Biomedical subjects

D H Walker

Publications and source records attributed to D H Walker.

At least 109 records · Page 6Linked to original sources

PubMed1994

Identification of the antigenic constituents of Ehrlichia chaffeensis.

Ehrlichia chaffeensis, the novel etiologic agent of human ehrlichiosis in the United States, was first isolated in 1990 and reported in 1991. To analyze the antigenic components of E. chaffeensis, we cultivated these obligate intracellular bacteria in DH82 cells, purified the ehrlichiae by renografin density gradient centrifugation, and examined the antigens by Western immunoblotting. Rabbit and human antisera to E. chaffeensis revealed more than 20 bands ranging from 20 to 200 kD. The distinct 22-kD protein was heat labile. The rest of the major immunoreactive components were heat stable. The immunoblots of E. chaffeensis were highly similar when probed with antisera to E. chaffeensis, E. canis, and E. ewingii, indicating the close antigenic relationships among the three species. The 22-kD protein cross-reacted only with anti-E. canis serum. The antibody against E. sennetsu reacted strongly with the 66-, 64-, 55-, and 44-kD antigens of E. chaffeensis. The E. risticii antisera reacted strongly with the 55- and 44-kD bands but only faintly with the 66-kD band. The major immunoreactive antigens of E. chaffeensis (66, 55, and 44 kD) showed cross-reactions with all the different antisera tested. The results indicated that E. chaffeensis is antigenically most closely related to E. canis, is less closely related to E. ewingii, and is only distantly related to E. sennetsu and E. risticii.

Animals↗

PubMed1993

Pathology of inhalational anthrax in 42 cases from the Sverdlovsk outbreak of 1979.

A large epidemic of anthrax that occurred in Sverdlovsk (now Ekaterinburg), Russia, in 1979 resulted in the deaths of many persons. A series of 42 necropsies, representing a majority of the fatalities from this outbreak, consistently revealed pathologic lesions diagnostic of inhalational anthrax, namely hemorrhagic necrosis of the thoracic lymph nodes in the lymphatic drainage of the lungs and hemorrhagic mediastinitis. Bacillus anthracis was recovered in bacterial cultures of 20 cases, and organisms were detected microscopically in the infected tissues of nearly all of the cases. A novel observation was primary focal hemorrhagic necrotizing pneumonia at the apparent portal of entry in 11 cases. Mesenteric lymphadenitis occurred in only 9 cases. This remarkably large series demonstrated the full range of effects of anthrax bacteremia and toxemia (edema especially adjacent to sites of extensive infection and pleural effusions) and hematogenously disseminated infection [hemorrhagic meningitis (21 cases) and multiple gastrointestinal submucosal hemorrhagic lesions (39 cases)].

Adult↗

PubMed1993

Antigens of Rickettsia conorii recognized by seropositive healthy people from Salamanca (central-west Spain).

The antigenic reactivity in Western immunoblotting assay of individual Rickettsia conorii components with sera of healthy people living in Salamanca Province, an endemic zone of Mediterranean spotted fever, is evaluated. Polypeptides of molecular weights 100 kDa (92.7%), 135 kDa (75.6%), 160 kDa (70.7%) and 115 kDa (48.8%) were recognized by a higher proportion of sera with indirect immunofluorescent antibody test titers > or = 1:80. Reaction with apparent rickettsial lipopolysaccharide was found in 15 (36.6%) of these samples. The involvement of different rickettsial strains, atypical routes of inoculation, varying content of the inoculum, and host factors may be determinants of the clinical expression of the spotted fever group rickettsial infection in people who produce antibodies reactive with Rickettsia conorii antigens.

Adult↗

PubMed1993

Human ehrlichiosis: hematopathology and immunohistologic detection of Ehrlichia chaffeensis.

