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D H Walker

Publications and source records attributed to D H Walker.

At least 91 records · Page 5Linked to original sources
PubMed1995

Comparative antigenic analysis of spotted fever group rickettsiae from Israel and other closely related organisms.

Spotted fever rickettsiosis in Israel has been considered as possibly somewhat more severe than boutonneuse fever, from which it also differs in having a very low proportion of cases with a tick-inoculation site eschar. This investigation was undertaken to determine whether the Israeli spotted fever group (SFG) rickettsiae differed sufficiently from Rickettsia conorii to be considered as a distinct species. Strains of Rickettsia conorii from Morocco and South Africa, four SGF rickettsial isolates from Israel, one from Russia, and one from Zimbabwe were compared by microimmunofluorescence serotyping, Western immunoblotting, monoclonal antibody reactivity, and polymerase chain reaction amplification of the repeat domain of the rickettsial outer membrane protein A (rOmpA). All are strains and isolates of R. conorii, yet there is considerable molecular and antigenic diversity of both rOmpA and rickettsial outer membrane protein B (rOmpB) among them. The rOmpA gene of the Israeli isolates and the Astrakhan strain from Russia is estimated to encode 15 rOmpA repeat units as compared with 10 for the South African strain and six for the strains from Morocco and Zimbabwe. The Israeli SFG rickettsial strains appear to be R. conorii, a species with substantial antigenic and genetic diversity. The Israeli strains appear to fall within the limit previously described for the genetic and antigenic diversity of R. conorii.

Animals↗
PubMed1995

Persistence of rickettsiae in the partially viable gangrenous margins of amputated extremities 5 to 7 weeks after onset of Rocky Mountain spotted fever.

A case of Rocky Mountain spotted fever complicated by gangrene of the extremities required amputation and showed persistent rickettsial infection in the margins of the gangrene at 5 and 7 weeks after onset of illness. Rickettsial organisms were demonstrated by immunoperoxidase staining in the viable endothelial cells in the ischemic margins of the gangrenous tissue. We conclude that the cellular, humoral, and pharmacologic components that lead to eradication of the rickettsiae were not delivered to the ischemic margins at effective antirickettsial concentrations.

Adult↗
PubMed1995

Misregulated expression of the cyclin dependent kinase 2 protein in human fibroblasts is accompanied by the inability to maintain a G2 arrest following DNA damage.

The misregulation of cell cycle checkpoints has been implicated in the onset of neoplasia. To thoroughly understand the differences in checkpoint regulation between normal and transformed cells, we have compared the cell cycle responses of normal and TAg-transformed fibroblasts to DNA damage by gamma-irradiation. Normal cell lines arrest in both G1 and G2 for in excess of 48 h after DNA damage. Surprisingly, both cyclin-dependent kinase 2 (CDK2) and cyclin A proteins were specifically down-regulated within 24 h of DNA damage. In contrast, TAg transformed cells did not down-regulate either cyclin A or CDK2 after DNA damage and showed a significantly shortened G2 arrest. To investigate the role CDK2 down-regulation plays in cell cycle arrests, we generated normal cell lines that constitutively overexpress CDK2. These cells fail to down-regulate both CDK2 protein and CDK2 protein kinase activity after DNA damage and also show a G2 checkpoint defect; although the cells are able to normally initiate both a G1 and a G2 arrest, they prematurely escape the G2-M arrest after DNA damage. The escape from G2 correlates with an illicit activation of cyclin B-associated protein kinase activity in these cells. These results suggest that the misregulation of CDK2 contributes to the failure of checkpoint control by allowing cells to enter mitosis prematurely.

CDC2-CDC28 Kinases↗
PubMed1995

Will pathologists play as important a role in the future as they have in the past against the challenge of infectious diseases.

Since the recognition less than 120 years ago that organisms visible only microscopically are capable of causing human diseases, pathologists have played a major role in identifying and characterizing the etiologic infectious agents and in elucidating the pathogenic mechanisms. In face of the opportunities and challenges presented by molecular technology, AIDS and other emerging infections, and the evolution of health care systems, it is worthwhile to question whether the field of pathology will continue in the future to make major contributions in the field of infectious diseases. The AIDS epidemic has awakened pathologists to the need to reemphasize infectious diseases in diagnostic anatomic and clinical pathology, basic and applied research, and medical and scientific education. The knowledge and skills of pathologists are uniquely critical to the achievement of efficient advances in infectious diseases, and will remain so provided that pathologists embrace molecular science and apply it as a principal component in their methodologic and conceptual armamentarium.

