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First published online September 3, 2007
British Journal of Radiology (2007) 80, 713-718
© 2007 British Institute of Radiology
doi: 10.1259/bjr/38377769
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Full paper

Iosimenol, a new non-ionic dimeric contrast medium, does not induce immunoreactivity in the popliteal lymph node assay

M J Fernandez Cabezudo, PhD 1 G Petroianu, MD, PhD 2 B Al-Ramadi, PhD, FRC 3 and R D Langer, MD, PhD 4

Departments of 1 Biochemistry, 2 Pharmacology, 3 Medical Microbiology and 4 Radiology, Faculty of Medicine and Health Sciences, United Arab Emirates University, PO Box 17666, Tawam Street, Al Ain, Abu Dhabi, UAE

Correspondence: Professor Ruth D Langer, MD, PhD, Professor, Associate Dean for Clinical Affairs; Professor of Radiology, Faculty of Medicine and Health Sciences, UAE University-FMHS, PO Box 17666, Tawam Street, Al Ain, Abu Dhabi, UAE. E-mail: rlanger{at}uaeu.ac.ae


   Abstract
Top
 Abstract
Introduction
 Methods and materials
 Results
 Discussion
 Conclusions
 References
 
Animal studies in mice were conducted to determine the potential immunoreactivity of the new non-ionic dimeric contrast medium (CM) iosimenol using the PLNA and flow cytometric analyses. Comparative studies were performed with iodixanol. The known immune-reactive substance strepozotocin (STZ) and vehicle injections served as positive and negative controls, respectively. Our experiments did not show any immunological effect of iosimenol, concluding that the new CM iosimenol may be beneficial for use in high-risk patients.


   Introduction
Top
 Abstract
 Introduction
Methods and materials
 Results
 Discussion
 Conclusions
 References
 
Intravascular contrast media (CM) are widely used for various conventional and cross-sectional radiological examinations. CT studies are performed at most medical centres in more than 90% of cases with intravenous (i.v.) contrast material administration; digital subtraction angiography (DSA) may require intraarterial (i.a.) high-dosage, high-speed CM injection. In patients receiving intravascular CM, side-effects have been reported to different extents [1–14].

In particular, the detection of immunogenic effects of pharmaceuticals, such as CM, is of special interest. Since non-ionic contrast agents have been introduced, toxicity as well as pseudoallergic reactions has been substantially reduced [1–5, 8, 14–20]. Most contrast materials commercially available are non-ionic monomeric CM; only one non-ionic dimeric contrast medium is approved and, at present, commercially available for intravascular application, i.e. iodixanol [8, 14, 17, 19, 21].

Compared with non-ionic monomeric contrast agents, non-ionic dimeric contrast materials are iso-osmolar to blood, plasma, liquor, and other body fluids, and thereby fulfil a basic requirement of a well-tolerated intravascular medium. Iso-osmolarity has been shown to reduce heat and pain in peripheral angiography, heat sensation following intravenous injection, and to some degree to improve cardiovascular tolerance [14, 17, 22, 23]. Although controversial, some clinical trials indicate that non-ionic dimeric contrast agents carry less risk of inducing transient creatinine elevation in patients suffering from renal insufficiency, most pronounced after intraarterial administration and also in intensive care patients [8–12]. It has been assumed that non-ionic dimeric contrast agents may be advantageous for diabetics and multiple myeloma patients with renal impairment [8–12].

However, limitations in intravascular application exist due to the high viscosity of iodixanol [17, 19, 21] and consecutive alterations of the microcirculation [24]. The new dimeric substance iosimenol shows a significantly lower viscosity than iodixanol and is likewise iso-osmolar.