Human ehrlichiosis is a recently described zoonosis caused by a rickettsia that infects leukocytes. Most patients have fever, headache, chills, and myalgias and develop leukopenia, thrombocytopenia, anemia, and elevations in serum hepatic aminotransferases. The cause of the peripheral leukopenia and thrombocytopenia is not known. We studied peripheral blood smears, bone marrow aspirates, and bone marrow biopsy specimens from patients with serologically proven ehrlichiosis to characterize the pathologic changes associated with leukopenia or thrombocytopenia, to detect the presence of immunohistologically demonstrable ehrlichiae, and to establish the infected host target cell(s). Specimens were obtained from 12 patients, and immunohistology for Ehrlichia chaffeensis was performed on tissue sections, aspirated bone marrow, and peripheral blood smears. Mean leukocyte and platelet counts available for nine patients were white blood cell count 3,300/microL (range, 1,100 to 10,300/microL) and platelets 61,000/microL (range, 40,000 to 82,000/microL). Findings included myeloid hyperplasia (eight cases), megakaryocytosis (seven cases), granulomas (eight cases), marrow histiocytosis (one case), myeloid hypoplasia (one case), pancellular hypoplasia (one case), and normocellular marrow (two cases). Morulae of E chaffeensis were detected in four of 10 cases examined by immunohistology. Most ehrlichiae were detected within histiocytes, although morulae were rarely present within lymphocytes. Leukopenia, thrombocytopenia, or pancytopenia apparently most often results from peripheral sequestration or destruction; however, hypoplasia of marrow elements is present occasionally. Immunohistologic demonstration of E chaffeensis offers a direct means for establishing the etiologic diagnosis. These observations show the relatively frequent occurrence of bone marrow granulomas and suggest that infection of cells of the reticuloendothelial system may participate in the pathogenesis of human ehrlichiosis.

Adult↗

PubMed1993

Rocky Mountain spotted fever complicated by gangrene: report of six cases and review.

Although mortality due to fulminant Rocky Mountain spotted fever (RMSF) is well appreciated, the ability of the disease to cause survivors to become permanently disabled is not as widely known. We report six cases of RMSF complicated by gangrene. Although four patients required multiple limb and/or digital amputations, only one death resulted. Our review of the English-language literature revealed 23 additional cases of RMSF complicated by gangrene. Pathophysiologically, gangrene is most likely related to small-vessel occlusion. Skin necrosis and gangrene in association with RMSF are the extreme end on a continuum from reversible to irreversible skin and tissue damage caused by Rickettsia rickettsii. Most patients with RMSF have a typical skin rash that resolves without sequelae. Some patients develop minute cicatrices marking the location of focal cutaneous necrosis; for other patients, digital ischemia occurs transiently or evolves to produce severe ischemic changes without gangrene that result in permanent impairment. At the far end of this clinical continuum are patients who develop gangrene requiring amputation.

Adult↗

PubMed1993

Persistent infection with Ehrlichia chaffeensis.

Although persistent infection of animals by members of the genus Ehrlichia is well known and may be associated with subsequent severe or fatal illness, persistent infection of humans with Ehrlichia chaffeensis has not been reported. Herein we report a typical case of serologically documented acute ehrlichiosis; despite therapy with tetracycline and chloramphenicol, the patient's condition progressively worsened and he suffered multiple secondary infections and gastrointestinal hemorrhage. He died 68 days after his initial hospitalization. Retrospective immunohistologic examination of both acute-phase bone marrow specimens (obtained day 12 of illness) and postmortem liver tissue specimens (obtained day 68 after onset of disease) revealed E. chaffeensis morulae in mononuclear cells, presumably macrophages and monocytes. Findings of this case provide the first definitive evidence that E. chaffeensis is capable of establishing persistent human infection and suggest a role for this obligate intracellular bacterium in the induction of immune compromise associated with a fatal outcome.

Acute Disease↗

PubMed1993

EIA with species-specific monoclonal antibodies: a novel seroepidemiologic tool for determination of the etiologic agent of spotted fever rickettsiosis.

All currently available assays for antibodies to the etiologic agents of spotted fever group rickettsioses detect reactivity with antigens of lipopolysaccharides and the major cell wall proteins, which contain epitopes that are shared among many spotted fever group rickettsiae. The hypothesis of this study was that a monoclonal antibody to a species-specific epitope of Rickettsia rickettsii would be blocked from binding to the rickettsial surface if the rickettsiae were incubated previously with serum containing species-specific antibodies to the epitope. In an EIA, binding of monoclonal antibody 5C10-F3 to R. rickettsii was blocked by convalescent sera from patients with Rocky Mountain spotted fever but not from those with Mediterranean spotted fever, endemic typhus, or Q fever or normal subjects. This assay should be useful in determining the origin of the high seroprevalence of spotted fever rickettsial antibodies in certain geographic regions and establishing a species-specific diagnosis in patients with undetermined rickettsial exposure.

