Low-Dose Volume-Perfusion CT of the Brain: Effects of Radiation Dose Reduction on Performance of Perfusion CT Algorithms.

PURPOSE: We aimed to compare different computed tomography (CT) perfusion post-processing algorithms regarding image quality of perfusion maps from low-dose volume perfusion CT (VPCT) and their diagnostic performance regarding the detection of ischemic brain lesions. METHODS AND MATERIALS: We included VPCT data of 21 patients with acute stroke (onset < 6h), which were acquired at 80 kV and 180 mAs. Low-dose VPCT datasets with 72 mAs (40 % of original dose) were generated using realistic low-dose simulation. Perfusion maps (cerebral blood volume (CBV); cerebral blood flow (CBF) from original and low-dose datasets were generated using two different commercially available post-processing methods: deconvolution-based method (DC) and maximum slope algorithm (MS). The resulting DC and MS perfusion maps were compared regarding perfusion values, signal-to-noise ratio (SNR) as well as image quality and diagnostic accuracy as rated by two blinded neuroradiologists. RESULTS: Quantitative perfusion parameters highly correlated for both algorithms and both dose levels (r ≥ 0.613, p < 0.001). Regarding SNR levels and image quality of the CBV maps, no significant differences between DC and MS were found (p ≥ 0.683). Low-dose MS CBF maps yielded significantly higher SNR levels (p < 0.001) and quality scores (p = 0.014) than those of DC. Low-dose CBF and CBV maps from both DC and MS yielded high sensitivity and specificity for the detection of ischemic lesions (sensitivity ≥ 0.82, specificity ≥ 0.90). CONCLUSION: Our results indicate that both methods produce diagnostically sufficient perfusion maps from simulated low-dose VPCT. However, MS produced CBF maps with significantly higher image quality and SNR than DC, indicating that MS might be more suitable for low-dose VPCT imaging.

Hapten-protein conjugates prepared by the mixed anhydride method. Cross-reactive antibodies in heterologous antisera.

Poly-L-lysine conjugates of three structurally unrelated mycotoxins were made by either a mixed anhydride intermediate (MA) or an activated N-hydroxysuccinimide ester intermediate (AE). Control conjugates, with no mycotoxin, were also prepared by each method. Antisera elicited by mycotoxin-albumin conjugates produced by the MA method bound to the three poly-L-lysine-MA mycotoxin conjugates and the MA control conjugate , but bound only to the poly-L-lysine-AE conjugates of the homologous mycotoxin. Binding of antisera to homologous poly-L-lysine conjugates was always inhibited by free hapten when the conjugate was prepared by the AE method but not by the MA method. The specific inhibition of antibody binding by various synthetic haptens indicated that the cross-reactions associated with the MA method were due to the undesired conjugation of isobutylformate during the mixed anhydride procedure.

Development of a tetracycline controlled gene expression system in the parasitic protozoan Giardia lamblia.

Giardia lamblia is a very common intestinal protozoan pathogen of humans. Recent development of gene transfection systems in G. lamblia has allowed constitutive expression of selected genes in the organism. To extend the uses of DNA transfection in G. lamblia, an inducible gene expression system was developed by integrating the bacterial tet operator-repressor elements into an episomal DNA transfection vector. Tetracycline-responsive promoters with insertions of multiple tet operator sequences in the vicinity of a synthetic ran promoter were tested for their inducibility of a luciferase reporter gene expression. Stable cell lines transfected with individual plasmid constructs were established under drug selection. By assaying luciferase activity in transfected cells in response to tetracycline, an inducible promoter with insertion of two tet operators downstream of the adjacent synthetic ran promoter was found to confer a 10-fold inducibility in gene expression with co-expression of the tet-repressor driven by a gdh promoter. To further improve its inducibility, several other synthetic promoter contexts were also tested to increase expression of the tet-repressor gene. An optimal inducibility of 50-fold was obtained when a synthetic alpha-giardin promoter was used. Fine tuning of luciferase expression was achieved by adjusting the concentration of tetracycline and duration of drug exposure. The inducible gene expression system provides us an easy way to manipulate the level of gene expression in G. lamblia in a controllable manner that could not previously be achieved.

