Pragmatic Considerations When Extracting DNA for Metagenomics Analyses of Clinical Samples.

Microbiome analyses are essential for understanding microorganism composition and diversity, but interpretation is often challenging due to biological and technical variables. DNA extraction is a critical step that can significantly bias results, particularly in samples containing a high abundance of challenging-to-lyse microorganisms. Taking into consideration the distinctive microenvironments observed in different bodily locations, our study sought to assess the extent of bias introduced by suboptimal bead-beating during DNA extraction across diverse clinical sample types. The question was whether complex targeted extraction methods are always necessary for reliable taxonomic abundance estimation through amplicon sequencing or if simpler alternatives are effective for some sample types. Hence, for four different clinical sample types (stool, cervical swab, skin swab, and hospital surface swab samples), we compared the results achieved from extracting targeted manual protocols routinely used in our research lab for each sample type with automated protocols specifically not designed for that purpose. Unsurprisingly, we found that for the stool samples, manual extraction protocols with vigorous bead-beating were necessary in order to avoid erroneous taxa proportions on all investigated taxonomic levels and, in particular, false under- or overrepresentation of important genera such as Blautia, Faecalibacterium, and Parabacteroides. However, interestingly, we found that the skin and cervical swab samples had similar results with all tested protocols. Our results suggest that the level of practical automation largely depends on the expected microenvironment, with skin and cervical swabs being much easier to process than stool samples. Prudent consideration is necessary when extending the conclusions of this study to applications beyond rough estimations of taxonomic abundance.

ESKAPEE Pathogen Biofilm Control on Surfaces with Probiotic Lactobacillaceae and Bacillus species.

Combatting the rapidly growing threat of antimicrobial resistance and reducing prevalence and transmission of ESKAPEE pathogens in healthcare settings requires innovative strategies, one of which is displacing these pathogens using beneficial microorganisms. Our review comprehensively examines the evidence of probiotic bacteria displacing ESKAPEE pathogens, with a focus on inanimate surfaces. A systematic search was conducted using the PubMed and Web of Science databases on 21 December 2021, and 143 studies were identified examining the effects of Lactobacillaceae and Bacillus spp. cells and products on the growth, colonization, and survival of ESKAPEE pathogens. While the diversity of study methods limits evidence analysis, results presented by narrative synthesis demonstrate that several species have the potential as cells or their products or supernatants to displace nosocomial infection-causing organisms in a variety of in vitro and in vivo settings. Our review aims to aid the development of new promising approaches to control pathogen biofilms in medical settings by informing researchers and policymakers about the potential of probiotics to combat nosocomial infections. More targeted studies are needed to assess safety and efficacy of different probiotic formulations, followed by large-scale studies to assess utility in infection control and medical practice.

Examining Different Analysis Protocols Targeting Hospital Sanitary Facility Microbiomes.

Indoor spaces exhibit microbial compositions that are distinctly dissimilar from one another and from outdoor spaces. Unique in this regard, and a topic that has only recently come into focus, is the microbiome of hospitals. While the benefits of knowing exactly which microorganisms propagate how and where in hospitals are undoubtedly beneficial for preventing hospital-acquired infections, there are, to date, no standardized procedures on how to best study the hospital microbiome. Our study aimed to investigate the microbiome of hospital sanitary facilities, outlining the extent to which hospital microbiome analyses differ according to sample-preparation protocol. For this purpose, fifty samples were collected from two separate hospitals-from three wards and one hospital laboratory-using two different storage media from which DNA was extracted using two different extraction kits and sequenced with two different primer pairs (V1-V2 and V3-V4). There were no observable differences between the sample-preservation media, small differences in detected taxa between the DNA extraction kits (mainly concerning Propionibacteriaceae), and large differences in detected taxa between the two primer pairs V1-V2 and V3-V4. This analysis also showed that microbial occurrences and compositions can vary greatly from toilets to sinks to showers and across wards and hospitals. In surgical wards, patient toilets appeared to be characterized by lower species richness and diversity than staff toilets. Which sampling sites are the best for which assessments should be analyzed in more depth. The fact that the sample processing methods we investigated (apart from the choice of primers) seem to have changed the results only slightly suggests that comparing hospital microbiome studies is a realistic option. The observed differences in species richness and diversity between patient and staff toilets should be further investigated, as these, if confirmed, could be a result of excreted antimicrobials.

Sinking Our Teeth in Getting Dental Stem Cells to Clinics for Bone Regeneration.

