Retention of Gadolinium in Brain Parenchyma: Pathways for Speciation, Access, and Distribution. A Critical Review.

The unexpected appearance of T1 hyperintensities, mostly in the dentate nucleus and the globus pallidus, during nonenhanced MRI was reported in 2014. This effect is associated with prior repeated administrations of gadolinium (Gd)-based contrast agents (GBCAs) in patients with a functional blood-brain barrier (BBB). It is widely assumed that GBCAs do not cross the intact BBB, but the observation of these hypersignals raises questions regarding this assumption. This review critically discusses the mechanisms of Gd accumulation in the brain with regard to access pathways, Gd species, tissue distribution, and subcellular location. We propose the hypothesis that there is early access of Gd species to cerebrospinal fluid, followed by passive diffusion into the brain parenchyma close to the cerebral ventricles. When accessing areas rich in endogenous metals or phosphorus, the less kinetically stable GBCAs would dissociate, and Gd would bind to endogenous macromolecules, and/or precipitate within the brain tissue. It is also proposed that Gd species enter the brain parenchyma along penetrating cortical arteries in periarterial pial-glial basement membranes and leave the brain along intramural peri-arterial drainage (IPAD) pathways. Lastly, Gd/GBCAs may access the brain parenchyma directly from the blood through the BBB in the walls of capillaries. It is crucial to distinguish between the physiological distribution and drainage pathways for GBCAs and the possible dissociation of less thermodynamically/kinetically stable GBCAs that lead to long-term Gd deposition in the brain. LEVEL OF EVIDENCE: 5. TECHNICAL EFFICACY STAGE: 3.

Mistargeting of peroxisomal EHHADH and inherited renal Fanconi's syndrome.

BACKGROUND: In renal Fanconi's syndrome, dysfunction in proximal tubular cells leads to renal losses of water, electrolytes, and low-molecular-weight nutrients. For most types of isolated Fanconi's syndrome, the genetic cause and underlying defect remain unknown. METHODS: We clinically and genetically characterized members of a five-generation black family with isolated autosomal dominant Fanconi's syndrome. We performed genomewide linkage analysis, gene sequencing, biochemical and cell-biologic investigations of renal proximal tubular cells, studies in knockout mice, and functional evaluations of mitochondria. Urine was studied with the use of proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. RESULTS: We linked the phenotype of this family's Fanconi's syndrome to a single locus on chromosome 3q27, where a heterozygous missense mutation in EHHADH segregated with the disease. The p.E3K mutation created a new mitochondrial targeting motif in the N-terminal portion of EHHADH, an enzyme that is involved in peroxisomal oxidation of fatty acids and is expressed in the proximal tubule. Immunocytofluorescence studies showed mistargeting of the mutant EHHADH to mitochondria. Studies of proximal tubular cells revealed impaired mitochondrial oxidative phosphorylation and defects in the transport of fluids and a glucose analogue across the epithelium. (1)H-NMR spectroscopy showed elevated levels of mitochondrial metabolites in urine from affected family members. Ehhadh knockout mice showed no abnormalities in renal tubular cells, a finding that indicates a dominant negative nature of the mutation rather than haploinsufficiency. CONCLUSIONS: Mistargeting of peroxisomal EHHADH disrupts mitochondrial metabolism and leads to renal Fanconi's syndrome; this indicates a central role of mitochondria in proximal tubular function. The dominant negative effect of the mistargeted protein adds to the spectrum of monogenic mechanisms of Fanconi's syndrome. (Funded by the European Commission Seventh Framework Programme and others.).

Application of laboratory and digital techniques for visual enhancement during the ultrastructural assessment of cilia.

Routine diagnostic electron microscopy of primary ciliary dyskinesia (PCD) is based on the findings of ultrastructural defects of axonemal components. Assessment of the typical abnormalities can be enhanced by improving the sample preservation status using tannic acid (TA) as additive in the biopsy fixation or processing steps. Another option is the implementation of computer-assisted image analysis tools. Advancements in high-resolution 3D visualization of the axonemal structure have been noted, with great potential for the future diagnosis of inherited cilia disorders.

Lipid-labeling facilitates a novel magnetic isolation procedure to characterize pathogen-containing phagosomes.

