Influence of segmented vessel size due to limited imaging resolution on coronary hyperemic flow prediction from arterial crown volume.

Computational predictions of the functional stenosis severity from coronary imaging data use an allometric scaling law to derive hyperemic blood flow (Q) from coronary arterial volume (V), Q = αV(ß) Reliable estimates of α and ß are essential for meaningful flow estimations. We hypothesize that the relation between Q and V depends on imaging resolution. In five canine hearts, fluorescent microspheres were injected into the left anterior descending coronary artery during maximal hyperemia. The coronary arteries of the excised heart were filled with fluorescent cast material, frozen, and processed with an imaging cryomicrotome to yield a three-dimensional representation of the coronary arterial network. The effect of limited image resolution was simulated by assessing scaling law parameters from the virtual arterial network at 11 truncation levels ranging from 50 to 1,000 µm segment radius. Mapped microsphere locations were used to derive the corresponding relative Q using a reference truncation level of 200 µm. The scaling law factor α did not change with truncation level, despite considerable intersubject variability. In contrast, the scaling law exponent ß decreased from 0.79 to 0.55 with increasing truncation radius and was significantly lower for truncation radii above 500 µm vs. 50 µm (P< 0.05). Hyperemic Q was underestimated for vessel truncation above the reference level. In conclusion, flow-crown volume relations confirmed overall power law behavior; however, this relation depends on the terminal vessel radius that can be visualized. The scaling law exponent ß should therefore be adapted to the resolution of the imaging modality.

Perfusion territories subtended by penetrating coronary arteries increase in size and decrease in number toward the subendocardium.

Blood flow distribution within the myocardium and the location and extent of areas at risk in case of coronary artery disease are dependent on the distribution and morphology of intramural vascular crowns. Knowledge of the intramural vasculature is essential in novel multiscale and multiphysics modeling of the heart. For this study, eight canine hearts were analyzed with an imaging cryomicrotome, developed to acquire high-resolution spatial data on three-dimensional vascular structures. The obtained vasculature was skeletonized, and for each penetrating artery starting from the epicardium, the dependent vascular crown was defined. Three-dimensional Voronoi tessellation was applied with the end points of the terminal segments as center points. The centroid of end points in each branch allowed classification of the corresponding perfusion territories in subendocardial, midmyocardial, and subepicardial. Subendocardial regions have relatively few territories of about 0.5 ml in volume having their own penetrating artery at the epicardium, whereas the subepicardium is perfused by a multitude of small perfusion territories, in the order of 0.01 ml. Vascular volume density of small arteries up till 400 µm was 3.2% at the subendocardium territories but only 0.8% in the subepicardium territories. Their higher volume density corresponds to compensation for flow impeding forces by cardiac contraction. These density differences result in different scaling law properties of vascular volume and tissue mass per territory type. This novel three-dimensional quantitative analysis may form the basis for patient-specific computational models on coronary perfusion and aid the interpretation of image-based clinical methods for assessing the transmural perfusion distribution.

Influence of cytotoxicity enhancers in combination with human serum on the activity of CD22-recombinant ricin A against B cell lines, chronic and acute lymphocytic leukemia cells.