Forecasting↗
PubMed1994

Human granulocytic ehrlichiosis in the upper Midwest United States. A new species emerging?

OBJECTIVE: To characterize the clinical presentation and course, laboratory findings, and treatment outcome of 12 patients with human granulocytic ehrlichiosis. SETTING: The 12 patients were male, ranged in age from 29 to 91 years, and contracted their illness in Wisconsin or Minnesota. METHODS: Cases were recognized by the presence of intracytoplasmic inclusions (morulae) in peripheral neutrophils of patients presenting with temperature of 38.5 degrees C or higher, chills, severe headache, and myalgias. All patients had a complete blood cell count and blood chemistry profile. Blood smears were examined by light microscopy. All available paired serum samples were analyzed for presence of indirect fluorescent antibodies against Ehrlichia chaffeensis, Ehrlichia phagocytophila, and Ehrlichia equi. Blood samples from 12 patients were subjected to polymerase chain reaction analysis using primers specific for the E phagocytophila/E equi group, primers that include the agent identified in our patients, as well as E chaffeensis. RESULTS: Varying combinations of leukopenia, anemia, and thrombocytopenia were found in all but one patient. All 12 patients demonstrated morulae in the cytoplasm of neutrophils, but not in mononuclear white blood cells. Serum assays failed to detect antibodies against E chaffeensis, but eight of 10 patients and seven of 10 patients tested had antibody titers of 1:80 or more for E phagocytophila and E equi, respectively. Polymerase chain reaction products obtained with primers for E phagocytophila, E equi, and the granulocytotropic Ehrlichia revealed that seven patients were infected with the same agent. The results of serological assays or polymerase chain reaction strongly suggest that all 12 patients were infected by E phagocytophila, E equi, or a closely related Ehrlichia species. Two of the 12 patients died. The other 10 patients improved rapidly with oral doxycycline treatment. CONCLUSIONS: We believe that all 12 patients have been infected with a granulocytic Ehrlichia species, reflecting a recently described new disease entity. The infective organism appears to be closely related to E phagocytophila and E equi. The geographic domain of human granulocytic ehrlichiosis is currently unknown. This novel granulocytic Ehrlichia species is capable of causing fatal infections in humans. Early detection and treatment with tetracycline drugs appear to offer the best chance for complete recovery.

Adult↗
PubMed1994

Sequence analysis of the 190-kDa antigen-encoding gene of Rickettsia conorii (Malish 7 strain).

The complete nucleotide (nt) sequence of the gene (rompA) encoding the 190-kDa immunodominant surface antigen (rOmpA) of Rickettsia conorii (Malish 7 strain) was determined. Sequence analysis revealed an ORF of 6063 nt encoding a deduced protein of 203,247 Da. Ten consecutive highly conserved repeat units, located at the 5' end of rompA, spanned 2.2 kb. Two types of repeats could be identified: type I of 225 bp, and type II of 216 bp. The order and number of repeats differed from those reported for R. conorii (Kenya tick typhus strain), R. akari (Kaplan strain) and R. rickettsii (R strain). Alignment of the R. conorii (Malish 7 strain) rompA gene with its R. rickettsii homolog revealed 95% nt sequence similarity. The conservation of rompA across several pathogenic spotted fever group (SFG) rickettsial species suggests that it may be a potential candidate for use as a subunit vaccine for SFG rickettsial diseases.

Amino Acid Sequence↗
PubMed1994

Identification of a natural cycle involving Rickettsia typhi infection of Monopsyllus sciurorum sciurorum fleas from the nests of the fat dormouse (Glis glis).