Immunogenic effects caused by administration of CM are not easy to predict; and it might be very difficult to detect immune responses in animal experiments. Although the search for antibodies is rarely helpful, the popliteal lymph node assay (PLNA) has become a useful technique for assessing the immune reaction of an organism, irrespective of the underlying mechanism. The PLNA has been reviewed in length as a testing system for immunosensitizing effects since the mid 1990s [25–32]. The PLNA facilitates the detection and analysis of immunotoxic effects in a quick and reproducible way, although the underlying reactions are not entirely clear [32]. The direct PLNA is performed by interdigital subcutaneous (s.c.) injection of the test substance into the footpad of one hind leg of a mouse (or rat), the defined draining area to the popliteal lymph nodes (PLNs). The defence reaction of a not immunocompromised organism is a proliferation of immunocompetent cells in the lymph node resulting in 'reactive hyperplasia', either by activation of a specific immune response, which involves B and T lymphocytes, or as activation of macrophages and other inflammatory cells, components of the non-specific innate immune system. Reactive hyperplasia is a reversible process and recedes when the triggering agent is removed [33, 34].

Potential immunological reactions of the new substance iosimenol have not yet been assessed and require further investigation. If a lack – or at least low level – of immunological effects and subsequently of pseudoallergic reactions can be proven, the contrast material iosimenol may have the potential of a wider use in high-risk patients.

The aim of the study is to determine whether and/or to what extent exposure to the new CM iosimenol provokes reactive hyperplasia in PLNs and/or other immunological reactions in mice by using the PLNA, aided by flow cytometric analysis for further elucidation of target cell populations. Potential immune-sensitizing properties of iosimenol will be compared with those of iodixanol. Streptozotocin (STZ), a known immune-reactive substance, will serve as a positive control. Vehicle injection into the contralateral hind leg will serve as negative controls in all experiments.


   Methods and materials
Top
 Abstract
 Introduction
 Methods and materials
Results
 Discussion
 Conclusions
 References
 
Mice
BALB/c mice were purchased from Harlan Olac (Bicester, UK), bred in the institutional animal facility, and used at 8–12 weeks of age. All studies involving animals were carried out in accordance with, and after approval by, the institutional Animal Research Ethics Committee (#AE/05/54).

Chemicals and reagents
The two contrast media used in this study, iosimenol and iodixanol, were obtained from Köhler Chemie (Alsbach-Hahnlein, Germany) and Amersham Buchler (Braunschweig, Germany), respectively. Both CM were supplied as sterile aqueous solutions. The stock solutions of iosimenol and iodixanol were at concentrations of 660 mg ml–1 and 652 mg ml–1, respectively, with iosimenol containing 340 mg ml–1 iodine and iodixanol 320 mg ml–1 iodine. Streptozotocin (Sigma Chemical Company, St Louis, MO) was used as a positive control in the PLNA, as published by Tuschl et al [35]. The molarities of the three tested substances were equivalent to 447 mM for iosimenol, 420 mM for iodixanol and 75 mM for streptozotocin.

Monoclonal antibodies
Monoclonal antibodies to the following cell surface antigens (all obtained from eBioscience, San Diego, CA) were used in the study: CD3, CD4, CD8, CD45/B220, T cell receptor Cbeta chain, F4/80, Ly-6A/E (Sca-1) and MHC class II I-Ad. All antibodies were pretitrated and used at saturating concentrations.

PLNA
Groups of mice (n = 9 for iosimenol and iodixanol groups; n = 6 for STZ group) were given a single s.c. injection of 50 µl of either iosimenol or iodixanol into the right hind footpad. As a negative control, an equal volume of normal saline was injected into the left hind footpad of each mouse. A third group of mice received a single s.c. injection of STZ (50 µl of 20 mg ml–1 stock solution) or vehicle (citrate buffer, negative control) into the right and left hind footpad, respectively. On day 5 after injection, the mice were killed; the draining popliteal lymph nodes (PLNs) were removed aseptically, teased gently to obtain a single cell suspension and counted. The right-side (experimental) and left-side (control) PLNs were processed and analysed separately. Three separate experiments were carried out for the iosimenol- and iodixanol-treated mice, and two separate experiments for the STZ groups.