Dental stem cells have been isolated from the medical waste of various dental tissues. They have been characterized by numerous markers, which are evaluated herein and differentiated into multiple cell types. They can also be used to generate cell lines and iPSCs for long-term in vitro research. Methods for utilizing these stem cells including cellular systems such as organoids or cell sheets, cell-free systems such as exosomes, and scaffold-based approaches with and without drug release concepts are reported in this review and presented with new pictures for clarification. These in vitro applications can be deployed in disease modeling and subsequent pharmaceutical research and also pave the way for tissue regeneration. The main focus herein is on the potential of dental stem cells for hard tissue regeneration, especially bone, by evaluating their potential for osteogenesis and angiogenesis, and the regulation of these two processes by growth factors and environmental stimulators. Current in vitro and in vivo publications show numerous benefits of using dental stem cells for research purposes and hard tissue regeneration. However, only a few clinical trials currently exist. The goal of this review is to pinpoint this imbalance and encourage scientists to pick up this research and proceed one step further to translation.

Histology and Gadolinium Distribution in the Rodent Brain After the Administration of Cumulative High Doses of Linear and Macrocyclic Gadolinium-Based Contrast Agents.

OBJECTIVES: Retrospective studies in patients with primary brain tumors or other central nervous system pathologies as well as postmortem studies have suggested that gadolinium (Gd) deposition occurs in the dentate nucleus (DN) and globus pallidus (GP) after multiple administrations of primarily linear Gd-based contrast agents (GBCAs). However, this deposition has not been associated with any adverse effects or histopathological alterations. The aim of this preclinical study was to systematically examine differences between linear and macrocyclic GBCAs in their potential to induce changes in brain and skin histology including Gd distribution in high spatial resolution. MATERIALS AND METHODS: Fifty male Wistar-Han rats were randomly allocated into control (saline, n = 10 rats) and 4 GBCA groups (linear GBCAs: gadodiamide and gadopentetate dimeglumine, macrocyclic GBCAs: gadobutrol and gadoteridol; n = 10 rats per group). The animals received 20 daily intravenous injections at a dose of 2.5 mmol Gd/kg body weight. Eight weeks after the last GBCA administration, the animals were killed, and the brain and skin samples were histopathologically assessed (hematoxylin and eosin; cresyl violet [Nissl]) and by immunohistochemistry. The Gd concentration in the skin, bone, brain, and skeletal muscle samples were analyzed using inductively coupled plasma mass spectroscopy (ICP-MS, n = 4). The spatial Gd distribution in the brain and skin samples was analyzed in cryosections using laser ablation coupled with ICP-MS (LA-ICP-MS, n = 3). For the ultra-high resolution of Gd distribution, brain sections of rats injected with gadodiamide or saline (n = 1) were assessed by scanning electron microscopy coupled to energy dispersive x-ray spectroscopy and transmission electron microscopy, respectively. RESULTS: No histological changes were observed in the brain. In contrast, 4 of 10 animals in the gadodiamide group but none of the animals in other groups showed macroscopic and histological nephrogenic systemic fibrosis-like skin lesions. The Gd concentrations observed in the skin/brain samples (in nanomole Gd per gram of tissue) for each agent were as follows: gadodiamide: 1472 ± 115/11.1 ± 5.1, gadopentetate dimeglumine: 80.8 ± 6.2/13.1 ± 7.3, gadobutrol: 1.1 ± 0.5/0.7 ± 0.4, and gadoteridol: 1.7 ± 0.8/0.5 ± 0.2. The average detected residual Gd concentration in the brain was approximately 15-fold higher for linear than for macrocyclic GBCAs. The highest amounts of Gd found in brain corresponded to less than 0.0002% of the injected dose per gram of tissue. Using LA-ICP-MS, high Gd concentrations in the deep cerebellar nuclei and in the granular layer of the cerebellar cortex were detected only for linear gadodiamide and gadopentetate dimeglumine but not for gadoteridol or gadobutrol. The energy dispersive x-ray spectroscopy analysis revealed Gd-containing spots in the skin of animals administered gadodiamide and gadopentetate dimeglumine. Transmission electron microscopy revealed several Gd-containing spots in the region of the dentate nuclei in the brain of 1 animal injected with gadodiamide. CONCLUSIONS: After repeated high dosing, nephrogenic systemic fibrosis-like macroscopic and histopathological lesions of the skin were observed only in some of the gadodiamide-treated animals. No histopathological findings were detected in the rodent brain. The administration of linear GBCAs was associated with significantly higher Gd concentrations in the brain and skin compared with macrocyclic GBCA administration. The results of LA-ICP-MS demonstrated local accumulation of Gd within the deep cerebellar nuclei and the granular layer only after the administration of linear agents. In summary, the detected low Gd concentrations in the skin and brain were well correlated with the higher kinetic stability of macrocyclic GBCA.