Stable DNA transfection of the primitive protozoan pathogen Giardia lamblia.

We have developed a stable DNA transfection vector pRANneo for genetic manipulation of the primitive protozoan Giardia lamblia. pRANneo was constructed by replacing the protein coding region of a Giardia ran gene with a bacterial neomycin phosphotransferase gene (neo). This plasmid was electroporated into G. lamblia, and the transfectants were selected by G418. pRANneo replicated episomally to approximately 80 copies per G. lamblia trophozoite as demonstrated by dot hybridizations, Southern hybridizations and transformations of the DpnI-treated plasmids into Escherichia coli. pRANneo/GDHluc was then constructed by incorporation of a luciferase expression system into pRANneo to persistently express firefly luciferase in G. lamblia under G418 selection. The NEO and luciferase proteins were detected in the transfected G. lamblia cells by Western blottings. The level of luciferase activity and the plasmid copy number correlated with the concentration of G418. Removal of G418 from the transfectant culture resulted in gradual loss of the plasmid and luciferase activity. The stable DNA transfection system should provide a valuable tool for genetic studies of G. lamblia.

Synthesis and antitumor activity of a series of [2-substituted-4,5-bis(aminomethyl)-1,3-dioxolane]platinum(II) complexes.

The synthesis, physical properties, antitumor activity, structure-activity relationships, and nephrotoxicity of a series of [2-substituted-4,5-bis(aminomethyl)-1,3-dioxolane]platinum(II) complexes are described. The 42 platinum(II) complexes having a seven-membered ring structure in this series have been prepared and characterized by 1H NMR, 13C NMR, IR, FAB-MS, and elemental analysis. All members of the series were designed to have a 1,3-dioxolane ring moiety in their carrier ligands to increase water solubility. The solubility of platinum complexes was related to the nature of leaving ligands and 2-substituents in the 4,5-bis(aminomethyl)-1,3-dioxolane carrier ligands. In general, compounds having two different R1 and R2 substituents in the 4,5-bis(aminomethyl)-1,3-dioxolane moiety were more water-soluble than those having the same substituents. Most members of this series showed the excellent antitumor activity against murine L1210 leukemia cells transplanted in mice and were superior to cisplatin and carboplatin. The (4R,5R)-stereoisomer 1a-h exhibited the higher antitumor activity than the corresponding (4S,5S)-stereoisomer 2a-h in the (1,1-cyclobutanedicarboxylato)platinum(II) complexes. The (glycolato)-platinum(II) complexes were highly cytotoxic toward four human stomach cancer cell lines, SNU-1, SNU-5, SNU-16, and NCI-N87, and among them, complexes 3d-g were even more cytotoxic than cisplatin. The (malonato)platinum(II) complex 1m and the (glycolato)platinum(II) complexes 3d-g were selected for further studies based on the greater in vivo and in vitro antitumor activity and desirable physical properties. The complexes 3e-g were almost equally cytotoxic to cisplatin toward human stomach cancer cell lines, KATO-III and MKN-45, and a human non-small cell lung cancer cell line, PC14. In contrast with cisplatin and carboplatin, five complexes selected significantly increased in life span in mice transplanted with cisplatin-resistant L1210 cells. Nephrotoxicity studies in ICR mice indicated that serum BUN and creatinine levels were not elevated when five complexes were given at a dose equal to 1.5 times the optimal dose determined in the in vivo L1210 screening system.

Pharmacokinetics and antitumor activity of a new platinum compound, cis-malonato[(4R,5R)-4,5-bis(aminomethyl)-2-isopropyl-1, 3- dioxolane]platinum(II), as determined by ex vivo pharmacodynamics.