In a mountainous area in the Dinaric Beech-Fir Forest of southern Slovenia, summer nests of the European fat dormouse (Glis glis) were collected. From these dormouse nests, 180 Monopsyllus sciurorum sciurorum fleas were examined by polymerase chain reaction with primers for the Rickettsia citrate synthase gene. Samples from one nest yielded the expected 381 base pair DNA product. The origin of the DNA product was identified as Rickettsia typhi by AluI restriction fragment length polymorphism analysis. Inoculation of the triturated positive fleas into Vero cell culture resulted in the cultivation of a rickettsia which reacted with polyclonal and species-specific monoclonal antibodies for R. typhi. The widespread distribution of this sylvatic flea species in nearly all of Europe as well as in the Middle East and its presence on other mammalian and avian hosts suggests that R. typhi might exist in unrecognised enzootic cycles. Further investigations are needed to determine the extent of these cycles in Europe and the potential occurrence of human infections.

Animals↗
PubMed1994

Identification of Rickettsia conorii infection by polymerase chain reaction in a soldier returning from Somalia.

A soldier developed characteristic manifestations of boutonneuse fever shortly after leaving Somalia. Rickettsial DNA was detected in a biopsy sample of the tache noire by a polymerase chain reaction (PCR) in which primers derived from the 190-kD antigen gene of Rickettsia rickettsii were used. The source of this DNA was identified as Rickettsia conorii by restriction fragment length polymorphism (RFLP) analysis of the PCR product. R. conorii was also isolated from the skin biopsy specimen. The patient did not develop a significant increase in specific antibodies, as assessed by indirect fluorescent antibody testing, until several weeks after the onset of symptoms. This case demonstrates that the PCR/RFLP technique can be used for the direct identification of rickettsiae from clinical specimens. To our knowledge, this is the first confirmed case of R. conorii infection in Somalia.

Adult↗
PubMed1994

Analysis of major surface polypeptides of Rickettsia japonica.

Major surface polypeptides of Rickettsia japonica migrated to the position of 120, 135, and 145 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, when the organisms were solubilized at room temperature. Two major bands at the position of 135 and 185 kDa were seen, when the organisms were solubilized by heating before electrophoresis. Heat-denaturation of the 120- and 145-kDa polypeptides in excised gel bands changed their mobility and caused them to migrate to 135- and 185-kDa positions, respectively. Two polypeptides at the 120-kDa position were demonstrated: one is a major heat-modifiable polypeptide and the other a minor heat-stable. Peptide mapping was performed to determine the identity between native and denatured polypeptides.

Bacterial Proteins↗
PubMed1994

Cloning, sequencing, and expression of the gene coding for an antigenic 120-kilodalton protein of Rickettsia conorii.

Several high-molecular-mass (above 100 kDa) antigens are recognized by sera from humans infected with spotted fever group rickettsiae and may be important stimulators of the host immune response. Molecular cloning techniques were used to make genomic Rickettsia conorii (Malish 7 strain) libraries in expression vector lambda gt11. The 120-kDa R. conorii antigen was identified by monospecific antibodies to the recombinant protein expressed on construct lambda 4-7. The entire gene DNA sequence was obtained by using this construct and two other overlapping constructs. An open reading frame of 3,068 bp with a calculated molecular mass of approximately 112 kDa was identified. Promoters and a ribosome-binding site were identified on the basis of their DNA sequence homology to other rickettsial genes and their relative positions in the sequence. The DNA coding region shares no significant homology with other spotted fever group rickettsial antigen genes (i.e., the R. rickettsii 190-, 135-, and 17-kDa antigen-encoding genes). The PCR technique was used to amplify the gene from eight species of spotted fever group rickettsiae. A 75-kDa portion of the 120-kDa antigen was overexpressed in and purified from Escherichia coli. This polypeptide was recognized by antirickettsial antibodies and may be a useful diagnostic reagent for spotted fever group rickettsioses.

Amino Acid Sequence↗
PubMed1994

Depletion of gamma interferon and tumor necrosis factor alpha in mice with Rickettsia conorii-infected endothelium: impairment of rickettsicidal nitric oxide production resulting in fatal, overwhelming rickettsial disease.