Here we describe a novel approach for the isolation and biochemical characterization of pathogen-containing compartments from primary cells: We developed a lipid-based procedure to magnetically label the surface of bacteria and visualized the label by scanning and transmission electron microscopy (SEM, TEM). We performed infection experiments with magnetically labeled Mycobacterium avium, M. tuberculosis and Listeria monocytogenes and isolated magnetic bacteria-containing phagosomes using a strong magnetic field in a novel free-flow system. Magnetic labeling of M. tuberculosis did not affect the virulence characteristics of the bacteria during infection experiments addressing host cell activation, phagosome maturation delay and replication in macrophages in vitro. Biochemical analyses of the magnetic phagosome-containing fractions provided evidence of an enhanced presence of bacterial antigens and a differential distribution of proteins involved in the endocytic pathway over time as well as cytokine-dependent changes in the phagosomal protein composition. The newly developed method represents a useful approach to characterize and compare pathogen-containing compartments, in order to identify microbial and host cell targets for novel anti-infective strategies.

Recessive HYDIN mutations cause primary ciliary dyskinesia without randomization of left-right body asymmetry.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder characterized by defective cilia and flagella motility. Chronic destructive-airway disease is caused by abnormal respiratory-tract mucociliary clearance. Abnormal propulsion of sperm flagella contributes to male infertility. Genetic defects in most individuals affected by PCD cause randomization of left-right body asymmetry; approximately half show situs inversus or situs ambiguous. Almost 70 years after the hy3 mouse possessing Hydin mutations was described as a recessive hydrocephalus model, we report HYDIN mutations in PCD-affected persons without hydrocephalus. By homozygosity mapping, we identified a PCD-associated locus, chromosomal region 16q21-q23, which contains HYDIN. However, a nearly identical 360 kb paralogous segment (HYDIN2) in chromosomal region 1q21.1 complicated mutational analysis. In three affected German siblings linked to HYDIN, we identified homozygous c.3985G>T mutations that affect an evolutionary conserved splice acceptor site and that subsequently cause aberrantly spliced transcripts predicting premature protein termination in respiratory cells. Parallel whole-exome sequencing identified a homozygous nonsense HYDIN mutation, c.922A>T (p.Lys307(∗)), in six individuals from three Faroe Island PCD-affected families that all carried an 8.8 Mb shared haplotype across HYDIN, indicating an ancestral founder mutation in this isolated population. We demonstrate by electron microscopy tomography that, consistent with the effects of loss-of-function mutations, HYDIN mutant respiratory cilia lack the C2b projection of the central pair (CP) apparatus; similar findings were reported in Hydin-deficient Chlamydomonas and mice. High-speed videomicroscopy demonstrated markedly reduced beating amplitudes of respiratory cilia and stiff sperm flagella. Like the hy3 mouse model, all nine PCD-affected persons had normal body composition because nodal cilia function is apparently not dependent on the function of the CP apparatus.

Inadequate corticosterone levels relative to arthritic inflammation are accompanied by altered mitochondria/cholesterol breakdown in adrenal cortex: a steroid-inhibiting role of IL-1ß in rats.

OBJECTIVES: In rheumatoid arthritis, inadequate cortisol secretion was observed relative to inflammation, but reasons are unknown. Human adrenal glands cannot be investigated due to ethical reasons. Thus, a model of arthritis was studied to test inadequate glucocorticoid secretion and adrenocortical alterations. METHODS: Arthritis in DA rats was induced by collagen type II. Plasma hormone (cytokine) levels were determined by ELISA or radioimmunoassay (Luminex). Adrenocortical cells were investigated making use of in vitro culture, immunohistochemistry and imaging techniques, cholesterol uptake studies and electron microscopical morphological analyses of adrenocortical lipid droplets and mitochondria. RESULTS: During the course of arthritis, corticosterone and adrenocorticotropic hormone (ACTH) levels were only elevated on day 1 after immunisation but were in the normal range from day 5 to 55. Serum levels of corticosterone relative to IL-1ß were markedly lower in arthritis than in controls. IL-1ß inhibited ACTH-stimulated corticosterone secretion from adrenocortical cells in vitro. Cholesterol uptake receptor SR-BI protein was unchanged. Number of altered swollen and cavitated mitochondria increased during the course of arthritis (maximum on day 55), and this was correlated to reduced breakdown of lipid droplets and increased Sudan III-positive lipid accumulation from day 28 to 55. Reduced lipid breakdown measured as a high number of homogenous lipid droplets negatively correlated with plasma corticosterone (p=0.022). Adrenocortical tissue density of normal mitochondria positively correlated with serum corticosterone levels. CONCLUSIONS: This study on inadequate adrenal glucocorticoid secretion in arthritis demonstrated altered mitochondria and altered lipid breakdown paralleled by low corticosterone levels in relation to inflammation. IL-1ß is a key cytokine.