Contrast Media for X-ray and Magnetic Resonance Imaging: Development, Current Status and Future Perspectives.

Over the last 120 years, the extensive advances in medical imaging allowed enhanced diagnosis and therapy of many diseases and thereby improved the quality of life of many patient generations. From the beginning, all technical solutions and imaging procedures were combined with dedicated pharmaceutical developments of contrast media, to further enhance the visualization of morphology and physiology. This symbiosis of imaging hardware and contrast media development was of high importance for the development of modern clinical radiology. Today, all available clinically approved contrast media fulfill the highest requirements for clinical safety and efficacy. All new concepts to increase the efficacy of contrast media have also to consider the high clinical safety standards and cost of goods of current marketed contrast media. Nevertheless, diagnostic imaging will contribute significantly to the progresses in medicine, and new contrast media developments are mandatory to address the medical needs of the future.

Reactive oxygen species and the bacteriostatic and bactericidal effects of isoconazole nitrate.

Bacterial superinfections often occur in dermatomycoses, resulting in greatly inflamed or eczematous skin. The objective of this study was to evaluate the antibacterial efficacy of isoconazole nitrate (ISN), a broad-spectrum antimicrobial imidazole, commonly used to treat dermatomycoses. Several gram-positive bacteria minimal inhibitory concentrations (MICs) for ISN (ISN solution or ISN-containing creams: Travogen or corticosteroid-containing Travocort) and ampicillin were obtained using the broth-dilution method. Speed of onset of the bactericidal effect was determined with bacterial killing curves. Reactive oxygen species (ROS) were visualised by staining cells with singlet oxygen detector stain. Compared with ampicillin MICs, ISN MICs for Bacillus cereus, Staphylococcus haemolyticus and Staphylococcus hominis were lower and ISN MICs for Corynebacterium tuberculostearicum and Streptococcus salivarius were similar. Incubation with ISN led to a 50% kill rate for Staphylococcus aureus and methicillin-resistant strains (MRSA). Post-ISN incubation, 36% (30 min) and 90% (60 min) of S. aureus cells were positive for ROS. Isoconazole nitrate has a broad bacteriostatic and bactericidal action, also against a MRSA strain that was not reduced by the corticosteroid in the Travocort cream. Data suggest that the antibacterial effect of ISN may be ROS dependent. An antifungal agent with robust antibacterial activity can provide a therapeutic advantage in treating dermatomycoses with suspected bacterial superinfections.

The osmolality of nonionic, iodinated contrast agents as an important factor for renal safety.

OBJECTIVE: Nonionic iodinated contrast agents (CAs) can be divided into monomeric, low-osmolar, and dimeric, iso-osmolar classes. In clinical practice, renal tolerance of CAs is a concern, especially in patients with impaired renal function. With regard to renal safety, we wanted to evaluate the role of osmolality and viscosity in renal tolerance. MATERIAL AND METHODS: We generated a formulation (iodixanol/mannitol) consisting of the dimeric iodixanol with an osmolality of the monomeric iopromide. Male Han-Wistar rats were intravenously injected with low-osmolar iopromide 300, iso-osmolar iodixanol 320, and iodixanol/mannitol. Saline and diatrizoate were used as controls. A total number of 227 rats were used in the following experiments. We compared the impact of osmolality on renal iodine retention using computed tomography 2 and 24 hours postinjection (p.i.). The animals were killed 2, 24, and 72 hours after injection, and the kidneys were excised for further investigations. Changes in renal cell proliferation were analyzed by 5-bromo-2'-deoxyuridine incorporation 48 hours p.i. as a degree of tissue regeneration after induced injury. To specify potential renal injury, we quantified the expression of acute kidney injury (AKI) markers (kidney injury marker-1 [KIM-1], neutrophil gelatinase-associated lipocalin [NGAL], and plasminogen activator inhibitor-1 [PAI-1]) by quantitative real-time polymerase chain reaction. Furthermore, the kidneys were analyzed histologically, including immunofluorescence analysis. RESULTS: After intravenous application of the CAs into Han-Wistar rats, renal iodine concentration was increased (3-fold) for iodixanol 2 hours p.i. and iodine retention was detected to be prolonged 24 hours p.i. compared with iopromide injection (iodixanol, 520 ± 50 Hounsfield Units [HU] vs iopromide, 42 ± 5 HU). The higher iodine concentration 2 hours p.i. upon iodixanol injection was reduced almost to the level of iopromide when injecting iodixanol/mannitol (iopromide: 289 ± 68 HU vs iodixanol/mannitol: 343 ± 68 HU). In addition, iodixanol application induced increased renal cell proliferation (2.7-fold vs saline), indicating renal injury, which was significantly lower in iopromide-treated animals (1.6-fold vs saline). More detailed analysis of markers for AKI revealed that iodixanol significantly induced the expression of PAI-1 (7.7-fold at 2 hours) as well as KIM-1 (2.1-fold) and NGAL (3.2-fold) at 2 and 24 hours when compared with saline treatment. In contrast, the expression of markers for AKI was low after iopromide (1.4-fold NGAL, 1.7-fold PAI-1, KIM-1 not significant) and iodixanol/mannitol (1.6-fold NGAL, 2.6-fold PAI-1, KIM-1 not significant) injection. CONCLUSION: The present results clearly show that prolonged iodine retention and the enhanced expression of kidney injury markers are caused mainly by the explicitly higher urine viscosity induced by iodixanol. We conclude that the osmolality of low-osmolar CAs such as iopromide induces a positive diuretic effect that is responsible for rapid iodine clearance and prevents increased expression of acute injury markers in the kidney.

