In vitro cultivation of the exoerythrocytic stage of Plasmodium berghei from sporozoites.

When inoculated with sporozoites of Plasmodium berghei, the human embryonic lung cell line WI38 supports the complete asexual developmental cycle of the exoerythrocytic stage. Cultured parasites were sensitive to primaquine, were shown to resemble parasites in living hosts by immunofluorescent antibody tests, and on subinoculation into mice induced a red blood cell infection, the gametocytes of which produced sporozoites in anopheline mosquitoes.

Predictive impact of rare genomic copy number variations in siblings of individuals with autism spectrum disorders.

Identification of genetic biomarkers associated with autism spectrum disorders (ASDs) could improve recurrence prediction for families with a child with ASD. Here, we describe clinical microarray findings for 253 longitudinally phenotyped ASD families from the Baby Siblings Research Consortium (BSRC), encompassing 288 infant siblings. By age 3, 103 siblings (35.8%) were diagnosed with ASD and 54 (18.8%) were developing atypically. Thirteen siblings have copy number variants (CNVs) involving ASD-relevant genes: 6 with ASD, 5 atypically developing, and 2 typically developing. Within these families, an ASD-related CNV in a sibling has a positive predictive value (PPV) for ASD or atypical development of 0.83; the Simons Simplex Collection of ASD families shows similar PPVs. Polygenic risk analyses suggest that common genetic variants may also contribute to ASD. CNV findings would have been pre-symptomatically predictive of ASD or atypical development in 11 (7%) of the 157 BSRC siblings who were eventually diagnosed clinically.

Scintigraphic screening prior to visceral arteriography in acute lower gastrointestinal bleeding.

UNLABELLED: We evaluated the effect on the diagnostic yield of visceral arteriography in patients with acute gastrointestinal bleeding of a protocol requiring a positive 99mTc-red blood cell scintiscan before the performance of arteriography (scintigraphic screening). METHODS: A retrospective review was conducted of 249 scintiscans and 271 arteriograms obtained over 99 mo, with scintigraphic screening implemented during the final 18 mo. RESULTS: Before the implementation of scintigraphic screening, arteriograms detected bleeding at a rate of 22%. After its implementation, 53% of the arteriograms detected bleeding. This represented a statistically significant increase (0.53 versus 0.22, p = 0.015). CONCLUSION: Scintigraphic screening appears to increase by a factor of 2.4 the diagnostic yield of arteriography by screening out patients who are not actively bleeding at the time of the examination, thus sparing them the risks and costs of a nondiagnostic invasive study.

Schistosoma mansoni: radiation dose and morphologic integrity of schistosomules as factors for an effective cryopreserved live vaccine.

The effectiveness of a cryopreserved, irradiated schistosomule vaccine against an homologous Schistosoma mansoni cercarial challenge was tested in C57B1/6 mice. Highly significant levels of protection developed consistently when mice were immunized with the vaccine irradiated at 10-20 Krad, i.e., doses below that considered optimal for irradiated cercariae (50 Krad). Cryopreserved schistosomules irradiated at 10 or 20 Krad induced greater levels of protection than did schistosomules irradiated at 2, 5, 30, or 50 Krad. Protective immunity developed as early as 3 weeks post-immunization. When immunizing inocula were injected at various times post-thaw, or when schistosomule subpopulations of normal-appearing, damaged or dead organisms were injected, those populations which had appeared to sustain the least degree of damage were those most capable of stimulating protective immunity. These findings highlight the hazards of extrapolating conditions considered standard for an irradiated cercarial vaccine to one in which cryopreservation, for storage of the schistosomules, is an added stress.

Entry of Plasmodium berghei sporozoites into cultured cells, and their transformation into trophozoites.