C3H/HeN mice infected intravenously with a dose of Rickettsia conorii (Malish 7 strain) that is sublethal for immunocompetent animals (1.1 x 10(3) PFU) developed disseminated infection of endothelial cells of the brain, lungs, heart, liver, kidney, testis, and testicular adnexa. In R. conorii-infected mice depleted of gamma interferon (IFN-gamma) and/or tumor necrosis factor alpha (TNF-alpha) by intravenous administration of neutralizing monoclonal antibodies on days 0, 2, and 4, the mortality rate was 100%. Death of the cytokine-depleted animals on days 5 and 6 was associated with overwhelming rickettsial infection documented by titration of rickettsial content in the brain and liver and by immunohistologic demonstration of massive quantities of R. conorii in endothelial cells of all organs examined, in macrophages of the liver and spleen, and in hepatocytes. Nondepleted, immunocompetent animals showed markedly reduced rickettsial content in the tissues on day 6, with rickettsial destruction in phagolysosomes not only in macrophages but also in endothelial cells and hepatocytes. All nondepleted, infected mice recovered and appeared completely healthy by day 9. Assay of liver infiltrated by lymphocytes and macrophages revealed mRNA of IFN-gamma and TNF-alpha, indicating that the host defenses were activated at the site of infection. Treatment of mice with an analog of L-arginine reduced the synthesis of nitric oxide and impaired rickettsial killing. Nitric oxide production was also impaired in cytokine-depleted infected mice. These observations support the hypothesis that IFN-gamma secreted by T lymphocytes and natural killer cells and TNF-alpha secreted by macrophages act in a synergistic, paracrine fashion on adjacent rickettsia-infected endothelial cells, hepatocytes, and macrophages to stimulate synthesis of nitric oxide, which kills intracellular R. conorii.

Animals↗
PubMed1994

Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease.

Six patients from northern Minnesota and Wisconsin with a febrile illness accompanied by granulocytic cytoplasmic morulae suggestive of ehrlichial infection were identified. Two patients died, and splenic granulocytes of one patient contained cytoplasmic vacuoles with organisms ultrastructurally characteristic of ehrlichiae. From one patient, a 1.5-kb DNA product was amplified by PCR with universal eubacterial primers of 16S rDNA. Analysis of the nucleotide sequence of the amplified product revealed 99.9 and 99.8% similarities with E. phagocytophila and E. equi, respectively, neither of which has previously been known to infect humans. From the variable regions of the determined sequence, a forward primer specific for three organisms (human granulocytic ehrlichia, E. phagocytophila, and E. equi) and a reverse primer for these ehrlichiae and E. platys were designed. By nested PCR with amplification by the universal primers and then reamplification with the specific primers described above, the expected 919-bp product was generated from the blood of the index patient and three additional patients. Blood from these four patients and two more patients with granulocytic morulae contained DNA which was amplified by nested PCR involving a combination of a universal primer and the human granulocytic ehrlichia-E. phagocytophila-E. equi-E. platys group-specific primer. This apparently vector-borne human granulocytic ehrlichia has only 92.5% 16S rDNA homology with E. chaffeensis. Nested PCR with group-specific primers did not amplify E. chaffeensis DNA, and E. chaffeensis-specific primers did not amplify DNAs of the human granulocytic ehrlichia. Thus, six patients were shown to be infected by an Ehrlichia species never previously reported to infect humans.

Aged↗
PubMed1994

Antigen-capture enzyme immunoassay: a comparison with other methods for the detection of spotted fever group rickettsiae in ticks.

To evaluate the prevalence of spotted fever group rickettsiae along the Adriatic Coast of Croatia, 832 ticks were examined by hemolymph test, direct immunofluorescence, antigen-capture enzyme immunoassay, and polymerase chain reaction. Very good agreement was observed among direct immunofluorescence, polymerase chain reaction, and antigen-capture enzyme immunoassay. Twelve ticks that were positive by hemolymph test and negative by both direct immunofluorescence and polymerase chain reaction presumably do not represent spotted fever group rickettsiae. By direct immunofluorescence, spotted fever group rickettsiae were present in 12% of Rhipicephalus bursa, 10.6% of Rh. sanguineus, and 7.8% of Dermacentor marginatus. From the 98 ticks containing rickettsia-like organisms by hemolymph test, seven spotted fever group rickettsial isolates were established in cell culture. Four isolates were identified as Rickettsia conorii. The antigen-capture enzyme immunoassay, which utilizes a monoclonal antibody to antigens of the 135-kD surface protein shared among many members of the spotted fever group, is recommended for primary screening of tick samples because it is reliable and yet less labor-intensive than the hemolymph and direct immunofluorescence tests. Although the polymerase chain reaction is too expensive for use as a screening method, it is recommended for confirmation of positive screening results. In addition to the technologic advance of the antigen-capture enzyme immunoassay, this study documented by contemporary methods that R. conorii is present along the eastern Adriatic Coast not only in the classic vector, Rh. sanguineus, but also in Rh. bursa and D. marginatus.