Endothelial dysfunction and altered mechanical and structural properties of resistance arteries in a murine model of graft-versus-host disease.

A putative involvement of the vasculature seems to play a critical role in the pathophysiology of graft-versus-host disease (GVHD). We aimed to characterize alterations of mesenteric resistance arteries in GVHD in a fully MHC-mismatched model of BALB/c mice conditioned with total body irradiation that underwent transplantation with bone marrow cells and splenocytes from syngeneic (BALB/c) or allogeneic (C57BL/6) donors. After 4 weeks, animals were sacrificed and mesenteric resistance arteries were studied in a pressurized myograph. The expression of endothelial (eNOS) and inducible nitric oxide (NO)-synthase (iNOS) was quantified and vessel wall ultrastructure was investigated with electron microscopy. The myograph study revealed an endothelial dysfunction in allogeneic-transplant recipients, whereas endothelium-independent vasodilation was similar to syngeneic-transplant recipients or untreated controls. The expression of eNOS was decreased and iNOS increased, possibly contributing to endothelial dysfunction. Additionally, arteries of allogeneic transplant recipients exhibited a geometry-independent increase in vessels strain. For both findings, electron microscopy provided a structural correlate by showing severe damage of the whole vessel wall in allogeneic-transplant recipient animals. Our study provides further data to prove, and is the first to characterize, functional and structural vascular alterations in the early course after allogeneic transplantation directly in an ex vivo setting and, therefore, strongly supports the hypothesis of a vascular form of GVHD.

Male infertility: assessment of juvenile testicular dysfunction and risk for malignancy in cryptorchid boys based on resin section evaluation.

Infertility is sometimes more a man's problem than a woman's, failure of one or both of the testes to descend (cryptorchidism) being the most frequent genital malformation in boys. Untreated, the undescended testis impairs germ cell development and significantly reduces adult fertility. A-dark spermatogonia are the stem cells for all future spermatozoa, and their depletion can be reliably estimated in resin semithin sections. Additionally, there is an increased risk of testicular preneoplasia in the form of carcinoma in situ (CIS) cells. The authors report how the pathologic biopsy examination of juvenile cryptorchid testes can assess infertility and malignancy risk.

Skin gadolinium following use of MR contrast agents in a rat model of nephrogenic systemic fibrosis.

PURPOSE: To detect the ultrastructural site of gadolinium retention in skin by using an animal model of nephrogenic systemic fibrosis and compare a linear, low-stability gadolinium chelate (formulated gadodiamide) with a macrocylic, high-stability gadolinium chelate (gadoterate meglumine). MATERIALS AND METHODS: Experimental procedures were performed according to rules and regulations laid down by the UK Home Office (Animal Procedures Act of 1986). Male Wistar rats were subjected to 5/6 subtotal nephrectomy (creatinine clearance, 25% normal). Gadolinium-based contrast agents, formulated gadodiamide (n = 9) and gadoterate meglumine (n = 11), were administered intravenously (2.5 mmol/kg for 5 days). After 28 days, skin was analyzed by means of morphometric and immunohistochemical techniques and electron microscopy. Data were compared with the Student t test. Skin gadolinium was located by means of energy-filtered transmission electron microscopy. RESULTS: Formulated gadodiamide produced a 40-fold greater increase in gadolinium in skin than did gadoterate meglumine. An electron-dense filamentous material, detected within extracellular matrix, displayed a "halo" appearance, associated with collagen fibrils and electron-dense intracellular fragments of collagen fibrils within activated fibroblasts. Both electron-dense features demonstrated the presence of gadolinium but were much less apparent following gadoterate meglumine administration, where the presence of gadolinium was not detected. Formulated gadodiamide increased dermal cell count, dermal thickness, and collagen bundle density with enhanced immunostain for CD34, fibroblast-specific protein 1,4-hydroxy-prolyl-hydroxylase, and factor XIIIa. Circular staining for α-smooth muscle actin indicated new blood vessel formation. Skin of rats receiving gadoterate meglumine remained unchanged. CONCLUSION: Gadolinium retention in skin following formulated gadodiamide administration was located to the collagen fibril, in both the extracellular matrix and within activated fibroblasts.