The attachment and entry of Plasmodium berghei sporozoites to cultured human lung fibroblast (WI38) or hepatoma (HepG2-A16) cells in vitro has been visualized using an immunoperoxidase technique coupled with light microscopy. Attachment and entry was substantially more frequent with HepG2-A16 cells, and appeared to be mediated by the Pb44 sporozoite surface protective antigen. When sporozoites were incubated with intact monoclonal antibodies to Pb44 or their monovalent Fab fragments, attachment was inhibited, suggesting that this technique may be an in vitro assay of protective immunity. Sporozoites appeared to enter cells actively, rather than by cell phagocytosis. Once within a membrane-bound vacuole, the sporozoite transformed into a trophozoite. It was suggested that Pb44 recognized a specific cell receptor, and that this technique may permit receptor characterization.

Cryopreservation of schistosomules of Schistosoma mansoni in quantity.

Large quantities of Schistosoma mansoni schistosomules were cryopreserved in 35% ethanediol, and attempts were made to improve their morphology and infectivity after thawing. Several variables affected the appearance of the thawed organisms. These included: 1) the particular in vitro method used to transform cercariae to schistosomules; 2) addition of serotonin to the thawing medium; 3) changing the thawing temperature; and 4) culturing schistosomules for extended post-thaw times before assessing their condition. Varying either the postemergence age of the cercariae before transformation or the concentration of organisms in the freezing suspension had no effect on their survival. Although damage to many of the thawed schistosomules became apparent only upon extended in vitro cultivation, the addition of fetal calf serum to the thawing medium usually retarded this deterioration. When injected into mice, approximately 5% of cryopreserved schistosomules matured to adult worms, representing 71% of the value for maturation of unfrozen schistosomules. These studies define conditions of importance for the successful preservation of very large quantities of schistosomules (up to 500,000 in 1 ml volume) in liquid nitrogen.

Serological reactivity of in vitro cultured exoerythrocytic stages of Plasmodium berghei in indirect immunofluorescent or immunoperoxidase antibody tests.

Exoerythrocytic (EE) stages of Plasmodium berghei were cultivated in vitro in WI38 cells inoculated with sporozoites, and examined for serological reactivity by indirect immunofluorescent or immunoperoxidase tests. At 24 hours post-inoculation, sporozoite and red blood cell (RBC) stage antigens were equally distributed, but by 48 hours, RBC stage antigens predominated. Merozoites, produced by 72 hours, reacted strongly with anti-RBC stage sera, but were weakly reactive with anti-sporozoite sera. Hybridoma-produced monoclonal antibodies to the surface protective antigen of P. berghei sporozoites also reacted with 24- and 48-hour EE parasites, and with RR merozoites, suggesting that the sporozoite-protective antigen may also be synthesized by the EE stage. Extra-EE parasite antigens associated with the host cell nucleus were detected as early as 24 hours using anti-sporozoite and anti-RBC stage sera, but did not contain the sporozoite-protective antigen.

Circumoval precipitin (COP) test in schistosomiasis with frozen Schistosoma mansoni eggs.

Frozen Schistosoma mansoni eggs were successfully employed in the circumoval precipitin (COP) test for schistosomiasis yielding results comparable with fresh eggs. This development may permit both standardization and kit production of the COP test.

In vitro infectivity of cryopreserved Plasmodium berghei sporozoites to cultured cells.

Plasmodium berghei sporozoites frozen in MEM (Eagle) medium supplemented with 10% hydroxyethyl starch and 50% normal mouse serum retained 0.5% infectivity to cultured hepatoma cells, compared to 6.8% before freezing. This demonstrates that frozen-thawed sporozoites can be used in in vitro investigations of the exoerythrocytic malarial parasite and means that when large numbers of sporozoites are available, they may be frozen and preserved for later use.

Percutaneous transluminal mesenteric angioplasty.

