Comparison of PCR with blood smear and inoculation of small animals for diagnosis of Babesia microti parasitemia.

The specific diagnosis of babesiosis, which is caused by the piroplasm Babesia microti, is made by microscopic identification of the organism in Giemsa-stained thin blood smears, detection of babesial antibody in acute-and convalescent-phase sera, or identification of the organism following the injection of patient blood into laboratory animals. Although rapid diagnosis can be made with thin blood smears, parasites are often not visualized early in the course of infection. PCR is a new, rapid diagnostic technique for the detection of Babesia spp. that has not yet been systematically evaluated. We conducted a blinded study of the sensitivity, specificity, and reproducibility of the PCR-based test with patients with babesiosis and a group of asymptomatic subjects residing in a region in southern New England where babesiosis is enzootic. Among 19 patients with recent babesial illness, we found that PCR was as sensitive and specific as the use of Giemsa-stained blood smears and inoculation of hamsters. Among asymptomatic subjects, the PCR result was positive for 3 persons with recent babesial infection and was negative for 41 persons without previous babesial infection. We conclude that the B. microti PCR procedure is sufficiently sensitive, specific, and reproducible for use in the diagnosis of acute babesiosis.

Mechanism of action of anti-IL-2R monoclonal antibodies. ART-18 prolongs cardiac allograft survival in rats by elimination of IL-2R+ mononuclear cells.

ART-18, a mouse IgG1 mAb recognizing the IL-2 binding domain of the rat p55 subunit IL-2R molecule, prevents graft rejection in various experimental models, although its mechanism of action in vivo, like that of anti-IL-2R mAb generally, remains elusive. These studies were designed to define whether IL-2R+ T effector cells were actually eliminated or their function merely inhibited by comparing directly the in vitro and in vivo efficacy of intact ART-18 and its F(ab)/F(ab')2 fragments. Addition of each mAb preparation profoundly suppressed MLR set up between naive LEW responders and x-radiated BN stimulators, suggesting that mAb fragments retained Ag binding functions in vitro. However, both ART-18 F(ab) and F(ab')2 were ineffectual in vivo as judged by their inability to affect acute (8 days) rejection of (LEW X BN)F1 cardiac allografts in LEW recipients (graft survival ca. 11 and 9 days, respectively, compared to ca. 21 days after therapy with intact ART-18, p less than 0.001). The sera levels of ART-18 and ART-18 F(ab')2 were 4 to 5 micrograms/ml, but only less than 0.5 micrograms/ml of F(ab) could be detected. The therapeutic failure of ART-18 fragments was unrelated to potential host sensitization, as rat antimouse F(ab) or F(ab')2 serum IgG titers remained in the same range as those against intact ART-18. The role of the Fc portion of Ig in the mode of action of ART-18 was then tested further by flow microfluorimetry analysis of host mononuclear spleen cells and immunoperoxidase stains of the graft infiltrate. IL-2R+ cells were abundant in rats treated with ART-18 fragments, comparable to acutely rejecting controls. In contrast, IL-2R expression was abolished in animals undergoing ART-18 therapy. The elimination of IL-2R+ cells is required to prolong cardiac allograft survival in rats after IL-2R targeted treatment with ART-18 mAb.

Secretion of mucin by explants of rabbit and human cervix in organ culture.

Small explants (2-3 mm3) of endocervix from virgin, estrous rabbits, and from hospitalized patients undergoing hysterectomy for nonneoplastic disease, were placed in organ culture and maintained in serum-free media for 4 days at 35 degrees C in a humid environment of 95% air/5% CO2. Waymouth's MB 752/1 with 10-5 M hydrocortisone succinate, 10-7 M retinyl acetate, and 1 microgram/ml insulin proved to be an excellent medium for maintaining these tissues, as judged by examination with light and scanning electron microscopy after incubation for 5 days. The explants incorporated the radiolabeled glycoprotein precursor, tritiated glucosamine, and secreted labeled mucin glycoproteins in vitro. Mucin released into the culture medium contained sialic acid and hexosamine in a molar ratio of approximately 0.5-0.8:1.0. Although some alterations occur in the morphology of secretory cells and their products after maintenance in culture for several days, the system can be utilized for studying various aspects of the cell biology of cervical mucin secretion.

Stimulatory effect of Pseudomonas aeruginosa on mucin secretion by the respiratory epithelium.

Cell-free filtrates of broth cultures from 11 of 18 strains of Pseudomonas aeruginosa isolated from patients with cystic fibrosis (CF) (or from patients without CF) increased substantially the secretion of mucin by explants of trachea from guinea pigs and human tracheal tissue obtained at autopsy. The effect was not related to the mucoid nature of the isolates, was not destroyed by heating at 90 degrees C for 30 minutes, and was not dependent on serotype. As P aeruginosa commonly infects the respiratory tracts of persons with CF, elaboration of an extracellular product that stimulates mucin secretion could play a role in the pathogenesis of the disease in the airways.

Effect of cytochalasin D on smooth muscle contraction.

Cylindrical segments of extraparenchymal pulmonary artery (essentially a preparation of smooth muscle with regard to contractile capability) were isolated from adult male rats. They were mounted in an isometric muscle bath in physiological salt solution (PSS) in an environment of 95% O2/5% CO2. After allowing 1 h for equilibration, the maximum force generated by the tissue in response to a depolarizing solution was determined. After relaxation, vessels were incubated for 1 h in one of several concentrations of cytochalasin D (CD) (0.01, 0.05, 0.5, 1, 10 micrograms/ml) and the response to stimulation retested immediately after returning to PSS, and then at 30 minute intervals up to 2 h. CD inhibited the ability of vascular smooth muscle to generate force (contract) in a concentration-dependent manner. The inhibitory effect was reversible within a short period of time. Quantitative electron microscopic examination of these vessels suggested that CD disrupts the integrity of myofilaments, especially at sites of "dense bodies." Our results indicate that a percentage of actin in smooth muscle cells is not permanently in the filamentous "F" form, but is part of the G:F actin system of the cell, labile to polymerization:depolymerization. The ability of smooth muscle cells to generate force could depend on the proper functioning of the F:G actin "treadmill."