Dissociation of the Jak kinase pathway from G-CSF receptor signaling in neutrophils.

Activation of the granulocyte colony-stimulating factor receptor (G-CSFR) induces rapid tyrosine phosphorylation of multiple intracellular substrates in proliferating cells and nonproliferating, terminally differentiated neutrophils. The kinases that couple ligand binding to tyrosine phosphorylation of cellular substrates by the G-CSFR with activation of specific functional programs are largely unknown. In this study, we examined early signaling events in proliferating and terminally differentiated cells following G-CSF stimulation to determine whether identical signaling cascades are activated. In murine Ba/F3 cells transfected with the human G-CSFR and NFS-60 cells constitutively expressing the murine G-CSFR, G-CSF induced tyrosine phosphorylation and activation of Jak1, Jak2, and Tyk2. Tyrosine phosphorylation of Stat3 and, to a lesser extent, Stat1 was also detected following G-CSF stimulation. Using a mitogenically incompetent human G-CSFR mutant in which Pro639 and Pro641 were substituted by alanine, the box 1 PDP motif was found to be required for activation of Jak kinases, tyrosine phosphorylation of the G-CSFR, and recruitment of Stat proteins. Notably, no activation of Jak1, Jak2, Tyk2, Stat1, or Stat3 was observed in neutrophils following G-CSF stimulation. In addition, there was no detectable activation in neutrophils of the recently cloned Jak3 kinase, which has been reported to be expressed at high levels as myeloid cells undergo terminal neutrophilic maturation. These results indicate a lack of involvement of Jak kinases in signaling by the G-CSFR in neutrophils, and suggest utilization of alternative signal transduction pathways distinct from those in proliferating cells. Activation of the Jak-Stat pathway correlates with proliferative signaling by the G-CSFR and requires the membrane-proximal box 1 PXP motif, which is conserved in members of the cytokine receptor superfamily.

Isoenzyme comparison of axenic Giardia lamblia strains.

We obtained isoenzyme patterns by polyacrylamide gradient gel electrophoresis (PGGE) of water-soluble protein fractions prepared from trophozoites of 11 axenic G. lamblia strains. The strains were isolated from animals and humans (both symptomatic and asymptomatic) from various geographic locations. Isoenzymes were also separated by isoelectric focusing. Of 12 enzymes attempted, eight exhibited well-defined and reproducible isoenzyme patterns by PGGE, based on which the strains were grouped into four zymodemes. Although the 11 strains were grouped into four zymodemes based on PGGE, no correlation between zymodeme and the known characteristics of the strains existed. Thus, a high degree of characteristic sharing appears to occur among genetically different G. lamblia strains.

Clinical evaluation of the cysticercosis enzyme-linked immunoelectrotransfer blot in patients with neurocysticercosis.

During the 3 years that the enzyme-linked immunoelectrotransfer blot (EITB) assay for the diagnosis of human cysticercosis has been in use at the Centers for Disease Control, 50 patients with both pathologically confirmed neurocysticercosis and computed tomographic (CT) or magnetic resonance imaging (MRI) scan results were identified. Of 32 patients with two or more lesions, 94% had detectable antibodies by EITB compared with 28% of 18 patients with single lesions. Patients with only calcified cysts (single or multiple) were less likely to have EITB-positive results than were those with noncalcified, enhancing lesions. Antibody was detectable more frequently in serum than in cerebrospinal fluid, regardless of the number or apparent condition of the cysts. These findings confirm that the EITB assay for cysticercosis antibodies is highly sensitive in patients with multiple, enhancing intracranial lesions but is less sensitive in patients with single lesions and in those with calcified lesions.

Viability of Acanthamoeba cysts in ophthalmic solutions.

Acanthamoeba keratitis is a chronic infection of the human cornea. Many people who have this infection wear soft contact lenses. Usually lens wearers clean and maintain their lenses with various ophthalmic solutions including homemade saline. Recently it has been shown that homemade saline solutions play a role in lens contamination and thus in Acanthamoeba keratitis. We therefore evaluated the viability of cysts of three species of Acanthamoeba by exposing them for various time periods to saline, cleaning, and disinfectant solutions generally used to care for these lenses. We found that the viability of the cysts in saline solutions ranged from a minimum of 14 days to 90 days of exposure. In cleaning solutions, the survival times ranged from a minimum of 1 day to 90 days of exposure. Disinfectants, as expected, were the most effective of all tested solutions in killing Acanthamoeba cysts. The survival times ranged from 6 h to 14 days. None of these products were effective in destroying Acanthamoeba cysts in less than 6 h of exposure, which exceeds the suggested time that any given solution should be used for lens care.

Evaluation of commercial serodiagnostic kits for toxoplasmosis.

To evaluate commercially available diagnostic kits for human immunoglobulin G to Toxoplasma gondii, we purchased three enzyme immunoassay (EIA) (Cordia-T, Toxo Bio-EnzaBead, and Toxoelisa), two indirect hemagglutination (IHA) (TPM-Test and ToxHAtest), one fluoroimmunoassay (Toxoplasma-G FIAX), and one latex agglutination (Eiken Toxotest-MT) kit from U.S. suppliers. A total of 100 serum specimens, including 27 that were negative (less than 1:16) and 73 that were positive for the various titers in the Toxoplasma indirect immunofluorescence (IIF) test, were tested once with each kit; serum samples with discrepant results were retested. Qualitatively, results obtained with the Toxo Bio-EnzaBead EIA, the TMP-Test IHA, and FIAX, and the Toxotest-MT latex agglutination kits agreed exactly with those of IIF. Although all IIF-positive serum samples were detected by the Cordia-T and the Toxoelisa EIAs, four samples determined to be negative by IIF were identified as positive with the Cordia-T kit, and six negative samples by IIF were determined to be positive with the Toxoelisa kit. Results of the ToxoHAtest IHA kit were extremely difficult to read. Quantitatively, the seven kits were difficult to compare because the expression of results was not standardized. Of the four kits that gave positive results in titers, Toxoplasma-G FIAX had the closest agreement with IIF, as determined by the Spearman rank correlation coefficient (0.9168), followed by the Eiken Toxotest-MT (0.8293), the Toxo Bio-EnzaBead (0.7553), and the TPM-Test (0.7206) kits.

The effect of the nitroimidazole drug dimetridazole on microaerophilic campylobacters.

Dimetridazole, a nitroimidazole drug reported to act only on obligately anaerobic micro-organisms, is widely used for the prevention and treatment of swine dysentery. Forty-four strains of the microaerophilic bacterium Campylobacter coli isolated from either healthy or diseased pigs, and a strain of Campylobacter fetus, were all sensitive to dimetridazole. The sensitivities (minimal inhibitory concentration less than 10 microng per ml) were similar to those of anaerobic bacteria. Dimetridazole inhibited growth of campylobacters in a shaken culture in air, but did not inhibit uptake of oxygen. Inhibition of growth appeared to result from an inhibition of nucleic-acid synthesis and does not seem to depend upon interference with electron transport in the catabolism of pyruvate.