Journal: Infection and Immunity

Article Title: ankA : an Ehrlichia phagocytophila Group Gene Encoding a Cytoplasmic Protein Antigen with Ankyrin Repeats

doi: 10.1128/iai.68.9.5277-5283.2000

Figure Lengend Snippet: FIG. 1. E. coli transformed with hge-d (left panel) or ee-o (right panel) expresses a 68- or 145-kDa antigen, respectively, as determined by protein im- munoblotting. The blots were reacted with the following primary antibodies: human anti-HGE agent (lanes 1), horse anti-HGE agent BDS strain (lanes 2), human anti-E. chaffeensis (lanes 3), dog anti-E. canis (lanes 4), human anti- Borrelia burgdorferi (lanes 5), and human anti-Babesia microti (lanes 6). Lanes 7 and 8 were reacted with normal human and horse sera, respectively. The location of the 68-kDa protein is indicated on the left.

Article Snippet: PCR was performed on DNA prepared from the following samples: four human HGE agent strains (Webster, Spooner, and 96HE-97 from Wisconsin and NY-8 from New York) in HL-60 cells, one HGE agent strain (97E12 from a Minnesota dog) in HL-60 cells, one blood sample from another Minnesota dog infected with the HGE agent, one blood sample from a Spanish goat with tick-borne fever (courtesy of Philippe Brouqui), neutrophils isolated from a cow with tick-borne fever in Sweden (courtesy of Philippe Brouqui and Anneli Bjöersdorff) containing different local strains of E. phagocytophila, E. sennetsu Miyayama strain in P388D1 cells (courtesy C. of Pretzman, Ohio State Department of Health), E. risticii HRC-IL strain (ATCC VR-986) in P388D1 cells, E. chaffeensis Arkansas strain in DH82 cells (courtesy of J. Dawson, Centers for Disease Control and Prevention, Atlanta, Ga.), E. chaffeensis 91HE17 strain in DH82 cells, and E. canis Oklahoma strain in DH82 cells (courtesy J. Dawson).

Techniques: Transformation Assay

Journal: Infection and Immunity

Article Title: ankA : an Ehrlichia phagocytophila Group Gene Encoding a Cytoplasmic Protein Antigen with Ankyrin Repeats

doi: 10.1128/iai.68.9.5277-5283.2000

Figure Lengend Snippet: FIG. 2. (A) Schematic representation and alignment of HGE agent (strain BDS) and E. equi (strain MRK) AnkA open reading frames. For comparison, the previously reported ank gene (24) is also shown at the top. hge-d in pBK-CMV refers to the initial clone, highlighting the SalI and HindIII sites used to subclone into the pMAL-c2 expression vector; the dotted line represents 15 nt of vector sequence. The SalI-BamHI fragment was the one used for probing Southern blots. The single line within the MRK sequences identifies the approximate position of the 81 nt coding for the second 27-amino-acid repeat, which is missing in AnkA from E. equi. (B) Schematic representation of AnkA. Closed boxes, 11 ankyrin repeats; open boxes, two contiguous 27-amino-acid repeats; hatched boxes, two 11-amino-acid repeats. Gene, clone, and protein designations are shown on the right.

Article Snippet: PCR was performed on DNA prepared from the following samples: four human HGE agent strains (Webster, Spooner, and 96HE-97 from Wisconsin and NY-8 from New York) in HL-60 cells, one HGE agent strain (97E12 from a Minnesota dog) in HL-60 cells, one blood sample from another Minnesota dog infected with the HGE agent, one blood sample from a Spanish goat with tick-borne fever (courtesy of Philippe Brouqui), neutrophils isolated from a cow with tick-borne fever in Sweden (courtesy of Philippe Brouqui and Anneli Bjöersdorff) containing different local strains of E. phagocytophila, E. sennetsu Miyayama strain in P388D1 cells (courtesy C. of Pretzman, Ohio State Department of Health), E. risticii HRC-IL strain (ATCC VR-986) in P388D1 cells, E. chaffeensis Arkansas strain in DH82 cells (courtesy of J. Dawson, Centers for Disease Control and Prevention, Atlanta, Ga.), E. chaffeensis 91HE17 strain in DH82 cells, and E. canis Oklahoma strain in DH82 cells (courtesy J. Dawson).