Absence of gadolinium deposits in the peritoneal membrane of patients with encapsulating peritoneal sclerosis.

UNLABELLED: Background. Encapsulating peritoneal sclerosis (EPS) is a severe complication of long-term peritoneal dialysis (PD) characterized by the development of an extensive fibrosis of the visceral peritoneum that may eventually lead to intestinal constriction. Its cause remains elusive. Nephrogenic systemic fibrosis (NSF), a disabling disease that can follow gadolinium-based contrast injection during magnetic resonance imaging, is characterized by systemic fibrosis of the skin, joints, liver, heart and vessels. Affected tissues are infiltrated by CD34+ and CD68+ fibroblasts. In the present study, we tested the hypothesis that EPS could have been triggered by a previous gadolinium injection. Methods. We performed histopathological analysis of the peritoneal membrane of two EPS and two control patients all exposed to long-term PD, including immunostaining with CD34 and CD68. The presence of gadolinium and other metals was also assessed by conventional and energy-filtered transmission electron microscopy. RESULTS: Numerous CD34+ and CD68+ cells were found in both the EPS and control patients within the vascular endothelium and in macrophages, respectively, but not in interstitial fibrocytes, as it could be expected in NSF. No trace of gadolinium deposits could be found in the four peritoneal samples; dispersed tiny iron inclusions were evidenced in the connective tissue of both EPS patients. CONCLUSIONS: These findings argue against the implication of gadolinium in the development of EPS in long-term PD patients.

Detection of intact rAAV particles up to 6 years after successful gene transfer in the retina of dogs and primates.

Gene transfer to the retina using recombinant adeno-associated viral (rAAV) vectors has proven to be an effective option for the treatment of retinal degenerative diseases in several animal models and has recently advanced into clinical trials in humans. To date, intracellular trafficking of AAV vectors and subsequent capsid degradation has been studied only in vitro, but the fate of AAV particles in transduced cells following subretinal injection has yet to be elucidated. Using electron microscopy and western blot, we analyzed retinas of one primate and four dogs that had been subretinally injected with AAV2/4, -2/5, or -2/2 serotypes and that displayed efficient gene transfer over several years. We show that intact AAV particles are still present in retinal cells, for up to 6 years after successful gene transfer in these large animals. The persistence of intact vector particles in the target organ, several years postadministration, is totally unexpected and, therefore, represents a new and unanticipated safety issue to consider at a time when gene therapy clinical trials raise new immunological concerns.

Cutaneous amyloidoses and systemic amyloidoses with cutaneous involvement.

Extracellular deposition of altered autologous protein (amyloid protein) within the dermis is the hallmark of cutaneous amyloidoses and systemic amyloidoses with cutaneous involvement. Amyloidoses may be acquired or hereditary in nature and subclassification differentiates between primary amyloidosis (no obvious predisposing disease) and secondary amyloidosis (specific underlying disease). More than 26 different proteins and peptides have been identified as amyloid precursors and these proteins are used to subclassify this heterogeneous group of diseases. The amyloid proteins show an anti-parallel beta-sheet conformation and form non-branching linear filaments of variable lengths and diameters of approximately 7.5 to 10 nm. However, the exact etiopathogenesis of amyloid formation still remains unclear. Depending on histoanatomical distribution and amount, amyloid may cause progressive and life-threatening organ dysfunction. Clinical presentation, histology, electron microscopy, and biochemical-immunological differentiation represent decisive tools for an accurate diagnosis.

Alterations in mechanical properties of mesenteric resistance arteries in experimental portal hypertension.