Flow cytometric analysis
In order to obtain sufficient cells for flow cytometric analysis, PLN cells were pooled into two groups for each treatment option. Processing of PLN cells for flow cytometric analysis was carried out as described previously [36]. Briefly, cells were resuspended in staining buffer (phosphate-buffered saline (PBS)/1% fetal calf serum (FCS)/0.1%NaN3) dispensed into wells of a round-bottom 96-well plate (1x106/well) and incubated with anti-CD16/CD32-specific mAb (clone 2.4G2) for 30 min on ice to block Fc{gamma} receptor sites. Cells were then stained with conjugated monoclonal antibodies for 30 min on ice, washed and analysed on a FACScan (Beckton Dickinson, Mountain View, CA). Data collected on 30 000 cells were analysed using CELLQUEST software.

Statistical analysis
Statistical significance was analysed by the unpaired Student's t-test using the statistical program of Graph Pad Prism software (San Diego, CA). Differences between experimental groups were considered significant when p-values were ≤0.05.


   Results
Top
 Abstract
 Introduction
 Methods and materials
 Results
Discussion
 Conclusions
 References
 
Total cellularity of PLN
To assess the potential immunoreactivity of iosimenol, the first step was comparing the effect of administration of a single dose of iosimenol with the currently used substance, iodixanol, on the total cellularity of PLNs. There was essentially no change in total cellularity of PLNs of mice treated with each contrast medium, iosimenol or iodixanol, in comparison with vehicle-injected mice. In iosimenol-treated mice, the average number of cells (±SEM) was 0.89±0.09x106, which represented no significant change from vehicle-injected PLNs (0.69±0.05x106) (mean of five individual determinations each from three separate experiments). Similarly, in iodixanol-treated mice, the means were 0.78±0.07x106 and 0.75±0.07x106 for iodixanol and vehicle-injected PLNs, respectively (mean of five individual determinations each from three separate experiments). In contrast, STZ-treated mice showed a statistically significant increase in total PLN cellularity, from a mean of 0.93±0.19x106 in the vehicle-injected group to 2.92±0.49x106 in the STZ-treated group (mean of triplicate determinations each from two separate experiments; p = 0.02)

Flow cytometric analyses
To identify potential changes in cellular phenotype in PLNs, flow cytometric analyses were undertaken. These experiments revealed that after s.c. injection of either the new contrast material iosimenol or iodixanol no effect on PLN cell composition (Figure 1a,bGo) could be observed compared with the effect of s.c. vehicle injection (negative control). In contrast, treatment with STZ resulted in a dramatic set of changes in cellular ratios (Figure 1cGo), characterized by a substantial increase in B cells (p = 0.013), and a significant decrease in total T cells (p = 0.033). The lower ratio of T cells in STZ-treated PLNs was mainly due to a preferential and significant loss of CD4+ T lymphocytes (p = 0.001). In contrast, the ratio of CD8+ T cells increased in STZ-treated mice significantly (Figure 1cGo; p = 0.015). PLN cells were also stained with a monoclonal antibody specific to macrophages. However, in all groups tested the proportion of macrophages in PLNs was extremely small (Figure 1a–cGo), indicating an absence of an overt splenic inflammatory response.


Figure 1
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  		Figure 1. Flow cytometric analysis of PLN cells following treatment. Mice were given a single s.c. injection of 50µl of iosimenol (a), iodixanol (b), or STZ (c) in the right hind footpad. As a control, an equal volume of saline or citrate buffer was injected into the left hind footpad. On day 5 post injection, PLNs were collected, pooled and processed for flow cytometric analysis. Cells were double-stained with monoclonal antibodies specific to the following proteins: CD45/B220 (total B cells), CD3 and TcR Cbeta chain (total T cells), CD4 (T helper cells), CD8 (T cytotoxic cells) and F4/80 (macrophages). Each datum point represents the mean±SEM of five determinations from three separate experiments (iosimenol and iodixanol groups) or three determinations from two individual experiments (STZ group). Asterisks denote statistically significant differences from respective cell types in control-treated PLNs (*p<0.05, **p<0.01). Data from a total of 30 000 cells per group were collected and analysed by CELLQUEST.