The pharmacokinetics and ex vivo pharmacodynamics studies on cis-malonato[(4R,5R)-4,5-bis(aminomethyl)-2-isopropyl-1, 3- dioxolane]platinum(II) (SKI 2053R, NSC D644591), cisplatin (CDDP), and carboplatin (CBDCA) were performed in beagle dogs. Equitoxic doses of SKI 2053R, CDDP, and CBDCA (7.5, 2.5, and 15.0 mg/kg, respectively) were given by i.v. bolus to three beagle dogs in a randomized crossover study. Plasma samples were analyzed for platinum by flameless atomic absorption spectrophotometry. Plasma concentrations of total and ultrafiltrable platinum for the three drugs declined in a biexponential fashion. The mean area under the concentration-time curve (AUC0-->infinity) determined for ultrafiltrable platinum derived from SKI 2053R, as an active component, was 7.72 +/- 2.74 micrograms h ml-1 (mean +/- SD), with an initial half-life of 0.37 +/- 0.20 h, a terminal half-life of 2.19 +/- 0.93 h, a total clearance of 16.83 +/- 4.76 ml min-1 kg-1, and a steady-state volume of distribution of 1.57 +/- 0.30 l/kg. The ex vivo antitumor activity of SKI 2053R was assessed using the ultrafiltrable plasma against two human lung-adenocarcinoma cell lines (PC-9 and PC-14) and five stomach-adenocarcinoma cell lines (MKN-45, KATO III, SNU-1, SNU-5, and SNU-16) by tetrazolium-dye (MTT) assay and was compared with that of CDDP and CBDCA using an antitumor index (ATI) determined from the ex vivo pharmacodynamic results of inhibition rates (%) versus time curves. The mean ATI value was shown to be ranked in the following order: SKI 2053R > CBDCA > CDDP. The mean ATI values recorded for SKI 2053R and CBDCA were significantly (P < 0.05) higher than that noted for CDDP; however, no statistically significant difference was observed between SKI 2053R and CBDCA, suggesting that the antitumor activity of SKI 2053R is superior to that of CDDP and is equivalent to that of CBDCA. These results suggest that SKI 2053R is a promising candidate for further development as a clinically useful anticancer drug.

Antitumor activity of cis-malonato[(4R,5R)-4,5-bis(aminomethyl)-2- isopropyl-1,3-dioxolane]platinum(II), a new platinum analogue, as an anticancer agent.

The in vitro and in vivo antitumor activity of a new antitumor platinum complex, cis-malonato[(4R, 5R)-4,5- bis(aminomethyl)-2-isopropyl-1,3-dioxolane]platinum(II) (SKI2053R, NSC D644591), were evaluated and compared with those of cisplatin (CDDP) and carboplatin (CBDCA) using murine tumors. SKI 2053R was highly active in vitro against both L1210 murine leukemia and its CDDP-resistant subline, L1210/DDP; the relative resistances were 20.0-, 14.5-, and 2.7-fold for CDDP, CBDCA, and SKI 2053R, respectively. SKI 2053R showed activity comparable with or superior to either CDDP or CBDCA in mice implanted with L1210. In mice implanted with L1210/DDP, as compared with CBDCA, SKI 2053R showed high values for the percentage of treated survivors relative to controls and for numbers of cured mice, whereas CDDP had virtually no activity. In mice implanted with P388, all three drugs were highly active, but the intensity of activity was shown to be ranked in the following order: SKI 2053R > CDDP > CBDCA. The antitumor activity of SKI 2053R against Lewis lung carcinoma was comparable with that of both CDDP and CBDCA. The antitumor activity of SKI 2053R was further investigated against two human tumor xenografts, KATO III (stomach adenocarcinoma) and WiDr (colon adenocarcinoma), implanted s.c. in nude mice and was compared with that of CDDP. In SKI 2053R-treated groups, the time required for a mean tumor weight of 1,000 mg was 33.1 days in KATO III xenografts and 35.0 days in WiDr xenografts as compared with 30.2 and 27.2 days in CDDP-treated groups, respectively. SKI 2053R achieved growth-inhibition rates comparable with those of CDDP against KATO III (65% versus 59%) and WiDr xenografts (64% versus 54%) on day 35. These results indicate that SKI 2053R is an attractive candidate for further development as a clinically useful anticancer drug.

Synergistic interaction between the trichothecene T-2 toxin and Salmonella typhimurium lipopolysaccharide in C3H/HeN and C3H/HeJ mice.

