Binding of a monoclonal antibody to sporozoites of Sarcocystis singaporensis enhances escape from the parasitophorous vacuole, which is necessary for intracellular development.

Early intracellular development in vitro of the cyst-forming protozoon Sarcocystis singaporensis and the influence of a monoclonal antibody on invasion, intracellular localization, and development of sporozoites were studied. As revealed by immunofluorescence using parasite-specific antibodies which labeled the parasitophorous vacuole membrane (PVM) and by ultrastructural analysis, sporozoites invaded pneumonocytes of the rat via formation of a parasitophorous vacuole (PV). About half of the sporozoites left this compartment within the first 8 h postinfection to enter the host cell cytosol. By semiquantitative analysis of acetyl-histone H4 expression of sporozoites, a marker linked to early gene expression of eukaryotic cells, we show (supported by ultrastructural analysis) that escape from the PV appears to be necessary for early intracellular development. More than 90% of sporozoites located in the cytosol expressed high levels of acetylated histone H4 in the nucleus, whereas only a quarter of the intravacuolar sporozoites exhibited a similar signal. As revealed by ultrastructural analysis, young schizonts all resided in the cytosol. Specific binding of a monoclonal antibody (11D5/H3) to sporozoites before invasion significantly enhanced their escape from the PV, whereas cell invasion itself remained unaffected. The antibody actually increased proliferation of the parasites in vitro, providing a further link between residence in the cytosol and successful intracellular development. Monoclonal antibody 11D5/H3 precipitated a major 58-kDa antigen from oocyst-sporocyst extracts and reacted with the cytoplasm and the surface of sporozoites in immunofluorescence assays. Collectively, the observed antibody-parasite interaction suggests the existence of a signaling event that influences intracellular development of Sarcocystis.

In vitro cultivation of the vascular phase of Sarcocystis singaporensis.

To establish an in vitro culture system for the precystic phase of Sarcocystis singaporensis, we initially tested various excysting fluids for sporocysts. An excysting fluid containing 2.5% bovine taurocholate and 10% bile of the specific intermediate host, Rattus norvegicus, in RPMI medium was the most suitable resulting in excystation of 80% of the sporozoites. Subsequently, we identified brain endothelial cells and pneumonocytes of the rat to promote growth of sporozoites to schizonts. Hepatoma, fibroblastic, or myoblastic cells were not suitable for the parasite's development. First-generation schizonts were seen at days 3-10 postinoculation (PI); a distinct second peak of schizogonic development only occurred in endothelial cells at days 14-18 PI. First-generation schizonts were 26.0 (+/- 3.8) microns in diameter and contained 32-50 merozoites, second-generation schizonts measured 34.4 (+/- 10.6) microns and contained 54-72 merozoites. Merozoite yield at large-scale culture conditions (75 cm2 flasks) using pneumonocytes as host cells was relatively low. Ultrastructurally, sporozoites and merozoites were quite similar to corresponding stages of other Sarcocystis species. With regard to host cell specificity and developmental kinetics, in vitro cultivation showed close similarities to the situation in vivo.

A highly sensitive assay for the simultaneous determination of morphine, morphine-3-glucuronide, and morphine-6-glucuronide in human plasma by high-performance liquid chromatography with electrochemical and fluorescence detection.

A novel, highly sensitive and specific bioanalytical method has been developed for the simultaneous determination of morphine and its major metabolites, morphine-3-glucuronide and morphine-6-glucuronide, in human plasma, using noroxymorphone as the internal standard. The analytes are isolated from human plasma using a nonpolar/polar C2 solid-phase extraction cartridge and analyzed by high-performance liquid chromatography with serial detection using electrochemical detection for morphine, morphine-6-glucuronide (M6G), and noroxymorphone and fluorescence detection for morphine-3-glucuronide (M3G). The limit of quantitation (sensitivity) using a 0.5-ml sample of plasma is 1 ng/ml, 10 ng/ml, and 5 ng/ml for morphine, M3G, and M6G, respectively. Standard curves were linear (correlation coefficients > 0.999) over the ranges 1-30 ng/ml, 10-500 ng/ml, and 5-100 ng/ml for morphine, M3G, and M6G, respectively. The overall interday accuracy of the method was -1.58% for morphine, 2.27% for M3G, and -5.34% for M6G. The assay is routinely used for the study of morphine, M3G, and M6G pharmacokinetics after oral administration of morphine.

Determination of codeine in human plasma by high-performance liquid chromatography with fluorescence detection.

A rapid, reliable and rugged assay for determining codeine in human plasma using reversed-phase high-performance liquid chromatography with fluorescence detection was developed. This analytical method utilized an ion-exchange/mixed-mode solid-phase extraction procedure. The chromatographic separation was achieved using a 150 x 4.6 mm I.D., 3-microns reversed-phase C8 (deactivated for basic analytes) column at ambient temperature. Fluorescence detection (excitation at 214 nm and emission above 345 nm) for codeine and nalorphine allowed for a detectable limit of 5 ng/ml. The results showed that the method was linear from 10 to 300 ng/ml. The method had good reproducibility, precision, accuracy and recoveries of 91 and 90% for codeine and nalorphine, respectively. This method has been applied to study the pharmacokinetics of codeine in normal human subjects.

Tubular ectasia within the mediastinum testis.

Eleven scrotal sonographic examinations showing a spectrum of findings within the mediastinum testis were collected over a 2 year period. Each case showed numerous small tubular or rounded anechoic structures within the mediastinum testis; often, the findings mimicked a hypoechoic mass. Findings were bilateral in eight of ten patients; one additional patient had only one testis because of orchiectomy. All patients had an associated extratesticular finding, in most cases a spermatocele. Tubular ectasia shares several features with testicular cysts and mechanisms of formation are postulated to be similar to those previously proposed for testicular cysts. Recognizing tubular ectasia is important to avoid unnecessary concern and potential surgery.

CT of parapelvic cystic renal cell carcinoma.

Parapelvic renal cysts are benign ubiquitous lesions of the renal sinus frequently demonstrated on CT and ultrasonography. Occasionally a renal parenchymal mass will grow so that its epicenter is within the renal sinus. We report two cases of surgically proven cystic renal cell carcinoma centered in the renal sinus with no apparent connection to the renal parenchyma. Cystic renal cell carcinoma must be excluded whenever a parapelvic cyst with atypical features is encountered on cross-sectional imaging studies.

Inhibition of vascular permeability changes in rats by captopril.

Systemic treatment of rats with captopril (50 mg/kg body wt per os), a specific competitive inhibitor of angiotensin l-converting enzyme, significantly inhibits vascular permeability changes induced by the intradermal injection of the vasoactive mediators histamine, bradykinin, serotonin, and compound 48/80. This effect of captopril is both dose- and time-dependent with approximately 60% inhibition of edema formation observed 7 h after captopril treatment (100 mg/kg body wt per os). The inhibitory effect of captopril on edema formation is temporally unrelated to the inhibition of serum angiotensin l-converting enzyme activity or serum prostaglandin E2 levels and is not inhibited by systemic treatment of rats with indomethacin. The data suggest that captopril may have potent antiinflammatory activity through as yet undefined mechanisms.