 
In addition to changes in cell number and phenotype, treatment with STZ, but not with iodixanol or iosimenol, resulted in a significant increase in the number of large proliferating lymphocytes (blasts). The ratio of blast cells within the PLNs of STZ-injected mice was 31.8%, which contrasted sharply with the ratios in control mice (5.5%), in iosimenol (3.6%), or iodixanol (5.3%) treated mice (data not shown).

Expression of Sca-1 antigen
Activation of immune system cells is often associated with upregulation of certain proteins on the cell surface. Sca-1, or Ly6A/E, is a protein expressed on all haematopoietic stem cells and is readily induced in diverse types of lymphoid and myeloid lineage cells by pro-inflammatory stimuli [37–39]. Therefore, we monitored the level of expression of Sca-1 antigen as a measure of cellular activation in PLN lymphocytes. As summarized in Figures 2a,bGo, only treatment with STZ resulted in a substantial increase in Sca-1 expression. The induced upregulation of Sca-1 surface expression was seen in both the B and T lymphocyte populations, and was highly significant in comparison with PLN cells of control mice (p<0.0001). However, treatment with either iosimenol or iodixanol resulted in no significant increase in Sca-1 expression in either of the two lymphocyte subpopulations. An example of the staining profiles seen in the four treatment groups is shown in Figure 2bGo. The shown data are representative from one of two independent experiments.


Figure 2
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  		Figure 2. Upregulation of cellular activation markers following treatment. The extent of activation of B and T lymphocytes was monitored by the level of expression of Ly6A/E (Sca-1) protein on the cell surface. (a) Pooled PLN cells were double-stained with a pair of direct conjugated monoclonal antibodies to B220 and Sca-1 (to identify activated B cells) or TcR Cbeta chain and Sca-1 (to identify activated T cells). The per cent Sca-1 positive B and T lymphocytes is shown in the bar graph. Each datum point represents the mean±SEM of three groups of pooled PLN cells from nine mice (iosimenol and iodixanol groups) or six mice (STZ group). Asterisks denote statistically significant differences from control (***p<0.001). (b) Representative dot blot profiles of PLN cells from the various treatment groups after staining with antibodies to B220 and Sca-1 antigens. Panels I to IV represent the staining profiles of PLN cells from control, STZ, iosimenol or iodixanol treated groups, respectively. The number shown in each of the quadrants denotes the percentage of cells in that quadrant. The dot blots represent analysis of one pooled PLN cells from each treatment group. Data from a total of 30 000 cells per group were collected and analysed by CELLQUEST. The data are representative of three (iosimenol and iodixanol groups) or two (STZ group) independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
Conclusions
 References
 
The new dimeric contrast material iosimenol for intravascular administration has not yet been tested with regard to its immune-sensitizing effects. As the substance is less viscous than iodixanol, faster injection is feasible. Moreover, benefits for its use in patients with atherosclerosis-induced changes in myocardial perfusion are predictable, as the decrease in the erythrocyte velocity in the peripheral capillaries [24, 40] should be less pronounced.

Compared with other preclinical methods, PLNA has been proven to be a reliable, fast and reproducible assay for testing immune reactions caused by various drugs [25–28, 31, 32].

The direct PLNA has been described as "a tool to predict the risk of drug induced systemic auto-immune like reactions" [31, 32]. Positive immunosensitivity studies in mice or rats using PLNA have been shown to cause similar effects in humans [32, 41].

So far, to our knowledge no studies assessing the cellularity and surface markers of PLNs have been carried out in non-ionic dimeric contrast materials. Preliminary tests were conducted by Abramjuk, Speck and coworkers using the direct PLNA in rats. In those experiments the weight indices (WIs) of the individual PLNs were obtained as originally described [25, 26, 32]. No increase in the WI of PLNs of rats exposed to iosimenol compared with saline treatment was observed (C Abramjuk and U Speck, personal communication, 2006).