The capacity of the trichothecene T-2 toxin to alter resistance to bacterial lipopolysaccharide (LPS) was examined in the mouse. Both LPS-susceptible (C3H/HeN) and LPS-resistant (C3H/HeJ) mouse strains exhibited markedly enhanced mortality when a single oral dose of T-2 toxin (1 mg/kg) was coadministered with a subacute i.p. dose of Salmonella typhimurium LPS. In the absence of LPS, T-2 toxin did not cause lethal effects when administered at this level. LD50 values for LPS decreased by 14-fold and 4.5-fold upon co-administration with T-2 toxin (1 mg/kg) in C3H/HeN and C3H/HeJ mice, respectively. Increased mortality was accompanied by an impaired splenic response to LPS in C3H/HeN mice. C3H/HeN mice pretreated with a sublethal dose of LPS 24 h prior to T-2 toxin administration also exhibited significantly increased susceptibility to T-2 toxin. Histopathological assessment revealed that the liver and spleen of mice exposed to T-2 toxin and LPS exhibited extensive cell death as compared to control mice treated with T-2 toxin or LPS only. The results suggest that bacterial LPS and trichothecenes such as T-2 toxin interact synergistically. This interaction may contribute to increased mortality that has been observed previously in animals challenged with Salmonella and T-2 toxin.

Identification of virus-specific vesicles in Giardiavirus-infected Giardia lamblia.

Giardiavirus (GLV), which infects the parasitic protozoan Giardia lamblia, is a nonsegmented double-stranded (ds) ribonucleic acid (RNA) virus. We previously purified two distinct types of related GLV from infected G. lamblia, and showed differential export of one of the viruses from infected cells. In the present study, fractionation of cell lysate was performed, revealing the presence of viruses in the membranous fraction. Distribution of viral antigens in the infected cells was examined by immunocytochemistry. The signal was enriched in certain regions of the cytoplasm, suggesting that a portion of GLV is confined to certain cellular compartments. A significantly reduced signal was also detected in the nuclei. We directly observed the viruses in the infected cells by electron microscopy. Consistent with previous observations, virus-like particles were clearly observed in some membranous vesicles in the cytoplasm at 48 h postinfection, and virus-like particles were again seen in the cytoplasm and then in the nuclei toward the late phase of virus infection. The virus-associated vesicles and some electron-dense nuclear structures were only observed in virus-infected cells, suggesting that virus infection may induce ultrastructural alteration of G. lamblia.

Impaired murine resistance to Salmonella typhimurium following oral exposure to the trichothecene T-2 toxin.

On orally exposing Salmonella-resistant C3H/HeN mice to the trichothecene T-2 toxin (1 mg/kg body weight), challenging with Salmonella typhimurium, and continuing to dose with T-2 toxin on alternate days for 3 wk, the LD50 for the organism decreased by five orders of magnitude, in comparison with control mice not treated with T-2 toxin. In the absence of S. typhimurium, T-2 toxin did not cause lethal effects when administered at this level. Increased mortality in response to S. typhimurium challenge was dependent on T-2 toxin dose in the range 0 to 1 mg/kg for this regimen. The toxin did not significantly affect intestinal infection but did increase splenic counts in mice challenged with a range of S. typhimurium doses and also accelerated body-weight loss in infected animals. Mice challenged with the organism exhibited similar mortality when T-2 toxin treatment was begun 1 day prior to infection or at 5 or 9 days after infection. A time-related decrease in mortality, relative to that found for the standardized co-challenge described above, was observed when T-2 toxin administration was begun at 9, 13 or 23 days after infection. The results indicated that, depending on the challenge dose of the organism, both early and late phase acquired immune response to S. typhimurium could be impaired by T-2 toxin. Markedly enhanced susceptibility to gram-negative bacterial infection is another manifestation of trichothecene toxicity and may be an important aetiological factor in animal health problems that are associated with these mycotoxins.

Effects of 8-week exposure of the B6C3F1 mouse to dietary deoxynivalenol (vomitoxin) and zearalenone.