Changes of renal water diffusion coefficient after application of iodinated contrast agents: effect of viscosity.

OBJECTIVE: X-ray contrast agents (CA) possess specific physicochemical properties and are excreted renally by glomerular filtration. Thereby, they may affect the diffusion of water molecules within the kidney. The aim of our preclinical study was to investigate potential changes in the apparent diffusion coefficient (ADC) of the kidney after administration of monomeric, low-osmolar, and dimeric, iso-osmolar CA by using diffusion-weighted magnetic resonance imaging (DWI). MATERIAL AND METHODS: First, the relationship between CA viscosity and the ADC of water was assessed by phantom measurements. Subsequently, Han Wistar rats (8 per group) received an intravenous injection of iso-osmolar CA (iodixanol) or low-osmolar CA (iopromide) at a dosage of 4 gI/kg body weight. The control group received saline (0.9% NaCl) at the same volume. The renal ADC was dynamically monitored up to 40 minutes postinjection (p.i.) by DWI using a 1.5-T clinical MR unit. After DWI, the animals were killed and the kidneys were removed for iodine measurements by x-ray fluorescence analysis. RESULTS: The in vitro measurements yielded an inverse relationship between increasing viscosity and decreasing water diffusion. In vivo, a slight increase in ADC was observed immediately after administration of the low-osmolar iopromide (ΔADC=80±78 µm²/s) and saline (ΔADC=89±53 µm²/s), which normalized to the baseline level at 40 minutes p.i. In contrast, a strong decrease in ADC was observed after administration of the iso-osmolar iodixanol. This was most prominent 12 minutes p.i. (ΔADC=-555±194 µm²/s) and persisted throughout the investigation. Concomitantly, the kidney iodine concentration 50 minutes p.i. was significantly higher after iodixanol (58.6±5.3 mgI/g kidney) compared with iopromide injection (18.4±4.5 mgI/g kidney). CONCLUSION: A significant difference in the renal ADC was observed between the low-osmolar CA/saline and the iso-osmolar CA. The in vitro measurements suggest that the substantial decrease in ADC observed after administration of the iso-osmolar CA is based on the high viscosity of the agent during renal passage. This, in turn, may explain the delayed iodine retention after administration of iso-osmolar CA and demonstrates the importance of the physicochemical properties of CA during their renal elimination.

Viscosity of iodinated contrast agents during renal excretion.