Antibodies, Bacterial↗

PubMed1993

Prevalence of antibodies to spotted fever group rickettsiae along the eastern coast of the Adriatic sea.

A seroepidemiological survey in coastal Croatia detected antibodies reactive with Rickettsia conorii in 4.2% of sera by immunofluorescence assay and in 5.0% of sera by enzyme immunoassay. Western immunoblotting demonstrated antibodies to the 120-kDa surface protein in all 20 positive serum samples examined and to rickettsial lipopolysaccharide in 3 of these serum samples. Humans in this area are clearly being exposed to spotted fever rickettsiae.

Antibodies, Bacterial↗

PubMed1993

Brazilian spotted fever in Espirito Santo, Brazil: description of a focus of infection in a new endemic region.

Although Brazilian spotted fever is known to occur in several adjacent states, infection with spotted fever group (SFG) rickettsiae has not previously been documented in the Brazilian state of Espirito Santo. We report a cluster of two proven and four suspected cases of Brazilian spotted fever in a small valley near Colatina, Espirito Santo. Four patients died without confirmatory serologic or pathologic studies. The two surviving patients had serologic and/or pathologic evidence of infection with SFG rickettsiae. Results of a survey done in 1991 confirmed the presence of antibodies reactive with SFG rickettsiae in the serum of dogs living in this region. Two of 25 dogs had elevated microimmunofluorescent antibody titers (1:64 and 1:256). Both seropositive dogs were pets of the index cases and lived in the valley where all six human cases were presumed to have acquired their infections. These cases and the results of the canine serosurvey suggest that a focus of virulent disease exists in an area not previously known to be endemic for SFG rickettsiae.

Adult↗

PubMed1993

Interferon-gamma and tumor necrosis factor-alpha exert their antirickettsial effect via induction of synthesis of nitric oxide.

How the host defenses control rickettsiae in the cytosol of nonphagocytic host cells, where they are not exposed to antibodies or phagocytes, has posed a difficult question. Rickettsia conorii infection of a mouse fibroblast cell line was inhibited in a dose-dependent manner by nitrogen oxide synthesized by eukaryotic host cells stimulated by interferon-gamma or tumor necrosis factor-alpha. L-arginine was the source of the nitric oxide as demonstrated by competitive inhibition by NG-monomethyl-L-arginine. Nitric oxide synthesis required host cell protein synthesis and had an approximately 48-hour lag phase following cytokine stimulation. At low doses of interferon-gamma and tumor necrosis factor-alpha, which had no detectable response as single agents, dramatic synergistic nitric oxide synthesis and antirickettsial effects were observed.

Animals↗

PubMed1993

Rickettsia australis infection: a murine model of a highly invasive vasculopathic rickettsiosis.

A mouse model of spotted fever group rickettsiosis, in which disease results from disseminated rickettsial infection of endothelial cells and vascular damage, was developed by intravenous inoculation of 6- to 8-week-old, male, Balb/c mice with Rickettsia australis. Animals developed progressively severe vasculitis, interstitial pneumonia, and multifocal hepatic necrosis. These lesions correlated with early disseminated infection of endothelial cells followed by growth and invasion of rickettsiae into perivascular cells. The dose of 2 x 10(6) organisms was uniformly lethal. Serum interleukin- (IL) 1, IL-6, and interferon (IFN) increased by day 3 and tumor necrosis factor (TNF) on day 5. TNF, IL-6, and IFN declined on day 7. Spleen cells responded to Rickettsia australis antigen by producing IFN, TNF, IL-1, and IL-6 on day 5, followed by lower quantities of these cytokines on day 7. Despite the production of antibodies, IFN, TNF, IL-1, and IL-6, a lethal outcome occurred frequently. A decreased ability to secrete IL-2 suggests an element of infection-associated immunosuppression.