Animals↗
PubMed1994

Rickettsia conorii infection of C3H/HeN mice. A model of endothelial-target rickettsiosis.

BACKGROUND: Rickettsial diseases result from disseminated intraendothelial cell infection. The clinically critical conditions, meningoencephalitis and interstitial pneumonia, are associated with multifocal rickettsial vascular injury. EXPERIMENTAL DESIGN: C3H/HeN mice inoculated intravenously with either 2.25 x 10(3) or 2.25 x 10(5) Rickettsia conorii (Malish 7 strain) were observed for illness with sacrifice of animals for evaluation of pathologic lesions and host responses by light and electron microscopy, rickettsial content and location by plaque assay, immunohistology, and electron microscopy, and immune response by cytokine analyses and serology. RESULTS: Mice inoculated with a high dose of rickettsiae established disseminated endothelial infection on day 1, became ill with progressive increase in rickettsiae on day 4, and died with vascular injury-based meningoencephalitis and interstitial pneumonia on day 5 or 6. Mice inoculated with the low rickettsial dose became ill on day 5 and recovered by day 10. Clearance of rickettsiae was associated with lymphohistiocytic perivasculitis. Rickettsial infection of Kupffer cells and hepatocytes led to the formation of transient hepatic granulomas. Infection-associated loss of the ability of spleen cells to secrete interleukin-2 on stimulation with concanavalin A suggested transient immunosuppression. CONCLUSIONS: This experimental infection provides the best available model for rickettsial disease with endothelial infection and injury, immune rickettsial clearance, regeneration of endothelium, and repair of the vascular lesions.

Animals↗
PubMed1994

Demonstration of heat-labile and heat-stable epitopes of Rickettsia japonica on ultrathin sections.

BACKGROUND: Spotted fever group rickettsiae including Rickettsia japonica have two major polypeptides and lipopolysaccharide (LPS)-like antigen on the surface. The major part of the antibodies to these polypeptides generated by immunization with live rickettsiae are reactive with heat-labile and conformation-dependent epitopes. EXPERIMENTAL DESIGN: To demonstrate and precisely localize the heat-labile and heat-stable epitopes on the major surface polypeptides and LPS-like antigen of R. japonica, the immunocolloidal gold method was performed on ultrathin sections with polyclonal and monoclonal antibodies. The antigenicity and fine structure were preserved by using periodate-lysine-paraformaldehyde as fixative, followed by embedding with the resin Lowicryl K4M at a polymerization temperature of -35 degrees C. RESULTS: Heat-labile and heat-stable epitopes on the two major surface polypeptides together with LPS-like antigen of R. japonica were demonstrated at the same location. These antigens were rather broadly distributed on the cell wall apart from the slime layer of the organism. The number of gold particles corresponding to the LPS-like antigen was much larger than that corresponding to the polypeptide antigen. CONCLUSIONS: Heat-labile epitopes of rickettsiae could be preserved and demonstrated by using the procedure cited here. Moreover, the precise localization of the major surface polypeptides and LPS-like antigen could be achieved in the cell wall by using ultrathin sections of infected cells instead of whole rickettsial cells. The LPS-like antigen was quantitatively predominant as judged by immunoelectron microscopy.

Antibodies, Monoclonal↗
PubMed1994

Diagnostic tests for Rocky Mountain spotted fever and other rickettsial diseases.

Rickettsial diseases continue to confound physicians because of their usually sporadic occurrence and nonspecific clinical presentation. In past years, diagnosis was confirmed only during convalescence; however, newer, more widely available methods, such as antigen detection and improved in vitro cultivation, have enabled a diagnosis at a time when therapeutic decisions are made. Molecular nucleic acid-based techniques have also expanded the spectrum of etiologic agents and illnesses caused by rickettsiae.

Angiomatosis, Bacillary↗