Splanchnic vasodilation is the pathophysiological hallmark in the development of the hyperdynamic circulatory syndrome in liver cirrhosis and portal hypertension. This has been attributed so far mainly to a marked vascular hyporeactivity to endogenous vasoconstrictors. However, myogenic tone and vessel stiffness have not been addressed in mesenteric arteries in liver cirrhosis. CCl(4)(-)-induced ascitic cirrhotic (LC) and age-matched control rats, portal vein-ligated (PVL) rats, and sham-operated rats were investigated. Third-order mesenteric resistance arteries were studied under no-flow conditions using a pressure myograph measuring media thickness and lumen diameter in response to incremental increases in intramural pressure, from which wall mechanics were calculated. Electron microscopy was used for investigation of wall ultrastructure, especially the fenestrae in internal elastic lamina (IEL). In PVL animals, no significant change in passive vessel strain, stress, media-to-lumen ratio, or cross-sectional area was noted. In contrast, in LC rats, vessel strain was markedly elevated compared with healthy control rats, indicating a marked reduction in vessel stiffness. In addition, the strain-stress curve was shifted to the right, and the elastic modulus in dependency on vessel stress decreased, demonstrating predominantly structure-dependent factors to be involved. The media-to-lumen quotient was not significantly altered, but cross-sectional area was highly increased in LC rats, indicating hypertrophic outward remodeling. These findings were paralleled by enlarged fenestrae in the IEL but no change in thickness of IEL or proportion of extracellular matrix or vascular smooth muscle in LC rats. We concluded that, in long-standing severe portal hypertension such as ascitic LC but not in short-term conditions such as PVL, mesenteric resistance arteries exhibit vascular remodeling and markedly less resistant mechanical properties, leading to decreased vessel stiffness accompanied by structural changes in the IEL. This may well contribute to the maintenance and severity of splanchnic arterial vasodilation in LC.

Electron microscopic documentation of late changes in permanent fillers and clinical management of granulomas in affected patients.

BACKGROUND: The manufacturers of permanent injectable fillers claim that their products are widely inert, biocompatible, atoxic, and nonimmunogenic. There are polymer gels without microparticles on the market and combination products that use collagen suspension or a hyaluronic acid gel as a vector to which polymer microspheres or polygonal particles are added. The filling effect of the polymer gels is based on the volume injected and, for the combination gels, partly on the volume injected and partly on the intended host foreign-body reaction to the microparticles. Foreign body reactions that are seen as inflammatory, sometimes disfiguring, nodules may develop years later at the injection sites. OBJECTIVES: Permanent fillers differ with respect to composition and chemical and biological characteristics. There have been reports that intend to explain how host tissue reacts with different permanent fillers and how adverse reactions differ depending on the filler used. The changes that some of the permanent fillers undergo during years of residence in human tissue have not been included in this discussion. These structural changes may be one of the reasons why adverse reactions to permanent fillers occur clinically with a delay of several years. METHODS: In a series of 10 patients who had been injected with a permanent filler of hydroxymethylmethacrylate and ethylmethacrylate (40%) in hyaluronic acid gel (60%) and had developed adverse reactions with inflammatory nodules after variable time elapsed, biopsies could be obtained for histologic and electron microscopic examinations. RESULTS: After 2 years in all specimens, changes of degradation of the filler material could be detected. Bacteria were not found in any of the specimen. In 40% of the particles, the size of the particles did not correspond to the size declared by the manufacturer (45-65 microm) and was smaller, thus being more susceptible to phagocytosis. CONCLUSIONS: Inflammatory nodules due to adverse reactions to permanent fillers containing microparticles with a hydrophobic surface were treated with good results with a regimen of allopurinol and intralesional injections with a mixture of fluorouracil and low-dose triamcinolon.

Multimodal Imaging Study of Gadolinium Presence in Rat Cerebellum: Differences Between Gd Chelates, Presence in the Virchow-Robin Space, Association With Lipofuscin, and Hypotheses About Distribution Pathway.