Techniques: Comparison, Expressing, Plasmid Preparation, Sequencing

Journal: Infection and Immunity

Article Title: ankA : an Ehrlichia phagocytophila Group Gene Encoding a Cytoplasmic Protein Antigen with Ankyrin Repeats

doi: 10.1128/iai.68.9.5277-5283.2000

Figure Lengend Snippet: FIG. 3. Southern blot analysis of restriction enzyme-digested DNA from the HGE agent, E. coli XLOLR, and HL-60 cells. Lane 1, lDNA BstEII marker; lane 2, 50 pg of hge-d linearized with BamHI; lanes 3 to 6, HGE agent genomic DNA digested with EcoRI (lane 3), HindIII (lane 4), XhoI (lane 5), and BglII (lane 6); lanes 7 to 9, PstI-digested genomic DNA from the HGE agent (lane 7), E. coli XLOLR (lane 8), and HL-60 cells (lane 9); lanes 10 to 12: BamHI-digested genomic DNA from the HGE agent (lane 10), E. coli XLOLR (lane 11), and HL-60 cells (lane 12); lanes 13 to 15: KpnI-digested genomic DNA from the HGE agent (lane 13), E. coli XLOLR (lane 14), and HL-60 cells (lane 15). Molecular sizes are shown on the left.

Article Snippet: PCR was performed on DNA prepared from the following samples: four human HGE agent strains (Webster, Spooner, and 96HE-97 from Wisconsin and NY-8 from New York) in HL-60 cells, one HGE agent strain (97E12 from a Minnesota dog) in HL-60 cells, one blood sample from another Minnesota dog infected with the HGE agent, one blood sample from a Spanish goat with tick-borne fever (courtesy of Philippe Brouqui), neutrophils isolated from a cow with tick-borne fever in Sweden (courtesy of Philippe Brouqui and Anneli Bjöersdorff) containing different local strains of E. phagocytophila, E. sennetsu Miyayama strain in P388D1 cells (courtesy C. of Pretzman, Ohio State Department of Health), E. risticii HRC-IL strain (ATCC VR-986) in P388D1 cells, E. chaffeensis Arkansas strain in DH82 cells (courtesy of J. Dawson, Centers for Disease Control and Prevention, Atlanta, Ga.), E. chaffeensis 91HE17 strain in DH82 cells, and E. canis Oklahoma strain in DH82 cells (courtesy J. Dawson).

Techniques: Southern Blot, Marker

Journal: Infection and Immunity

Article Title: ankA : an Ehrlichia phagocytophila Group Gene Encoding a Cytoplasmic Protein Antigen with Ankyrin Repeats

doi: 10.1128/iai.68.9.5277-5283.2000

Figure Lengend Snippet: FIG. 4. Protein immunoblots of recombinant MBP-AnkA (A), total lysate of E. coli transformed by ee-o (B), and whole HGE agent (C). (A) Recombinant MBP-AnkA probed with the following primary antibodies: mouse polyclonal anti-MBP-AnkA (lane 1), mouse polyclonal anti-HGE agent (BDS strain) (lane 2), nonimmune mouse serum (lane 3), rabbit anti-MBP (lane 4), nonimmune rabbit serum (lane 5), mouse monoclonal IE3 [IgG1(k)] to MBP-AnkA (lane 6), and mouse monoclonal IgG1(k) control antibody (lane 7). (B) Lysates of E. coli transformed by ee-o (lanes 1 and 3) or not transformed (lanes 2 and 4) were processed as described for panel A and probed with the following primary antibodies: mouse monoclonal IE3 [IgG1(k)] to MBP-AnkA (lanes 1 and 2) and mouse polyclonal anti-HGE agent (lanes 3 and 4). (C) Whole HGE agent was processed as for panels A and B and probed with the following primary antibodies: rabbit anti-HGE agent (Webster strain) (lane 1), rabbit anti-MBP (lane 2), nonimmune rabbit serum (lane 3), mouse polyclonal anti-HGE agent (BDS strain) (lane 4), mouse polyclonal anti-MBP-AnkA (lane 5), and nonimmune mouse serum (lane 6). The numbers on the right represent the molecular masses (MWs) of HGE agent proteins that contain AnkA antigens (150, 90, 75, and 51 kDa) and the 42-kDa immunodominant HGE agent antigen that reacts only with polyclonal HGE agent antibodies.

