANTES plays a role in immunity to rickettsiae. Human cells are capable of controlling rickettsial infections intracellularly, the most relevant location in these infections, by one or a combination of three mechanisms involving nitric oxide synthesis, hydrogen peroxide production, and tryptophan degradation.

Cells, Cultured↗

PubMed2000

Molecular cloning and characterization of the 120-kilodalton protein gene of Ehrlichia canis and application of the recombinant 120-kilodalton protein for serodiagnosis of canine ehrlichiosis.

The 120-kDa outer membrane protein (p120) is a potential adhesin of Ehrlichia chaffeensis, and recombinant p120 is very useful for serodiagnosis of human monocytotropic ehrlichiosis. The analogous gene of p120 in Ehrlichia canis was cloned, sequenced, and expressed. Like the E. chaffeensis p120, the E. canis p120 contains tandem repeat units. However, neither the repeat number nor the amino acid sequences in the repeats are identical in the two Ehrlichia species. The repeat units are hydrophilic and by probability analysis are predicted to be surface exposed in both species. The repeat regions of the p120s of the two species have common amino acid sequences that are predicted to be surface exposed. The overall amino acid sequence of the E. canis p120 is 30% homologous to that of E. chaffeensis p120. Protein immunoblotting demonstrated that the recombinant E. canis p120 reacted with convalescent sera from dogs with canine ehrlichiosis. These results indicate that the recombinant p120 is a potential antigen for the serodiagnosis of canine ehrlichiosis.

Amino Acid Sequence↗

PubMed2000

Phylogenetic analysis of the rompB genes of Rickettsia felis and Rickettsia prowazekii European-human and North American flying-squirrel strains.

The rickettsial outer membrane protein B (rompB) gene encodes the major surface antigens of Rickettsia species. We undertook sequencing and molecular analysis of the rompB gene of Rickettsia felis and a comparison with its homologs in spotted fever group (SFG) and typhus group (TG) rickettsiae, including the complete sequences of two North American flying squirrel strains and two European human strains of Rickettsia prowazekii. We sequenced 5,226 base pairs (bp) of the R. felis rompB, encoding a protein of 1,654 amino acids. We also sequenced 5,015 bp of rompB of the flying squirrel strains, encoding a protein of 1,643 amino acids. Analysis of the R. felis rompB gene sequence showed 10-13% divergence from SFG rickettsiae and 18% divergence from the TG rickettsiae. The rompB of all sequenced strains of R. prowazekii showed an overall similarity of 99.7-99.9%.

Animals↗

PubMed1999

The incidence of ehrlichial and rickettsial infection in patients with unexplained fever and recent history of tick bite in central North Carolina.

We examined the clinical and laboratory findings of a consecutive series of patients from central North Carolina presenting with fever and a history of tick bite within the preceding 14 days. Evidence of a tick-transmitted pathogen was detected in 16 of 35 patients enrolled over a 2-year period. Nine patients were infected with Ehrlichia chaffeensis, and 6 were infected with a spotted fever group rickettsia; 1 patient had evidence of coinfection with E. chaffeensis and a spotted fever group rickettsia. Four patients had detectable antibodies against the human granulocytic ehrlichiosis agent; however, only 2 had a 4-fold antibody titer rise without detectable antibodies against E. chaffeensis. The other 2 were thought to have cross-reacting antibodies to E. chaffeensis. We conclude that ehrlichial infections may be as common as spotted fever group rickettsial infections in febrile patients from central North Carolina with a recent history of tick bite.

Adult↗

PubMed1999

Hemostatic changes in Rocky Mountain spotted fever and Mediterranean spotted fever.

Rocky Mountain spotted fever and Mediterranean spotted fever are rickettsial infections primarily of endothelial cells that normally have a potent anticoagulant function. As a result of endothelial cell infection and injury, the hemostatic system is perturbed and shows changes that vary widely from a minor reduction in the platelet count (frequently) to severe coagulopathies, such as deep venous thrombosis and disseminated intravascular coagulation (rarely). Changes favoring a hypercoagulable state include endothelial injury and release of procoagulant components, activation of the coagulation cascade with thrombin generation, platelet activation, increased antifibrinolytic factors, consumption of natural anticoagulants, and possibly high levels of coagulation-promoting cytokines. Yet, most studies have been performed on endothelial cell cultures that provide nonphysiologic, reductionistic, experimental conditions. The lack of flow, platelets, and WBCs makes these experiments far from simulating the response of endothelial cells in the human body. Coagulopathies and thrombotic events should be considered as potential complications of severe Rocky Mountain spotted fever and Mediterranean spotted fever.

Blood Coagulation↗

PubMed1999

Molecular cloning of the gene for a conserved major immunoreactive 28-kilodalton protein of Ehrlichia canis: a potential serodiagnostic antigen.

A gene encoding a 28-kDa protein of Ehrlichia canis was cloned, sequenced, and expressed, and a comparative molecular analysis with homologous genes of E. canis, Cowdria ruminantium, and Ehrlichia chaffeensis was performed. The complete gene has an 834-bp open reading frame encoding a protein of 278 amino acids with a predicted molecular mass of 30.5 kDa. An N-terminal signal sequence was identified, suggesting that the protein undergoes posttranslational modification to a mature 27.7-kDa protein (P28). The E. canis p28 gene has significant nucleic acid and amino acid sequence homologies with the E. chaffeensis outer membrane protein-1 (omp-1) gene family, with the Cowdria ruminantium map-1 gene, and with other E. canis 28-kDa-protein genes. Southern blotting revealed the presence of at least two additional homologous p28 gene copies in the E. canis genome, confirming that p28 is a member of a polymorphic multiple-gene family. Amino acid sequence analysis revealed that E. canis P28 has four variable regions, and it shares similar surface-exposed regions, antigenicity, and T-cell motifs with E. chaffeensis P28. The p28 genes from seven different E. canis isolates were identical, indicating that the gene for this major immunoreactive protein is highly conserved. In addition, reactivity of sera from clinical cases of canine ehrlichiosis with the recombinant P28 demonstrated that the recombinant protein may be a reliable serodiagnostic antigen.

Amino Acid Sequence↗