Weanling female B6C3F1 mice were fed semi-purified diets containing 0, 0.5, 2.0, 5.0, 10.0 or 25.0 ppm (mg/kg) deoxynivalenol (DON) over 8 wk and were assessed for effects on feed intake, body-weight gain, terminal organ weights, histopathology, haematology and serum immunoglobulin levels. To determine whether DON effects were potentiated by the oestrogen zearalenone (ZEA), a mycotoxin frequently found to occur with DON in cereals, two additional groups of mice were fed diets containing either 10 ppm ZEA or 10 ppm ZEA plus 5 ppm DON. The rate of body-weight gain was significantly reduced (P less than 0.01) for all mice consuming feed containing 2.0 ppm or more of DON, whereas only the mice ingesting the diet containing 25 ppm DON showed a significantly decreased (P less than 0.01) rate of feed consumption. Gross and histopathological evaluation of thymus, spleen, liver, kidney, uterus, small intestine, colon, heart, brain, lungs and bone marrow from control and all mycotoxin-exposed mice revealed that these tissues were normal in appearance and in histological architecture. DON-amended diets did however, cause dose-dependent decreases in the terminal organ weights recorded (thymus, spleen, liver, kidney and brain). In the DON-treated groups, statistically significant dose-dependent decreases in the counts of total circulating white blood cells were associated with an increase in polymorphonuclear neutrophils and a decrease in lymphocytes and monocytes. Dietary DON caused a dose-dependent decrease in serum IgM but, in contrast, a dose-dependent increase in serum IgA. In none of the above instances was 10 ppm ZEA shown to act synergistically or antagonistically with 5 ppm DON. Since dietary DON at levels as low as 2.0 ppm exerted significant effects on the growing B6C3F1 female mouse, future approaches should include studies of the mechanisms by which this mycotoxin affects nutrient utilization and modifies the normal immune response.

Comparison of acute toxicities of deoxynivalenol (vomitoxin) and 15-acetyldeoxynivalenol in the B6C3F1 mouse.

The acute toxic effects of deoxynivalenol (DON) and 15-acetyldeoxynivalenol (15-ADON) were compared in the B6C3F1 female mouse after oral and intraperitoneal exposure. Using the abbreviated procedure of Lorke (Archs Toxicol. 1983, 54, 275), LD50 values for DON were estimated to be 78 mg/kg (oral) and 49 mg/kg (ip) whereas the LD50 values for 15-ADON were 34 mg/kg (oral) and 113 mg/kg (ip). Acute doses of these toxins resulted in extensive necrosis of the gastro-intestinal tract, bone marrow and lymphoid tissues, and focal lesions in kidney and cardiac tissue. The minimum doses required for these histopathological effects were consistent with LD50 estimations. The results indicate that 15-ADON was more or less toxic than DON depending on the route of administration. Risk assessments for DON should therefore consider the potential for 15-ADON occurrence and toxicity in food and feed.

Suppression of immune response in the B6C3F1 mouse after dietary exposure to the Fusarium mycotoxins deoxynivalenol (vomitoxin) and zearalenone.

The effect that dietary exposure to the naturally-occurring Fusarium graminearum toxins deoxynivalenol (DON) and zearalenone (ZEA) may have on immune function was assessed in the B6C3F1 mouse. Dietary DON depressed the plaque-forming response to sheep red blood cells, the delayed hypersensitivity response to keyhole limpet haemocyanin and the ability to resist Listeria monocytogenes. Listerial resistance was similarly decreased in control mice fed restricted diets comparable to the dietary restriction caused by DON-induced feed refusal, whereas equivalent food restriction did not decrease the plaque or delayed hypersensitivity responses. ZEA ingestion decreased resistance to L. monocytogenes but did not affect splenic plaque-forming or delayed hypersensitivity responses. Resistance to Listeria was reduced to a greater extent by co-administration of DON and ZEA than by DON alone, whereas the ability of DON to inhibit the delayed hypersensitivity response was significantly lessened in the presence of ZEA. While effects on resistance to Listeria and delayed hypersensitivity were detectable in mice ingesting the mycotoxins for 2-3 wk, these effects disappeared upon extension of the feeding period to 8 wk. In contrast, some effect on the plaque-forming response was detectable with both the 2- and the 8-wk period of mycotoxin ingestion. Immunosuppression can thus result from ingestion of F. graminearum-infected agricultural staples, the suppression being attributable to interactions between direct immunotoxic effects of DON and ZEA and nutritional effects associated with DON-induced food refusal.

Transmissible gastroenteritis virus induces apoptosis in swine testicular cell lines but not in intestinal enterocytes.