The endovascular treatment of chronic mesenteric ischemia remains in its infancy. This state is most certainly related to the small patient population affected by this devastating condition and the surgeon's limited desire to pursue percutaneous options for this group. Surgical trepidation is not unwarranted because in many facets mesenteric angioplasty is similar to percutaneous carotid intervention. Interventions in these vessels represent manipulation of terminal and unduplicated arterial systems. Complications compromising flow to either vascular bed can yield rapid grave outcomes. Despite this risk, early experience with mesenteric angioplasty for chronic ischemia suggests that it is a viable treatment alternative and that it can be performed with relative safety. A randomized prospective trial of mesenteric angioplasty with operative bypass grafting appears to be the next logical step.

Angioplasty and intravascular stents in peripheral vascular disease.

Advances in the technique as well as the technology of percutaneous angioplasty over the past 30 years have allowed the percutaneous treatment of many vascular lesions that previously required operative management. PTA, and more recently intravascular stents, have been shown to be effective, safe, and, in some cases, more cost effective than surgical options. Although patients need to be appropriately selected to optimize the treatment plan in individual cases, it is clear that percutaneous treatment, either alone or in combination with surgical procedures, offers a significant advancement in patient care. The success in treating iliac disease is perhaps the most convincing, but evidence is rapidly building to support infrapopliteal procedures, and, as further advances are made in technology, the role of such procedures in distal vessels may be expanded to include more patients. These procedures need to be prospectively evaluated not only for their efficacy, long-term result, and safety, but also for their cost-effectiveness and cost relative to the proven surgical options.

Intravascular stents in the management of acute superior vena cava obstruction of benign etiology.

The purpose of our study was to report our experience with percutaneous placement of intravascular stents to relieve venous occlusion in patients with acute superior vena cava syndrome resulting from benign etiologies. Six patients ranging in age from 39 to 66 years received thrombolysis followed by placement of stents within the superior vena cava or received stent placement alone as emergency treatment for symptoms of acute superior vena cava obstruction. Treatment was successful in all patients, with establishment of a patent lumen angiographically, and patients experienced prompt symptomatic relief. Follow-up examination at intervals of 5 months to 2 years has demonstrated no evidence of reocclusion. Three patients have subsequently had central lines placed across the stented vena cava for vascular access. Percutaneous placement of intravascular stents to treat acute occlusion offered a safe and effective method of treatment in patients with superior vena cava syndrome resulting from benign causes.

Iodixanol and ioxaglate in adult aortography and peripheral arteriography: a phase III clinical trial.

RATIONALE AND OBJECTIVES: This study compared the safety and efficacy of iodixanol with those of ioxaglate in adult patients undergoing aortography or peripheral arteriography. METHODS: Forty-six patients were enrolled in this controlled, randomized, double-blind, two-center study. The patients were monitored for adverse events, injection-related discomfort, and trends in laboratory data or vital signs. Diagnostic efficacy was assessed by the quality of contrast enhancement. RESULTS: All procedures were diagnostic, with no significant difference in quality of visualization (p = .205). No significant difference was seen in the number of patients experiencing mild or moderate adverse effects (p = .106). Patients receiving ioxaglate reported a significantly greater incidence of injection-related pain (p = .025). CONCLUSION: The results of this study support the conclusion that iodixanol at 320 mg I/kg is safe and effective for adult aortography and peripheral arteriography and causes significantly less injection-related pain than ioxaglate.

Comparison of iodixanol and ioxaglate for adult aortography and renal/visceral angiography: a phase III clinical trial.