OBJECTIVE: Modern iodinated non-ionic contrast agents (CAs) can be classified based on their molecular structure into monomeric and dimeric CAs and have at comparable iodine concentrations a different viscosity and osmolality. During their renal excretion, CAs are concentrated in the renal tubuli which might enhance the viscosity difference between monomeric and dimeric CAs. The viscosity of a CA might have an underestimated importance for renal safety, as suggested by recent publications. In this study, we investigated the viscosities of CAs at the concentrations expected to be present in renal tubules. This concentration process was simulated in vitro using dialysis. Furthermore, we investigated urine viscosity and urine flow in rodents after administration of several non-ionic monomeric and dimeric CAs. MATERIALS AND METHODS: To estimate the viscosity of the CAs in vivo, we performed an in vitro dialysis of monomeric and dimeric CAs at various physiological osmolalities of the renal tubulus (290, 400, 500, 700 and 1000 mOsm/kg H2O). Following the dialysis, the iodine concentrations and the viscosities of the CAs were determined. Furthermore, to investigate the concentration process in vivo, we measured the urine viscosity and the urine flow in Han Wister rats after the administration of Iopromide, Iohexol, Ioversol, Iomeprol, Iodixanol, and Iosimenol at comparable iodine concentrations. As a control, saline was injected at the same volume. RESULTS: In vitro dialysis of the dimeric CA increased the iodine concentration and strongly increased the viscosity at all tested osmolalities. In contrast, for the monomeric agents an increase in concentration and viscosity was observed only at 700 as well 1000 mOsm/kg H2O but to a lesser extent. In summary, dialysis strongly enhanced the viscosity differences between the non-ionic monomeric and dimeric CAs. The administration of dimeric CAs leads to a strong increase in urine viscosity; this was not observed for the monomeric CAs. In contrast, a significantly higher urine flow was measured after the administration of the monomeric CAs as compared to the dimeric CAs. CONCLUSION: We demonstrated that the viscosity differences between monomeric and dimeric CAs are strongly enhanced due to a concentration process of the CAs upon increasing osmolalities, a process which is likely to take place in a similar manner in the tubular system. This result suggests that the viscosity of the dimeric agents increases dramatically in vivo and gives a plausible explanation for measured enhancement of urine viscosity upon dimeric CA administration. On the other hand, the higher osmolality of the monomeric agents causes an osmodiuresis, indicated by a higher urine flow, which leads to a faster elimination of the CAs from the kidney.

The role of residual gadolinium in the induction of nephrogenic systemic fibrosis-like skin lesions in rats.

OBJECTIVE: Nephrogenic systemic fibrosis (NSF) is an acquired, idiopathic disorder. Most of the cases are observed in patients with end stage renal disease (ESRD). The objective of this nonclinical animal study was to test the hypothesis that gadolinium (Gd) deposits play a role in the induction of NSF lesions. In addition, we evaluated whether an acute response to Gd exposure can initiate a process that results in fibrosis of the skin. MATERIALS AND METHODS: Han-Wistar rats were administered 3 intravenous injections of Gd-DTPA-BMA formulated without Gd-free excess ligand (Gadodiamide without Caldiamide) at a dose of 2.5 mmol/kg of body weight (b.w.) per injection given at 24-hour or 14-, 28-, or 56-day intervals. The occurrence and development of NSF-like fibrosing dermopathy lesions were followed. The Gd concentration was determined by Inductively Coupled Plasma Mass Spectrometry in skin biopsies taken during the study and organ samples taken at the end of the study.In a separate study, after injection of a single intravenous dose of 2.5 mmol/kg b.w. Gd-DTPA-BMA administered to Han-Wistar rats, the expression of cytokines and signaling molecules in serum and skin tissue was determined by quantitative RT-PCR and Luminex technology 6 hours or 14, 28, or 56 days. RESULTS: The occurrence of NSF-like macroscopic skin lesions differed between the injection groups. Shorter injection intervals resulted in more severe skin reactions. In contrast, the injection interval did not influence the long-term presence and level of accumulation of Gd concentration in tissue. The single injection of Gd-DTPA-BMA was followed by a rapid and transient induction of signaling molecules in the serum (MCP1, MCP3, IL1, IP-10, Osteopontine SCF and Timp1) as well as in the skin (MCP1 and TGFb). CONCLUSION: The presence of NSF-like fibrosing dermopathy in rats was found to be dependent on the injection interval and not on the amount of Gd in tissue. Our findings suggest the possibility of a more acute intrinsic reaction on administration of Gd-DTPA-BMA that triggers events leading to the development of skin lesions. The finding that single injections of Gd-DTPA-BMA were accompanied by a fast and transient induction of signaling molecules that are known to be involved in several fibrotic events provides additional support for this hypothesis. The study findings, however, do not support the theory that the long-term presence of Gd plays a relevant role in the development of NSF.

Efficacy and safety of lanthanoids as X-ray contrast agents.