Evidence of apoptosis caused by infection with the Purdue strain of transmissible gastroenteritis virus (TGEV) was sought in vitro (in infected swine testicular [ST] cells) and in vivo (in the intestinal tissues of infected piglets). The methods used were (1) DNA electrophoresis for detection of DNA fragmentation, and (2) terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-fluorescein nick and labelling (TUNEL). DNA "laddering" was detected in TGEV-infected ST cells only. Numerous signs of apoptosis were detected in TGEV-infected ST cells by TUNEL assay, the positive (dark brown) staining reaction being present in the majority of cell nuclei, without background staining. No such staining was seen in TGEV-infected enterocytes at various times after inoculation of piglets. Thus, it would appear that apoptosis does not occur in the enterocytes of piglets infected with TGEV.

Apoptosis in rabbit haemorrhagic disease.

Rabbit haemorrhagic disease virus (RHDV) causes an acute hepatitis and disseminated intravascular coagulation. Six rabbits were inoculated experimentally with RHDV to investigate any potential relationship between infection and apoptosis in the liver. Two rabbits were killed at 12 h post-inoculation (PI) and two at 24 h PI. The remaining two rabbits died at 30 h and 31 h PI. Immunohistochemical labelling for RHDV antigen-positive cells, TUNEL assay for apoptotic cells, and DNA analysis were performed on samples of liver. The four rabbits that died or were killed 24-31 h PI had acute hepatitis with infiltration of heterophils and necrotic hepatocytes. RHDV antigen-positive cells and apoptotic cells appeared in the centriacinar areas at 12 h PI; subsequently they spread to periacinar areas and increased in number, but the viral antigen-positive cells outnumbered apoptotic cells. At 24-31 h PI, few apoptotic cells were recognized in the areas infiltrated with lymphocytes and heterophils. The results suggested an association between RHDV infection and apoptosis of hepatocytes.

The economic value of screening haemodialysis patients for methicillin-resistant Staphylococcus aureus in the USA.

Methicillin-resistant Staphylococcus aureus (MRSA) can cause severe infections in patients undergoing haemodialysis. Routine periodic testing of haemodialysis patients and attempting to decolonize those who test positive may be a strategy to prevent MRSA infections. The economic value of such a strategy has not yet been estimated. We constructed a Markov computer simulation model to evaluate the economic value of employing routine testing (agar-based or PCR) at different MRSA prevalence, spontaneous clearance, costs of decolonization and decolonization success rates, performed every 3, 6 or 12 months. The model showed periodic MRSA surveillance with either test to be cost-effective (incremental cost-effectiveness ratio ≤$50 000/quality-adjusted life-year) for all conditions tested. Agar surveillance was dominant (i.e. less costly and more effective) at an MRSA prevalence ≥10% and a decolonization success rate ≥25% for all decolonization treatment costs tested with no spontaneous clearance. PCR surveillance was dominant when the MRSA prevalence was ≥20% and decolonization success rate was ≥75% with no spontaneous clearance. Routine periodic testing and decolonization of haemodialysis patients for MRSA may be a cost-effective strategy over a wide range of MRSA prevalences, decolonization success rates, and testing intervals.

T-2 toxin impairment of murine response to Salmonella typhimurium: a histopathologic assessment.

T-2 toxin and other trichothecene mycotoxins experimentally impair normal immune function and may predispose humans and animals to infectious disease. In this study, the histopathologic effects of Salmonella typhimurium challenge concurrently with sublethal T-2 toxin exposure were examined in the Salmonella-resistant C3H/HeN mouse. Oral administration of T-2 toxin (1 mg/kg) every other day for 10 d had little effect on the tissues examined when compared to control animals. Mice challenged with S. typhimurium and then treated with T-2 toxin every other day for 10 d had markedly larger and more bacterial-related lesions in the spleens, kidneys, and livers than animals challenged with S. typhimurium alone. Differences in bone marrow, Peyer's patches and ileal tissues were less discernable between S. typhimurium and S. typhimurium plus T-2 toxin treated groups. These results were consistent with previous findings that T-2 toxin compromised murine resistance to S. typhimurium infection and ultimately caused death in animals challenged with a sublethal dose of the organism.