Despite the strong in vitro activity of some immunotoxins (ITs), clinical application did not result in complete cure. The outcome of therapy may be improved by combining ITs with IT-cytotoxicity enhancing agents. We studied the effect of various agents that influence the intracellular routing of ITs on the activity of the anti-B cell IT CD22-recombinant (rec) ricin A. In protein synthesis inhibition assays the carboxylic ionophores monensin and nigericin enhanced the activity of the IT 117- and 382-fold, respectively, against the cell line Daudi, and 81- and 318-fold, respectively, against the cell line Ramos. IT activity to Daudi and Ramos was enhanced to a lesser extent by the lysosomotropic amines chloroquine (14- and 11-fold, respectively) and NH4Cl (nine- and 10-fold, respectively). However, the combination of NH4Cl and chloroquine induced more than an additive effect (145- and 107-fold, respectively). Cytotoxicity was not influenced by brefeldin A, all-trans retinoic acid (ATRA), verapamil and perhexiline maleate. Bacitracin enhanced the IT cytotoxicity in contrast to the other protease inhibitors aprotinin, leupeptin and soybean trypsin inhibitor, albeit enhancement was weak (two-fold). The enhancers exerted only a negligible effect on bone marrow progenitor cells. We recently developed a flow cytometric cytotoxicity assay in which cell elimination can be assessed. In order to detect enhancement in this assay, we used 5 x 10(-11) M IT (approximately the 50% protein synthesis inhibiting dose (ID50)). This concentration killed 41% of the Daudi cells and 42% of the Ramos cells. In the presence of 10 nM monensin the IT killed 74% and 99% and in the presence of 10 nM nigericin 96% and 99% of the Daudi and Ramos cells, respectively. At 10(-8) M, CD22-rec ricin A eliminated malignant cells originating from three patients with B-CLL (0.42 log) and two with B-ALL (0.19 log) patients. Cytotoxicity to malignant cells was enhanced by NH4Cl, chloroquine, monensin and nigericin. The combination of NH4Cl and chloroquine enhanced the activity most effectively (up to 2.06 log). To determine the applicability of the IT in combination with enhancers in vivo we investigated the effect of human serum. Human serum inhibited IT activity which could not be restored by monensin and nigericin because of complete inhibition of these enhancers by serum. In contrast, chloroquine partially restored the activity of CD22-rec ricin A in the presence of human serum. We conclude that monensin, nigericin and the combination of NH4Cl and chloroquine can be used instead of NH4Cl to potentiate CD22-rec ricin A activity in purging autologous bone marrow transplants contaminated with malignant B cells. Chloroquine might be a promising enhancer of CD22-rec ricin A for treating patients in vivo.

Relationship of the CD22 immunotoxin dose and the tumour establishment in a SCID mice model.

Immunotoxins (ITs) may be very potent to erradicate tumour growth in vivo. We investigated the influence of the IT-dose, in relation to the establishment of the tumour, on the anti-tumour activity of CD22-recombinant (rec) ricin A for a disseminated tumour (Ramos) in SCID mice. Furthermore, the enhancement of the IT cytotoxicity in vivo by chloroquine was assessed. CD22-rec ricin A appeared to be highly effective. Paralysis of the hind legs was significantly delayed by a very low IT-dose of 2 microg administered intravenously (i.v.) 7 days after i.v. inoculation of the tumour cells. Even a dose of 30 microg administered 21 days after inoculation of the target cells significantly delayed the onset of paralysis up to 8 days compared with the median paralysis time (MPT) of the control group. The efficacy of treatment was obviously influenced by the establishment of the tumour, the tumour load and localisation. The anti-tumour activity of 10 and 30 microg IT diminished when the IT was administered after increasing the time lag following inoculation of tumour cells. Delaying IT administration resulted in growth of solid tumours. This implies that cells migrate to sanctuaries protected from the IT indicating that the anti-tumour activity was influenced by the accessibility of the IT to the target cells. The in vivo anti-tumour activity of CD22-rec ricin A could not be enhanced by simultaneously administered chloroquine, despite the continuous infusion with an intraperitoneally (i.p.) implanted mini-osmotic pump. Ex vivo experiments revealed that the maximally tolerated serum concentration (3.9 microM) was too low to be effective. In conclusion, CD22-rec ricin A is highly effective for in vivo treatment of B-cell malignancies, in particular if treatment is started when the tumour load is low and before migration takes place to poorly accessible sanctuaries.

Cytotoxic potency of CD22-ricin A depends on intracellular routing rather than on the number of internalized molecules.

Cytotoxicity of immunotoxins (ITs) varies considerably depending on factors like the capability of the target antigen to internalize IT molecules, intracellular processing and routing of the IT. We studied factors that may influence cytotoxicity of CD22-ricin A IT to several B cell lines. The antigen density varied from 5.9 x 10(3) to 6.0 x 10(4) molecules/cell. The ID50, determined by protein synthesis inhibition, varied from 2.1 x 10(-12) to 3.8 x 10(-11) M IT in absence and from 2.8 x 10(-14) M to 5.2 x 10(-12) M IT in presence of the cytotoxicity enhancer NH4Cl (6 mM). In absence as well as in presence of NH4Cl no correlation could be found between antigen density and ID50. No relation was observed either with the rate of cytotoxicity. Even in cell lines with a low antigen density, such as KM3, protein synthesis was quickly inhibited. In order to investigate whether the cytotoxicity was dependent on the number of internalized molecules the kinetics of internalization and exocytosis of degraded 125I-labelled CD22 molecules were studied. After 24 h the number of internalized CD22 molecules was highest in Ramos (154,500), followed by Daudi (110,300) and KM3 (69,900). However, despite the higher internalization rate of Daudi the rate of cytotoxicity of 10(-8) M IT was comparable with KM3. NH4Cl did not influence the number of internalized molecules but postponed degradation of CD22. In conclusion, CD22-ricin A is a very potent and fast acting IT even for elimination of target cells that express low numbers of antigen. These results may have implication for treatment of different B cell malignancies with CD22-ricin A.