Beyond this pilot study we could demonstrate for the first time that there is no increase in the cellularity of B cells or T cells in the PLNs after treatment with either iosimenol or iodixanol.

Our phenotyping data, counting of B cells, (total) CD3 T cells, the subpopulations of CD4– and CD8 T cells, and the development of blast cells, did not reveal any significant changes in PLNs of mice treated with either CM compared with negative controls. Similarly, there was no increase in the surface expression of the Sca-1 activation marker on B and T lymphocytes. Taken together, and based on the PLNA, our data suggest that the new contrast medium iosimenol is immunologically inert. In contrast, injection of STZ resulted in a significant increase in PLN cellularity and selective CD4 T cell depletion, which is in accordance with previously published data [35]. Moreover, STZ administration led to a dramatic upregulation in Sca-1 expression on both B and T lymphocytes, which is indicative of the pro-inflammatory milieu induced by STZ in these mice [35, 38].

In 2003 a multicentre study, published by Aspelin et al [8], demonstrated that in particular intraarterial administration of dimeric non-ionic CM is advantageous over monomeric non-ionic CM in high-risk diabetic patients. A recent study, conducted by Huber et al [12], laid emphasis on the high incidence of renal impairment in elderly intensive care patients with additional hypertension, multiple myeloma or pre-existing proteinuria after i.a. CM injection of doses of >100 ml. Therefore, an iso-osmolar non-ionic dimeric CM may be desirable for that small subgroup of high-risk patients.

So far only a few articles have been published on the late side effects of non-ionic dimeric CM for intravascular application [14, 20, 23, 42]. The publication by Boehm et al [20] revealed slight changes in cytokine release in humans without clinically visible side-effects in a small trial of nine patients after i.v. CM injection in CT. It was concluded that late allergy-like adverse reactions might be induced by cytokine liberation [20]. On the other hand, late reactions to CM have been discussed as probable T-cell-mediated allergic reactions [42]. The prospective randomized clinical trial, published by Schild et al in 2006 [14], investigated three different groups of patients with i.v. CM administration in CT and unenhanced CT. Except for cutaneous symptoms the results did not show significant differences in the incidence of late adverse reactions after non-ionic monomeric and dimeric CM; however, a significant lower incidence of acute adverse effects was recorded for non-ionic dimeric CM. These results also emphasize potential benefits of non-ionic dimeric CM for high-risk patients.

In summary, the data of our experiments did not show any cellular response or activation of surface markers after either iosimenol or iodixanol injection, indicating no immunological effect of iosimenol.

Owing to iso-osmolarity and low viscosity, iosimenol appears to be a promising CM that may be used intravenously and intraarterially at higher concentrations and faster injection rates than iodixanol. Benefits may be also expected in individuals with atherosclerotic coronary disease and in patients with peripheral occlusive disease owing to a decrease in the abovementioned side effects [14, 17, 24]. Iosimenol is furthermore deemed advantageous for use in CT-angiography (CTA) because of fewer involuntary movements as a result of heat sensation [14, 17].

Limitations
All experiments were carried out as PLNA in mice. Although PLNA results are considered valid for human immune response, it remains unclear if our data are applicable for administration of iosimenol or iodixanol in humans.


   Conclusions
Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
 Conclusions
References
 
Based on the results of our experiments, we conclude that the new contrast agent iosimenol may well prove to be not immunogenic, similar to the approved and currently available CM iodixanol.

As a result of the iso-osmolarity and low viscosity of iosimenol, benefits for i.v. and i.a. use in high-risk patients may be expected if our results are applicable for humans.


   Acknowledgments
 
The authors would like to express their sincere appreciation to the Research Grant Committee at the Faculty of Medicine and Health Sciences, United Arab Emirates University, for financial support of this project under fund grant no. NP/05/05. The investigators wish also to thank A Ullah and S M Nurulain for excellent technical assistance and Prof. N Nagelkerke for advice on and review of statistical analyses.

Received for publication November 1, 2006. Revision received December 10, 2006. Accepted for publication December 12, 2006.


   References
Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
 Conclusions
 References
 

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