Article Snippet: PCR was performed on DNA prepared from the following samples: four human HGE agent strains (Webster, Spooner, and 96HE-97 from Wisconsin and NY-8 from New York) in HL-60 cells, one HGE agent strain (97E12 from a Minnesota dog) in HL-60 cells, one blood sample from another Minnesota dog infected with the HGE agent, one blood sample from a Spanish goat with tick-borne fever (courtesy of Philippe Brouqui), neutrophils isolated from a cow with tick-borne fever in Sweden (courtesy of Philippe Brouqui and Anneli Bjöersdorff) containing different local strains of E. phagocytophila, E. sennetsu Miyayama strain in P388D1 cells (courtesy C. of Pretzman, Ohio State Department of Health), E. risticii HRC-IL strain (ATCC VR-986) in P388D1 cells, E. chaffeensis Arkansas strain in DH82 cells (courtesy of J. Dawson, Centers for Disease Control and Prevention, Atlanta, Ga.), E. chaffeensis 91HE17 strain in DH82 cells, and E. canis Oklahoma strain in DH82 cells (courtesy J. Dawson).

Techniques: Western Blot, Recombinant, Transformation Assay, Control

Journal: Infection and Immunity

Article Title: ankA : an Ehrlichia phagocytophila Group Gene Encoding a Cytoplasmic Protein Antigen with Ankyrin Repeats

doi: 10.1128/iai.68.9.5277-5283.2000

Figure Lengend Snippet: FIG. 5. Immunoelectron microscopic visualization of AnkA with monoclonal antibody IE3 in HL-60 cells infected with HGE agent (Webster strain). Bars, 0.5 mm. (A) The label is localized in the cytoplasm of the ehrlichiae, both reticulate (r) and dense-cored (d) cells, and on condensed chromatin of the host cell nucleus (arrowheads). (B) In reticulate cells, the cytoplasm is labeled and many gold particles are aligned along the DNA fibrils of the nucleoid (arrowheads). (C) In an apoptotic cell, condensed chromatin of the apoptotic nucleus is heavily labeled.

Article Snippet: PCR was performed on DNA prepared from the following samples: four human HGE agent strains (Webster, Spooner, and 96HE-97 from Wisconsin and NY-8 from New York) in HL-60 cells, one HGE agent strain (97E12 from a Minnesota dog) in HL-60 cells, one blood sample from another Minnesota dog infected with the HGE agent, one blood sample from a Spanish goat with tick-borne fever (courtesy of Philippe Brouqui), neutrophils isolated from a cow with tick-borne fever in Sweden (courtesy of Philippe Brouqui and Anneli Bjöersdorff) containing different local strains of E. phagocytophila, E. sennetsu Miyayama strain in P388D1 cells (courtesy C. of Pretzman, Ohio State Department of Health), E. risticii HRC-IL strain (ATCC VR-986) in P388D1 cells, E. chaffeensis Arkansas strain in DH82 cells (courtesy of J. Dawson, Centers for Disease Control and Prevention, Atlanta, Ga.), E. chaffeensis 91HE17 strain in DH82 cells, and E. canis Oklahoma strain in DH82 cells (courtesy J. Dawson).

Techniques: Infection, Labeling

Journal: Infection and Immunity

Article Title: Expression of Interleukin-1β, Tumor Necrosis Factor Alpha, and Interleukin-6 in Human Peripheral Blood Leukocytes Exposed to Human Granulocytic Ehrlichiosis Agent or Recombinant Major Surface Protein P44