PURPOSE: The aim of this study was to investigate, based on in-depth multimodal imaging, the presence of Gd deposits, their ultrastructure, location, and co-location with endogenous elements, in the cerebellum, after repeated administrations of gadolinium-based contrast agents (GBCAs). METHODS: Rats sensitized by subtotal nephrectomy received 20 daily intravenous injections of 0.6 mmol Gd/kg for 5 weeks of commercial forms of either gadoterate, gadobenate or gadodiamide, or saline (n = 2/group). The study was randomized and blinded. Magnetic resonance imaging examination was performed weekly. One month after the last injection, electron microscopy analysis of the deep cerebellar nuclei, the granular layer of cerebellar cortex, and the choroid plexus was performed. Elemental analysis of deposits was carried out by electron energy loss spectroscopy. Secondary ion mass spectroscopy was used for complementary chemical mapping. RESULTS: A T1 hypersignal was evidenced in the deep cerebellar nuclei of rats treated with linear GBCAs, and Gd deposits were identified in all the studied cerebellar structures with gadobenate and gadodiamide (except in the granular layer in gadobenate-treated rats). No such effect was found with the macrocyclic GBCA gadoterate. Most of the Gd deposits revealed a characteristic spheroid "sea urchin-like" morphology, rich in phosphorus, and were localized in the basal lamina of microvessels, in the perivascular Virchow-Robin space, and in the interstitium. Gd was also identified in the glial cells, associated with lipofuscin pigments, for these same groups. CONCLUSIONS: Transmission electron microscopy analysis of cerebellums of renally impaired rats repeatedly injected with gadobenate and gadodiamide revealed the presence of Gd. Spheroid Gd depositions consisting of a filamentous meshwork were observed in the wall of microvessels, in perivascular Virchow-Robin space, and in the interstitium. Gd was also found in choroid plexus and was associated with pigments (likely lipofuscin) in glial cells. This is consistent with the involvement of the glymphatic distribution pathway for GBCAs. No insoluble Gd deposits were detected in rats injected with the macrocyclic GBCA gadoterate and controls.

Primary and metastatic high-grade pleomorphic sarcoma/malignant fibrous histiocytoma of the gastrointestinal tract: an approach to the differential diagnosis in a series of five cases with emphasis on myofibroblastic differentiation.

Primary and metastatic so-called malignant fibrous histiocytoma/undifferentiated high-grade pleomorphic sarcoma (MFH) is rare in the gastrointestinal (GI) tract with approximately 50 primary and five metastatic cases reported so far. We evaluated two primary gastric and three metastatic intestinal high-grade pleomorphic sarcomas with features of storiform-pleomorphic MFH. Gastric tumours occurred in a 79-year-old man and a 68-year-old woman. One patient died post-operatively, and the other was disease-free at 6 months. Three patients presented with GI metastasis 24, 60 and 0 months after diagnosis of MFH of the heart (n = 1) and the thigh (n = 2). Metastases were located in the small (n = 1) and large bowel (n = 2) and were characteristically pedunculated and polypoid with oedematous haemorrhagic stroma. Concurrent metastases (brain, lung, bone) were present in all three cases. Tumours expressed alpha-smooth muscle actin (four of five), platelet-derived growth factor receptor (PDGFR) alpha (three of three) and PDGFRbeta (two of three) but were negative for CD117, CD34 and other lineage-specific markers. Ultrastructural examination revealed myo/fibroblastic features. Both gastric MFH were wild type for KIT and PDGFRalpha. In conclusion, primary and metastatic MFH of the GI tract commonly express PDGFRalpha and show a myo/fibroblastic phenotype. They should be distinguished from a variety of primary and metastatic pleomorphic neoplasms, in particular high-grade sarcomatous GI stromal tumours (GIST), pleomorphic leiomyosarcoma, sarcomatoid carcinoma and other mimics.

Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural killer cells.

Detergent-soluble membrane vesicles are actively released by human pancreas (Colo-/Colo+) and colon (CX-/CX+) carcinoma sublines, differing in their capacity to present heat shock protein 70 (Hsp70)/Bag-4 on their plasma membranes. Floating properties, acetylcholine esterase activity, and protein composition characterized them as exosomes. An enrichment of Rab-4 documented their intracellular transport route from early endosomes to the plasma membrane. After solubilization, comparable amounts of cytosolic proteins, including tubulin, Hsp70, Hsc70, and Bag-4, but not ER-residing Grp94 and calnexin, were detectable in tumor-derived exosomes. However, with respect to the exosomal surface, only Colo+/CX+ but not Colo-/CX- derived exosomes were Hsp70 membrane positive. Therefore, concomitant with an up-regulated cell surface density of activation markers, migration and Hsp70 reactivity of natural killer (NK) cells was stimulated selectively by Hsp70/Bag-4 surface-positive exosomes, but not by their negative counterparts and tumor cell lysates. Moreover, the exosome-mediated lytic activity of NK cells was blockable by Hsp70-specific antibody. As already shown for TKD stimulation, NK cells preincubated with Hsp70 surface-positive exosomes initiated apoptosis in tumors through granzyme B release. In summary, our data provide an explanation how Hsp70 reactivity in NK cells is induced by tumor-derived exosomes.

Microwave-assisted tissue processing for same-day EM-diagnosis of potential bioterrorism and clinical samples.