Animals↗

PubMed1993

[The western immunoblotting technique in atypical situations of Rickettsia conorii infection. Presentation of 2 cases].

BACKGROUND: During the last few years the application of Western immunoblotting in the study of human infection by Rickettsia conorii has led to the development of a new method of serologic diagnosis of the Mediterranean exanthematous fever. METHODS: The pattern of reactivity of serum samples sequentially obtained in the course of infection versus R. conorii antigens (Malish 7 strain) purified in discontinued density gradient and separated by SDS-PAGE was analyzed by Western immunoblotting. RESULTS: The presence of typical profiles of rickettsial infection similar to those observed in the common forms of presentation of the Mediterranean exanthematous fever was demonstrated in a case of accidental infection transmitted via aerosol and followed by an incomplete clinical picture after the early administration of specific antibiotic therapy and in a patient with signs and symptoms characteristic of Mediterranean exanthematous fever in whom the results of indirect immunofluorescence tests were repeatedly negative. CONCLUSIONS: Western immunoblotting may be a specially useful technique as a complementary procedure in the laboratory to guide in the diagnosis of Mediterranean exanthematous fever in special situations.

Acute Disease↗

PubMed1992

Cdc25 regulates the phosphorylation and activity of the Xenopus cdk2 protein kinase complex.

The Xenopus cdk2 gene encodes a 32-kDa protein kinase with sequence similarity to the 34-kDa product of the cdc2 gene. Previous studies have shown that the kinase activity of the protein product of the cdk2 gene oscillates in the Xenopus embryonic cell cycle with a high in M-phase and a low in interphase. In the present study cdk2 was found not to be associated with any newly synthesized proteins during the cell cycle, but the enzyme did undergo periodic changes in phosphorylation. Upon exit from metaphase, cdk2 became increasingly phosphorylated on both tyrosine and serine residues, and labeling on these residues increased progressively until entry into mitosis, when tyrosine residues were markedly dephosphorylated. Phosphopeptide mapping of cdk2 demonstrated the major sites of phosphorylation were in a phosphopeptide with a pI of 3.7 that contained both phosphoserine and phosphotyrosine. This phosphopeptide accumulated in egg extracts blocked in S-phase with aphidicolin and was not evident in cdc2 immunoprecipitated under the same conditions. Under the same conditions cdc2 was phosphorylated primarily on a phosphopeptide containing both phosphothreonine and phosphotyrosine residues, most likely threonine 14 and tyrosine 15. Affinity-purified human GST-cdc25 was able to dephosphorylate and activate cdk2 isolated from interphase cells. Phosphopeptide mapping demonstrated that the phosphate was specifically removed from the same phosphopeptide identified as the major in vivo site of phosphorylation. These results demonstrate that cdk2 is regulated in the cell cycle by phosphorylation and dephosphorylation on both serine and tyrosine residues. Moreover, the increased phosphorylation of cdk2 in aphidicolin-blocked extracts and the ability of cdc25 to mediate cdk2 dephosphorylation in vitro suggest the possibility that cdk2 is part of the mechanism ensuring mitosis is not initiated until completion of DNA replication. It also implies cdc25 may have other functions in addition to the regulation of cdc2 kinase activity.

Animals↗

PubMed1992

Use of western blot to analyze the reactivity of sera from patients with Mediterranean spotted fever.

The reactivity of antigenic components of Rickettsia conorii with sequentially obtained sera from 20 adult Spanish patients with Mediterranean spotted fever was analyzed by Western blot. The major rickettsial antigens reacting with the serum samples corresponded to molecular weights of 135 and 115 kDa. These antigens constantly exhibited higher staining intensity than the other antigens, and reacted with 100% and 86.7%, respectively, of acute sera and with 100% of convalescent phase samples. Rickettsial lipopolysaccharide antigens reacted with 94.7% of sera collected in the fourth and fifth week after onset of symptoms. Other major antigens reactive in the blots had molecular sizes of 160, 100, 90 and 60 kDa, and a relatively frequent humoral immune response was also seen to antigens of 80, 73 and 55 kDa.

Adult↗

PubMed1992

Multiple roles for protein phosphatase 1 in regulating the Xenopus early embryonic cell cycle.