OBJECTIVE: It has been suggested that elements from the lanthanoid (Ln) series may be well suited for use as absorbing elements in X-ray contrast agents (CA). Because gadolinium, an element of the lanthanoid series, has been identified as being possibly associated with nephrogenic systemic fibrosis (NSF), a rare but potentially severe disease, we sought to determine if other lanthanoids might possess a similar potential. MATERIALS AND METHODS: By computed tomography (CT), we compared the X-ray attenuation of all lanthanoids to that of iodine in vitro. In addition, we injected Han-Wistar rats on five consecutive days with 2.5 mmol Ln/kg bodyweight intravenously to test several Ln-DTPA-BMA complexes (praseodymium, europium, gadolinium, and holmium). Saline solution and a Ca-DTPA-BMA group served as controls. Ln concentrations in the skin and organs were determined by inductively coupled plasma mass spectrometry (ICP-MS). This method measures the total Ln content and cannot differentiate between chelated and unchelated Ln. In addition, serum cytokine levels were measured by Luminex technology. The complex stability of the Ln-DTPA-BMA complexes was also assessed in vitro. RESULTS: Lanthanoids showed up to 50% higher X-ray attenuation than iodine in CT. The highest X-ray attenuation was observed with holmium and erbium. Differences in the in vitro complex stability of Pr-, Eu-, Gd-, and Ho-DTPA-BMA complexes were observed. The complex stability differences were also reflected by differences in the concentrations in tissue of the lanthanoids in vivo. Injections of Ln complexes caused NSF-like skin lesions in rats and a rapid upregulation of pro-fibrotic and inflammatory serum cytokines. The Ca-DTPA-BMA complex did not to induce pro-fibrotic cytokines or skin lesions. Pr-DTPA-BMA appeared to be toxic; all Pr-DTPA-BMA treated animals died within the first four days of the experiment and were therefore excluded from further analyses. CONCLUSION: Lanthanoids are very well suited for higher X-ray tube voltages, particularly CT examinations. However, Ln-specific induction of NSF-like skin lesions and rapid elevation of pro-fibrotic serum cytokines levels were observed in rats following multiple administrations of high doses of Ln-DTPA-BMA complexes. The results of this animal study suggest that the stability of lanthanoid complexes may be an important consideration in evaluating the potential for in vivo safety. Furthermore the results suggest a potential of the entire class of lanthanoids to have the potential to trigger NSF-like skin lesions in rats rather than only some of the specific elements of this series.

A review of preclinical safety data for magnevist (gadopentetate dimeglumine) in the context of nephrogenic systemic fibrosis.

OBJECTIVES: Magnevist (gadopentetate dimeglumine, Bayer Healthcare, Bayer) is a gadolinium-based contrast agent (GBCA) for magnetic resonance imaging approved for clinical use in various indications since 1988. A possible association between the administration of GBCAs to patients with severe kidney impairment, and a condition first identified in 2000 and later described as "nephrogenic systemic fibrosis" (NSF), was published in early 2006. In the light of this reported association and the published histologic findings of certain NSF patients, Bayer, with support of external experts, reassessed the preclinical safety data from in vivo studies in healthy rats and dogs that were conducted with Magnevist during the drug development process in the mid-80s. These studies, which were performed according to standard regulatory requirements as defined in pertinent guidelines and which were conducted before the reported association between GBCAs and NSF, were not specifically designed to detect NSF-like lesions. Instead, the intention of this reassessment was to analyze whether the acquired knowledge on NSF would lead to a revised interpretation of the original preclinical data. MATERIALS AND METHODS: Studies on repeat-dose toxicity of Magnevist performed in the mid-80s with healthy rats and dogs were re-evaluated, with special emphasis on the retrospective analysis of morphologic findings which have come to be considered potentially suggestive of NSF. In particular, histologic slides of the skin of repeat-dose toxicity studies were re-examined by Bayer pathologists, with a special focus on the occurrence of morphologic lesions that have subsequently been identified as consistent with NSF. In addition, slides from selected studies were also subjected to a blinded external peer review by an independent international Pathology Working Group. RESULTS: A review of the preclinical data from the repeated-dose toxicity studies provided no evidence for toxicological effects after administration of Magnevist, which could be construed as suggestive of or consistent with NSF. More specifically, histopathology peer reviews of skin samples from repeated-dose toxicity studies with rats and dogs revealed no signs of skin lesions even after repeated high-dose administration to rats of 5.0 mmol Gd/kg of Magnevist (50 times the standard diagnostic dose). CONCLUSIONS: No findings were observed in any of the preclinical studies with Magnevist in healthy rats and dogs which could be characterized as similar to the types of morphologic lesions that have subsequently been identified as consistent with NSF. This preclinical assessment is in contrast to the reported clinical evidence of rare NSF cases in patients with severe kidney impairment after Magnevist administration. The differences between the preclinical models and their predictive limitations with regard to the clinical situation of renally impaired patients are discussed.

The impact of the viscosity and osmolality of iodine contrast agents on renal elimination.