The purpose of this study was to explore the turnaround times, section and image quality of a number of more "difficult" specimens destined for rapid diagnostic electron microscopy (EM) after microwave-assisted processing. The results were assessed and compared with those of conventionally processed samples. A variety of infectious agents, some with a potential for bioterrorism, and liver biopsies serving as an example for routine histopathology samples were studied. The samples represented virus-producing cell cultures (such as SARS-coronavirus, West Nile virus, Orthopox virus), bacteria suspensions (cultures of Escherichia coli and genetically knockout apathogenic Bacillus anthracis), suspensions of parasites (malaria Plasmodium falciparum, Leishmania major, Microsporidia cuniculi, Caenorhabditis elegans), and whole Drosophila melanogaster flies infected with microsporidia. Fresh liver samples and infected flies were fixed in Karnovsky-fixative by microwaving (20 min), all other samples were fixed in buffered glutaraldehyde or Karnovsky-fixative overnight or longer. Subsequently, all samples were divided to evaluate alternative processing protocols: one part of the sample was OsO4-postfixed, ethanol-dehydrated, Epon-infiltrated (overnight) in an automated tissue processor (LYNX, Leica), and polymerized at 60 degrees C for 48 h; in parallel the other part was microwave-assisted processed in the bench microwave device (REM, Milestone), including post-osmication and the resin block polymerization. The microwave-assisted processing protocol required at minimum 3 h 20 min: the respective epon resin blocks were uniformly polymerized allowing an easy sectioning of semi- and ultrathin sections. Sections collected on non-coated 200 mesh grids were stable in the electron beam and showed an excellent preservation of the ultrastructure and high contrast, thus allowing an easy, unequivocal and rapid assessment of specimens. Compared with conventional routine methods, microwave technology facilitates a significant reduction in sample processing time from days to hours without any loss in ultrastructural details. Microwave-assisted processing could, therefore, be a substantial benefit for the routine electron microscopic diagnostic workload. Due to its speed and robust performance it could be applied wherever a rapid electron microscopy diagnosis is required, e.g., if bioterrorism or emerging agents are suspected. Combining microwave technology with digital image acquisition, the 1-day diagnosis based on ultrathin section electron microscopy will become possible, with crucial or interesting findings being consulted or shared worldwide with experts using modern telemicroscopy tools via Internet.

Distribution profile of gadolinium in gadolinium chelate-treated renally-impaired rats: role of pharmaceutical formulation.

While not acutely toxic, chronic hepatic effect of certain gadolinium chelates (GC), used as contrast agent for magnetic resonance imaging, might represent a risk in renally-impaired patients due to free gadolinium accumulation in the liver. To answer this question, this study investigated the consequences of the presence of small amounts of either a soluble gadolinium salt ("free" Gd) or low-stability chelating impurity in the pharmaceutical solution of gadoteric acid, a macrocyclic GC with high thermodynamic and kinetic stabilities, were investigated in renally-impaired rats. Renal failure was induced by adding 0.75% adenine in the diet for three weeks. The pharmaceutical and commercial solution of gadoteric acid was administered (5 daily intravenous injections of 2.5 mmol Gd/kg) either alone or after being spiked with either "free" gadolinium (i.e., 0.04% w/v) or low-stability impurity (i.e., 0.06 w/v). Another GC, gadodiamide (low thermodynamic and kinetic stabilities) was given as its commercial solution at a similar dose. Non-chelated gadolinium was tested at two doses (0.005 and 0.01 mmol Gd/kg) as acetate salt. Gadodiamide induced systemic toxicity (mortality, severe epidermal and dermal lesions) and substantial tissue Gd retention. The addition of very low amounts of "free", non-chelated gadolinium or low thermodynamic stability impurity to the pharmaceutical solution of the thermodynamically stable GC gadoteric acid resulted in substantial capture of metal by the liver, similar to what was observed in "free" gadolinium salt-treated rats. Relaxometry studies strongly suggested the presence of free and soluble gadolinium in the liver. Electron microscopy examinations revealed the presence of free and insoluble gadolinium deposits in hepatocytes and Kupffer cells of rats treated with gadoteric acid solution spiked with low-stability impurity, free gadolinium and gadodiamide, but not in rats treated with the pharmaceutical solution of gadoteric acid. The presence of impurities in the GC pharmaceutical solution may have long-term biological consequences.