doi: 10.1128/iai.68.6.3394-3402.2000

Figure Lengend Snippet: FIG. 1. Cytokine mRNA expression in human PBLs exposed to the HGE agent or rP44. (A) Cytokine mRNA expression in human PBLs exposed to the HGE agent (100 bacteria/cell), rP44 (1 mg/ml), or E. coli LPS (1 mg/ml) for 2 h. Total RNA was extracted and subjected to RT-PCR. The cDNAs, in quantities normalized against G3PDH mRNA levels in corresponding samples, were amplified for 25 cycles (20 cycles for IL-8 mRNA), and PCR products were resolved on agarose gels containing EtBr. DNA size markers (HaeIII fragments of fX174 RF DNA) were run in the leftmost lanes. The data presented (donor no. 2 in Table 1) are representative of six donors (Table 1) who had similar results for IL-1b, TNF-a, IL-6, and LPS. (B) Linearity of RT-PCR. Different amounts of cDNA from human PBLs (107 cells) incubated with E. coli LPS (1 mg/ml) for 2 h were amplified for 25 cycles. The PCR products were resolved on agarose gels containing EtBr. DNA size markers (HaeIII fragments of fX174 RF DNA) were run in the leftmost lanes (M). The data presented (donor no. 1 in Table 1) are representative of two independent experiments (donor no. 1 and 2) that gave similar results. The lower panel shows a plot of the relative band densities of the PCR products, recorded by a gel video system and analyzed by an image analysis software, against the amounts of cDNA present in the PCR.

Article Snippet: HGE agent HZ (24) was propagated in HL-60 cells (American Type Culture Collection, Manassas, Va.) in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, Ga.), 1% minimal essential medium nonessential amino acid mixture (GIBCO-BRL, Grand Island, N.Y.), 1 mM minimal essential medium sodium pyruvate (GIBCO-BRL), and 2 mM L-glutamine (GIBCO-BRL).

Techniques: Expressing, Bacteria, Reverse Transcription Polymerase Chain Reaction, Incubation, Software

Journal: Infection and Immunity

Article Title: Expression of Interleukin-1β, Tumor Necrosis Factor Alpha, and Interleukin-6 in Human Peripheral Blood Leukocytes Exposed to Human Granulocytic Ehrlichiosis Agent or Recombinant Major Surface Protein P44

doi: 10.1128/iai.68.6.3394-3402.2000

Figure Lengend Snippet: FIG. 2. Dose-dependent induction of IL-1b, TNF-a, and IL-6 mRNA expression in human PBLs. (A) PBLs were incubated for 2 h with different amounts of the HGE agent. Total RNA was extracted and subjected to the competitive RT-PCR. The PCR products were resolved on agarose gels containing EtBr. DNA size markers (HaeIII fragments of fX174 RF DNA) were run in the leftmost lanes (M). (B) Relative amounts of cytokine mRNAs expressed in human PBLs in response to the HGE agent. Band densities were recorded by a gel video system and analyzed by an image analysis software, and ratios of target to MIMIC PCR products were plotted against the estimated HGE agent numbers per cell. The amounts of cDNAs were normalized against G3PDH mRNA levels in corresponding samples. The data presented (donor no. 2 in Table 1) are representative of two independent experiments (donor no. 2 and 3 in Table 1) that gave similar results.

Article Snippet: HGE agent HZ (24) was propagated in HL-60 cells (American Type Culture Collection, Manassas, Va.) in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, Ga.), 1% minimal essential medium nonessential amino acid mixture (GIBCO-BRL, Grand Island, N.Y.), 1 mM minimal essential medium sodium pyruvate (GIBCO-BRL), and 2 mM L-glutamine (GIBCO-BRL).

Techniques: Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction, Software

Journal: Infection and Immunity

Article Title: Expression of Interleukin-1β, Tumor Necrosis Factor Alpha, and Interleukin-6 in Human Peripheral Blood Leukocytes Exposed to Human Granulocytic Ehrlichiosis Agent or Recombinant Major Surface Protein P44

doi: 10.1128/iai.68.6.3394-3402.2000

Figure Lengend Snippet: FIG. 3. Time course analysis of IL-1b, TNF-a, and IL-6 mRNA expression in human PBLs in response to the HGE agent (100 bacteria/cell), rP44 (1 mg/ml), or E. coli LPS (1 mg/ml). (A) Human PBLs were incubated for the indicated time periods. Total RNA was extracted and subjected to the competitive RT-PCR. The PCR products were resolved on agarose gels containing EtBr. DNA size markers (HaeIII fragments of fX174 RF DNA) were run in the leftmost lanes. (B) Relative amounts of cytokine mRNAs expressed. Band densities were recorded by a gel video system and analyzed by an image analysis software, and the ratios of target to MIMIC PCR products were plotted against the incubation time. The amounts of cDNAs were normalized against G3PDH mRNA levels in corresponding samples. The data presented (donor no. 3 in Table 1) are representative of two independent experiments (donor no. 2 and 3) that gave similar results.