OBJECTIVE: The iodinated contrast agents (CAs) that are currently used in radiographic procedures possess special physicochemical properties and a high safety profile; however, according to a large retrospective study (Swedish registry), the viscosity of CAs may have an underestimated impact on renal failure. The aim of our study was to investigate the possible consequences of CA viscosity differences, such as CA retention in the kidney. MATERIAL AND METHODS: Five Göttingen minipigs were each intravenously injected in a crossover setting at intervals of at least 7 days with monomeric (Iopromide) and dimeric (Iodixanol) CAs at 2 doses (1 and 2 g iodine/kg bodyweight), and the retention of the CA in the kidneys was determined during the first 6 hours postinjection using a 64-slice computed tomography scanner. Additionally we performed in vitro dialysis of the monomeric and dimeric CAs across the various physiological osmolalities of the renal tubulus (300, 600, 800, and 1200 mOsm/kg H(2)O) to estimate CA viscosity in vivo. Following the dialyzes, iodine concentrations and CA viscosities were determined. RESULTS: A different exposure of the kidneys to iodine and a different elimination kinetics from the kidneys was observed after the administration of monomeric and dimeric CAs. The monomeric agent was observed to clear from the kidney immediately after administration. In contrast, after administration of the dimeric CA an increase in iodine concentration in the kidney was observed up to 180 minutes postinjection, before the CA was observed to begin clearing; however, no difference was observed between the plasma half-lives of the 2 investigated CAs. In vitro dialysis of the dimeric CA increased iodine concentrations and strongly increased viscosity at all of the tested osmolalities. In contrast, the monomeric agent only demonstrated increases in iodine concentration and viscosity at 800 and 1200 mOsm/kg, and these changes were smaller than those observed for the dimeric CA. In summary, dialysis strongly enhanced the viscosity differences between the 2 investigated CAs. CONCLUSION: The viscosity differences between the investigated monomeric and dimeric CAs are strongly enhanced by concentration processes, such as the process taking place in the tubular system. These viscosity differences may be the cause of the prolonged retention and the different elimination kinetics from the kidney observed after application of the dimeric CA relative to the monomeric CA.

Intra-individual comparison of different contrast media concentrations (300 mg, 370 mg and 400 mg iodine) in MDCT.

OBJECTIVE: To compare intra-individual contrast enhancement in multi-detector-row computed tomography (MDCT) using contrast media (CM) containing 300, 370 and 400 mg iodine per ml (mgI/ml). METHODS: Six pigs underwent repeated chest MDCT using three different CM (iopromide 300, iopromide 370, iomeprol 400). An identical iodine delivery (IDR) rate of 1.5 gI/s and a constant total iodine dose of 300 mg/kg body weight were used. Dynamic CT were acquired at the level of the pulmonary artery, and the ascending and descending aorta. After the time enhancement curves were computed, the pulmonary and aortic peak enhancement, time to peak and plateau time above 300 HU were calculated. RESULTS: Intra-individual peak contrast enhancement was significantly higher for the 300 mgI/ml contrast medium compared with the 370 and 400 mgI/ml media: pulmonary trunk 595 HU vs 516 HU (p = 0.0093) vs 472 HU (p = 0.0005), and aorta 505 HU vs 454 HU (p = 0.0008) vs 439 HU (p = 0.0001), respectively. Comparison of time to peaks showed no significant difference. Plateau times were significantly longer for the 300 mgI/ml than for the 370 and 400 mgI/ml CM at all anatomical sites. CONCLUSION: Given normalised IDR and total iodine burden, the use of CM with a standard concentration with 300 mg iodine/ml provides improved contrast enhancement compared with highly concentrated CM in the chest.

Gadolinium-based contrast agents and NSF: evidence from animal experience.

Nephrogenic systemic fibrosis (NSF) is a potentially severe systemic disease typically characterized by fibrosis of the skin and connective tissues. The etiology of NSF is still unknown but is likely to be multifactorial. Specific triggers under scientific evaluation have included surgery and/or the occurrence of thrombosis or other vascular injury, proinflammatory state, the administration of high doses of erythropoietin, and more recently the use of gadolinium-based contrast agents (GBCAs). The aim of this review is to summarize knowledge regarding the pathogenesis of NSF and the potential role of GBCAs in its pathology, with a focus on animal experiments. The potential role of complex stability of GCBAs will be highlighted by results from several in vitro and in vivo experiments in rodent models of NSF. J. Magn. Reson. Imaging 2009;30:1268-1276. (c) 2009 Wiley-Liss, Inc.

Evaluating the role of zinc in the occurrence of fibrosis of the skin: a preclinical study.