Highly potent CD22-recombinant ricin A results in complete cure of disseminated malignant B-cell xenografts in SCID mice but fails to cure solid xenografts in nude mice.

The highly specific cytotoxic action of ribosome-inactivating protein (RIP) containing immunotoxins (ITs) makes IT therapy a promising approach to eliminating residual malignant cells. We investigated the cytotoxicity of the IT CD22-recombinant ricin A to the B-cell line Ramos in vitro and in vivo. Cytotoxicity of CD22-recombinant ricin A in vitro was very high as expressed by the very low 50% inhibition dose (ID50) of 3.5 x 10(-11) M. Cytotoxicity was increased 7 times in the presence of the cytotoxicity enhancer NH4Cl. The ultimate kill of Ramos cells by CD22-recombinant ricin A was high (2.7-log kill) and was increased strongly in the presence of NH4Cl (4.2-log kill). Anti-tumor activity in vivo was investigated by i.v. treatment of solid s.c. Ramos xenografts in nude BALB/c mice. A single dose did not inhibit tumor growth. Treatment on 5 consecutive days resulted in evident tumor reduction. In one mouse, tumor could no longer be detected on the 6th day after starting treatment. However, after 8 days tumor volumes increased again. Antitumor activity was more pronounced in a disseminated tumor model in SCID mice. IT treatment (i.v.) 7 days after i.v. inoculation with Ramos cells resulted in cure of all mice. Non-specific toxicity was low. Alanine aminotransferase (ALAT) levels in serum were elevated temporarily. Serum values of gamma-glutamyl transferase (gamma-GT), bilirubin and creatinin did not change. Body weight was also transiently reduced. The LD50 in SCID mice after i.v. administration was high (0.626 mg IT per mouse). The clearance rate in SCID mice, as determined by ELISA, was biphasic.

C5 does not play a major role in the immune response of mice to SRBC in vivo.

The role of complement component C5 in the immune response of mice to sheep red blood cells (SRBC) was investigated. Congenic C5-sufficient and C5-deficient B10. D2 mice and genetically C5-deficient DBA/2 mice, as such or supplemented with C5-sufficient serum, were used as experimental animals. C5-substitution of the C5-deficient mice resulted in measurable C5 levels for days. The functional half-life of C5 in C5-deficient DBA/2 mice was about 21 h. No significant differences between the IgM-responses of C5-bearing and naive C5-deficient animals were observed. This suggests that C5 does not play a major role in the primary humoral immune response of mice in vivo, although C5 seems to do so in in vitro experiments, even with the same antigen. Antigen-induced C5-production by C5-deficient mice as one of the explanations of the in vitro/in vivo discrepancy could not be confirmed experimentally.

Iron-dependent stability of the ferredoxin I transcripts from the cyanobacterial strains Synechococcus species PCC 7942 and Anabaena species PCC 7937.

The effect of iron on ferredoxin I specific mRNA levels was studied in the cyanobacterial strains Synechococcus sp. PCC 7942 (Anacystis nidulans R2) and Anabaena sp. PCC 7937 (Anabaena variabilis ATCC 29413). In both strains addition of iron to iron-limited cells resulted in a rapid increase in ferredoxin mRNA levels. To investigate the possible role of the ferredoxin promoter in iron regulation, a vector for promoter analysis in Synechococcus PCC 7942 strain R2-PIM9 was constructed, which contains the ferredoxin promoter fused to the gene encoding beta-glucuronidase (GUS) as reporter. Neither the Synechococcus nor the Anabaena ferredoxin promoter was able to direct iron-regulated GUS activity in Synechococcus R2-PIM9, indicating that transcription initiation is not responsible for the iron-dependent ferredoxin mRNA levels. Determination of the half-life of the ferredoxin transcript in iron-supplemented and iron-limited cells revealed that, in both strains, the ferredoxin transcript is much more stable in iron-supplemented cells than in iron-limited cells. These results lead to the conclusion that in these strains, iron-regulated expression of the ferredoxin I gene is mediated via differential mRNA stability.