Using cytostatic factor metaphase II-arrested extracts as a model system, we show that protein phosphatase 1 is regulated during early embryonic cell cycles in Xenopus. Phosphatase 1 activity peaks during interphase and decreases shortly before the onset of mitosis. A second peak of activity appears in mitosis at about the same time that cdc2 becomes active. If extracts are inhibited in S-phase with aphidicolin, then phosphatase 1 activity remains high. The activity of phosphatase 1 appears to determine the timing of exit from S-phase and entry into M-phase; inhibition of phosphatase 1 by the specific inhibitor, inhibitor 2 (Inh-2), causes premature entry into mitosis, whereas exogenously added phosphatase 1 lengthens the interphase period. Analysis of DNA synthesis in extracts treated with Inh-2, but lacking the A- and B-type cyclins, shows that phosphatase 1 is also required for the process of DNA replication. These data indicate that phosphatase 1 is a component of the signaling pathway that ensures that M-phase is not initiated until DNA synthesis is complete.

Animals↗

PubMed1992

Periodic changes in phosphorylation of the Xenopus cdc25 phosphatase regulate its activity.

The cdc25 tyrosine phosphatase is known to activate cdc2 kinase in the G2/M transition by dephosphorylation of tyrosine 15. To determine how entry into M-phase in eukaryotic cells is controlled, we have investigated the regulation of the cdc25 protein in Xenopus eggs and oocytes. Two closely related Xenopus cdc25 genes have been cloned and sequenced and specific antibodies generated. The cdc25 phosphatase activity oscillates in both meiotic and mitotic cell cycles, being low in interphase and high in M-phase. Increased activity of cdc25 at M-phase is accompanied by increased phosphorylation that retards electrophoretic mobility in gels from 76 to 92 kDa. Treatment of cdc25 with either phosphatase 1 or phosphatase 2A removes phosphate from cdc25, reverses the mobility shift, and decreases its ability to activate cdc2 kinase. Furthermore, the addition of okadaic acid to egg extracts arrested in S-phase by aphidicolin causes phosphorylation and activation of the cdc25 protein before cyclin B/cdc2 kinase activation. These results demonstrate that the activity of the cdc25 phosphatase at the G2/M transition is directly regulated through changes in its phosphorylation state.

Amino Acid Sequence↗

PubMed1992

Rickettsia japonica sp. nov., the etiological agent of spotted fever group rickettsiosis in Japan.

We propose the name Rickettsia japonica sp. nov. (with type strain YH [= ATCC VR-1363]) for a serologically specific species of spotted fever group rickettsiae that are pathogenic for humans (J. Infect. Dis. 159:1122-1126, 1989; J. Clin. Microbiol. 28:1177-1180, 1990). The biologic and genomic characteristics of the organism (G+C content, 31.2 +/- 0.7 mol%) are essentially the same as those of other pathogenic spotted fever group rickettsiae, although the R. japonica isolates cause a persistent infection in Vero cells for many subcultures.

Animals↗

PubMed1992

Characterization of rickettsial attachment to host cells by flow cytometry.

The mechanisms of rickettsial attachment have been studied by measuring quantitative changes in rickettsial binding to host cells by flow cytometry after different treatments of the rickettsiae and host cells. Time-dependent binding of Rickettsia conorii to host cells was demonstrated by the increasing intensity of host cell surface fluorescence of rickettsia-host cell combinations when examined with a rickettsia-specific monoclonal antibody. More than 70% of host cells had intensity of fluorescence above the threshold value after 10 min of incubation, owing to rickettsiae bound to the cell surface, and the greatest fluorescence intensity indicative of binding occurred at 20 min. The binding kinetics was rickettsial dose dependent. The binding of rickettsiae to host cells was greatly decreased when host cells or rickettsiae were treated with 1% paraformaldehyde for 30 min or 0.25% trypsin for 5 or 15 min, respectively. Rickettsiae that were heated at 56 degrees C for 15 min lost more than 80% of their ability to attach to host cells. R. rickettsii, an organism closely related to R. conorii, competitively inhibited the attachment of R. conorii (51% inhibition when mixed in equal numbers). These results indicate that the rickettsial binding structures are trypsin and heat sensitive and likely to be surface proteins.

Animals↗