RATIONALE AND OBJECTIVES: We investigated whether iodixanol offers improved safety or tolerance compared with ioxaglate and evaluated whether iodixanol-enhanced radiographs are diagnostically comparable or superior to those produced with ioxaglate. Iodixanol is a new isosmotic hexa-iodinated nonionic contrast agent being evaluated for intravascular use. METHODS: Fifty-four adult patients undergoing renal or visceral angiography, aortography, or both were enrolled in a prospective, randomized, double-blind, two-center study in which iodixanol and ioxaglate were compared. Subjects were monitored for adverse events, injection-associated discomfort, and changes in laboratory parameters and vital signs. Efficacy was measured by the overall quality of angiographic enhancement. RESULTS: No serious adverse events occurred during this trial. Mild-to-moderate adverse events were more common in patients receiving ioxaglate (p = .041). Injection-associated pain was reported by three patients receiving ioxaglate and none receiving iodixanol (p = .093). Clinical laboratory and vital-sign data showed no differences between groups, and there was no difference in the overall quality of angiographic visualization (p = .711). CONCLUSION: Iodixanol resulted in improved safety and patient tolerability while providing images of equivalent diagnostic efficacy compared with ioxaglate.

The influence of cell type and culture medium on the in vitro cultivation of exoerythrocytic stages of Plasmodium berghei.

Plasmodium berghei sporozoites successfully entered and developed into exoerythrocytic schizonts in a variety of cell types cultured in vitro, but segmentation and release of merozoites was only observed in human embryonic lung cells. Exoerythrocytic development was generally not influenced by the culture medium, and NCTC-135 was used routinely. In vitro infectivity of P. berghei sporozoites was unaffected by the serum type used for isolation.

Free-flow electrophoretic separation of Plasmodium berghei sporozoites.

Sporozoites of the rodent malaria, Plasmodium berghei, were obtained from infected Anopheles stephensi by grinding mosquitoes, prepurifying the material in a discontinuous Hypaque gradient and further purifying by means of continuous free-flow electrophoresis. Bacteria, debris, mitochondria, mitoplasts, and other contaminants were removed in the electric field. The isolated sporozoites were morphologically intact and were positive in indirect immunofluorescence assay. They were infective to mice prior to and following free-flow electrophoretic separation. The surface of the sporozoites exhibited a polysaccharide-rich layer. The predominant surface protein labelled after surface iodination had a molecular weight between 42,000 and 46,000 daltons.

Insulin-mimetic effect of trypsin on the insulin receptor tyrosine kinase in intact adipocytes.

It has previously been demonstrated that the insulin-mimetic agent trypsin stimulates autophosphorylation of purified insulin receptors and activates the insulin receptor tyrosine kinase in vitro. We now report the effects of trypsin on whole cell tyrosine kinase activation and insulin receptor autophosphorylation. Trypsin treatment of intact adipocytes produces a time-dependent stimulation of tyrosine kinase activity as measured in lectin extracts containing the insulin receptor, or specifically immunoprecipitated insulin receptor samples. Trypsin treatment of adipocytes also results in a loss of insulin binding capacity, and a linear correlation exists between loss of binding and stimulation of tyrosine kinase activity. Exposure of adipocytes to trypsin is known to result in a time- and dose-dependent activation of intracellular glycogen synthase. Examination of the time courses of stimulation of tyrosine kinase and glycogen synthase activation in our system indicates that the stimulation of tyrosine kinase activity by trypsin occurs with sufficient rapidity and magnitude to be consistent with a role of phosphorylation in the activation of glycogen synthase. Trypsin has further been demonstrated to stimulate autophosphorylation of the beta-subunit of the insulin receptor in intact adipocytes. Cells prelabeled with [32P]PO4 for 2 h were exposed to trypsin, and receptors were partially purified over wheat germ agglutinin-agarose columns. Receptors were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the beta-subunit was identified by autoradiography. The protein was extracted and hydrolyzed, and the phosphoamino acids were separated by electrophoresis and quantitated. Two- and five-fold increases in phosphotyrosine were observed with 3 and 10 min of trypsin treatment, respectively. We conclude that trypsin-induced cleavage of the insulin receptor alpha-subunit is relevant to the ability of trypsin to activate the insulin receptor tyrosine kinase in intact adipocytes. We further conclude that autophosphorylation of the insulin receptor and activation of its tyrosine kinase by trypsin may be important to the insulin-mimetic anabolic effects of trypsin.