Article Snippet: HGE agent HZ (24) was propagated in HL-60 cells (American Type Culture Collection, Manassas, Va.) in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, Ga.), 1% minimal essential medium nonessential amino acid mixture (GIBCO-BRL, Grand Island, N.Y.), 1 mM minimal essential medium sodium pyruvate (GIBCO-BRL), and 2 mM L-glutamine (GIBCO-BRL).

Techniques: Expressing, Bacteria, Incubation, Reverse Transcription Polymerase Chain Reaction, Software

Journal: Infection and Immunity

Article Title: Expression of Interleukin-1β, Tumor Necrosis Factor Alpha, and Interleukin-6 in Human Peripheral Blood Leukocytes Exposed to Human Granulocytic Ehrlichiosis Agent or Recombinant Major Surface Protein P44

doi: 10.1128/iai.68.6.3394-3402.2000

Figure Lengend Snippet: FIG. 5. Influences of various treatments on expression of IL-1b, TNF-a, and IL-6 mRNAs in human PBLs exposed to the HGE agent (100 bacteria/cell), rP44 (1 mg/ml), or E. coli LPS (1 mg/ml). To examine which components of the HGE agent are responsible for expression of the three proinflammatory-cytokine mRNAs, PBLs (107 cells) were incubated for 2 h with HGE agent that had been subjected to different treatments as described in Materials and Methods. Total RNA was extracted and subjected to RT-PCR. The amounts of cDNAs used were normalized against G3PDH mRNA levels in corresponding samples. The PCR products were resolved on agarose gels containing EtBr. DNA size markers (HaeIII fragments of fX174 RF DNA) were run in the leftmost lanes. The data presented (donor no. 4 in Table 1) are representative of three independent experiments (donor no. 3 to 5) that gave similar results.

Article Snippet: HGE agent HZ (24) was propagated in HL-60 cells (American Type Culture Collection, Manassas, Va.) in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, Ga.), 1% minimal essential medium nonessential amino acid mixture (GIBCO-BRL, Grand Island, N.Y.), 1 mM minimal essential medium sodium pyruvate (GIBCO-BRL), and 2 mM L-glutamine (GIBCO-BRL).

Techniques: Expressing, Bacteria, Incubation, Reverse Transcription Polymerase Chain Reaction

Journal: Infection and Immunity

Article Title: Expression of Interleukin-1β, Tumor Necrosis Factor Alpha, and Interleukin-6 in Human Peripheral Blood Leukocytes Exposed to Human Granulocytic Ehrlichiosis Agent or Recombinant Major Surface Protein P44

doi: 10.1128/iai.68.6.3394-3402.2000

Figure Lengend Snippet: FIG. 6. Cytokine mRNA expression in human neutrophils and monocytes. (A) Human neutrophils purified (.95%) by Ficoll-Paque and Percoll gradient centrifugation were used for IL-1b, TNF-a, and IL-6 mRNA expression. Neutrophils (107 cells) were incubated for 2 h with the HGE agent (100 bacteria/cell), rP44 (1 mg/ml), or E. coli LPS (1 mg/ml). The data presented (donor ID 4 in Table 1) are representative of two independent experiments (donor no. 4 and 5) that gave similar results. (B) Purified (.95%) monocytes (107 cells) were incubated for 2 h under the stimulation conditions used for the neutrophils. Total RNA was extracted and subjected to RT-PCR. The amounts of cDNAs used were normalized against G3PDH mRNA levels in corresponding samples. The PCR products were resolved on agarose gels containing EtBr. DNA size markers (HaeIII fragments of fX174 RF DNA) were run in the leftmost lanes. The data presented (donor ID 5 in Table 1) are representative of two independent experiments (donor no. 5 and 6) that gave similar results.

Article Snippet: HGE agent HZ (24) was propagated in HL-60 cells (American Type Culture Collection, Manassas, Va.) in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, Ga.), 1% minimal essential medium nonessential amino acid mixture (GIBCO-BRL, Grand Island, N.Y.), 1 mM minimal essential medium sodium pyruvate (GIBCO-BRL), and 2 mM L-glutamine (GIBCO-BRL).

Techniques: Expressing, Gradient Centrifugation, Incubation, Bacteria, Reverse Transcription Polymerase Chain Reaction