PURPOSE: To investigate the possible role of Zn as a trigger for NSF we were using a previously established preclinical model. The depletion of endogenous Zinc ions (Zn) caused by the administration of gadolinium-based contrast agents (GBCAs) has been suggested as a possible pathomechanism for nephrogenic systemic fibrosis (NSF). MATERIALS AND METHODS: In the Zn supplementation study, rats were injected with Gadodiamide, Omniscan, and Magnevist with or without Zn supplementation. In the Zn depletion study, animals were kept on a Zn-deficient diet or a special control diet and received injections of Omniscan, OptiMARK, Magnevist, Gadovist, and Gd-EDTA. Gd, Zn, and Cu concentrations in tissue were measured and histology of the skin was performed. RESULTS: As seen in earlier studies, a difference in Gd concentration in the skin was observed following treatment with the different GBCAs. High Gd concentration in the skin correlated with the occurrence of NSF-like skin lesions. We observed no differences in the occurrence of skin lesions between the Zn supplementation and the Zn-deficient groups compared to their respective control groups. CONCLUSION: We found no significant effect of Zn on the initiation of NSF-like skin lesions. The results further support data from previous studies highlighting the importance of complex stability of the investigated GBCAs.

Impact of renal impairment on long-term retention of gadolinium in the rodent skin following the administration of gadolinium-based contrast agents.

OBJECTIVE: Several publications have suggested a possible association between Gd-based contrast agents (GBCAs) and the development of nephrogenic systemic fibrosis, a rare but serious disease. To date, nephrogenic systemic fibrosis has been observed only in patients with severe renal insufficiency.The aim of this study was to determine the impact of a prolonged circulation time of GBCAs caused by reduced renal clearance on the long-term retention of Gd in the skin of rats after administration of different GBCAs. MATERIAL AND METHODS: Renally impaired Han Wistar Rats (5/6-nephrectomized rats) were injected with Omniscan, OptiMARK, Magnevist, or Gadovist. The contrast agents were administered once daily for 5 consecutive days into the tail vein at a dose of 2.5 mmol Gd/kg b.w. Skin biopsies were taken at various time points, and the gadolinium (Gd) concentration was determined by inductive coupled plasma mass spectrometry (ICP-MS) over an observation period of 168 days post injection (p.i.). RESULTS: Differences in the skin Gd concentrations were observed between the 4 investigated GBCAs. For the nonionic linear compounds, Omniscan and OptiMARK, high Gd concentrations were maintained in the skin over the observation period of up to 168 days p.i. For the ionic linear compound, Magnevist, comparatively lower Gd retention in the skin was observed over time. For the macrocyclic compound, Gadovist, the Gd values in the skin were even lower, and significantly lower than Gd values in the skin in Omniscan and OptiMARK treated animals. CONCLUSION: The results of this preclinical study support the use of 5/6-nephrectomized rats as a model for prolonged circulation time of GBCAs as seen in patients with severe renal impairment. Surgically induced severe renal impairment resulted in delayed clearance of the administered GBCAs in the study animals. The highest amount of Gd was observed in the skin after treatment with the nonionic linear GBCAs, whereas the lowest Gd values were observed after treatment with the macrocyclic agent. This suggests that the difference in the Gd values observed in rat skin tissue after treatment with the different GBCAs is caused of a different propensity of the different GBCAs to release Gd in vivo. However, the analytical method used does not distinguish between chelated and unchelated Gd.

Long-term retention of gadolinium in the skin of rodents following the administration of gadolinium-based contrast agents.

Several publications suggest a potential association between the administration of Gadolinium-based contrast agents (GBCAs) and the onset of a rare but serious disease, Nephrogenic Systemic Fibrosis (NSF). The aim of this study was to determine the elimination time-course of Gadolinium (Gd) from skin tissue after application of GBCAs in rats. Seven different marketed GBCAs were injected on five consecutive days at a dose of 2.5 mmol/kg bodyweight into the tail vein of Han-Wistar rats and the Gd concentrations were determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) in skin biopsies taken at various time-points up to a year after the last injection. Most of the administered Gd was eliminated from the skin within a time-period of about 2 months. However, the repeated administration of linear GBCAs resulted in long-term retention of a small portion of the administered Gd in the skin tissue of rats, with substantially higher values observed in animals treated with non-ionic linear agents than in those that received ionic linear GBCAs. Following treatment with macrocyclic GBCAs, Gd values in the skin were in the same range as observed in the controls from day 24 post-injection onwards. In summary, we observed a correlation between the complex stability of GBCAs and the amount of residual Gd in the skin up to a year after application of GBCAs.