Testicular biodistribution of 450 nm fluorescent latex particles after intramuscular injection in mice.

International audienceThe significant expansion in the use of nanoparticles and submicron particles during the last 20 years has led to increasing concern about their potential toxicity to humans and particularly their impact on male fertility. Currently, an insufficient number of studies have focused on the testicular biodistribution of particles. The aim of our study was to assess the distribution of 450 nm fluorescent particles in mouse testes after intramuscular injection. To this end, testes were removed from 5 groups of 3 mice each at 1 h (H1), 4 days (D4), 21 days (D21), 45 days (D45) and 90 days (D90) after the injection of 7.28 × 10(9) particles in the tibialis anterior muscles of each mouse. We examined histological sections from these samples by epifluorescence microscopy and confocal microscopy and identified testicular biodistribution of a small number of particles in groups H1, D4, D21, D45 and D90. Using CD11b immunostaining, we showed that particles were not carried into the testis by macrophages. The intratesticular repartition of particles mainly followed testicular vascularization. Finally, we found some particles in seminiferous tubules but could not determine if the blood-testis barrier was crossed

Astrocytes reverted to a neural progenitor-like state with transforming growth factor alpha are sensitized to cancerous transformation.

International audienceGliomas, the most frequent primitive central nervous system tumors, have been suggested to originate from astrocytes or from neural progenitors/stem cells. However, the precise identity of the cells at the origin of gliomas remains a matter of debate because no pre-neoplastic state has been yet identified. Transforming growth factor (TGF)-alpha, an epidermal growth factor family member, is frequently overexpressed in the early stages of glioma progression. We previously demonstrated that prolonged exposure of astrocytes to TGF-alpha is sufficient to trigger their reversion to a neural progenitor-like state. To determine whether TGF-alpha dedifferentiating effects are associated with cancerous transforming effects, we grafted intracerebrally dedifferentiated astrocytes. We show that these cells had the same cytogenomic profile as astrocytes, survived in vivo, and did not give birth to tumors. When astrocytes dedifferentiated with TGF-alpha were submitted to oncogenic stress using gamma irradiation, they acquired cancerous properties: they were immortalized, showed cytogenomic abnormalities, and formed high-grade glioma-like tumors after brain grafting. In contrast, irradiation did not modify the lifespan of astrocytes cultivated in serum-free medium. Addition of TGF-alpha after irradiation did not promote their transformation but decreased their lifespan. These results demonstrate that reversion of mature astrocytes to an embryonic state without genomic manipulation is sufficient to sensitize them to oncogenic stress

Clinical Relevance of Tumor Cells with Stem-Like Properties in Pediatric Brain Tumors

BACKGROUND: Primitive brain tumors are the leading cause of cancer-related death in children. Tumor cells with stem-like properties (TSCs), thought to account for tumorigenesis and therapeutic resistance, have been isolated from high-grade gliomas in adults. Whether TSCs are a common component of pediatric brain tumors and are of clinical relevance remains to be determined. METHODOLOGY/PRINCIPAL FINDINGS: Tumor cells with self-renewal properties were isolated with cell biology techniques from a majority of 55 pediatric brain tumors samples, regardless of their histopathologies and grades of malignancy (57% of embryonal tumors, 57% of low-grade gliomas and neuro-glial tumors, 70% of ependymomas, 91% of high-grade gliomas). Most high-grade glioma-derived oncospheres (10/12) sustained long-term self-renewal akin to neural stem cells (>7 self-renewals), whereas cells with limited renewing abilities akin to neural progenitors dominated in all other tumors. Regardless of tumor entities, the young age group was associated with self-renewal properties akin to neural stem cells (P = 0.05, chi-square test). Survival analysis of the cohort showed an association between isolation of cells with long-term self-renewal abilities and a higher patient mortality rate (P = 0.013, log-rank test). Sampling of low- and high-grade glioma cultures showed that self-renewing cells forming oncospheres shared a molecular profile comprising embryonic and neural stem cell markers. Further characterization performed on subsets of high-grade gliomas and one low-grade glioma culture showed combination of this profile with mesenchymal markers, the radio-chemoresistance of the cells and the formation of aggressive tumors after intracerebral grafting. CONCLUSIONS/SIGNIFICANCE: In brain tumors affecting adult patients, TSCs have been isolated only from high-grade gliomas. In contrast, our data show that tumor cells with stem cell-like or progenitor-like properties can be isolated from a wide range of histological sub-types and grades of pediatric brain tumors. They suggest that cellular mechanisms fueling tumor development differ between adult and pediatric brain tumors

CD133, CD15/SSEA-1, CD34 or side populations do not resume tumor-initiating properties of long-term cultured cancer stem cells from human malignant glio-neuronal tumors

<p>Abstract</p> <p>Background</p> <p>Tumor initiating cells (TICs) provide a new paradigm for developing original therapeutic strategies.</p> <p>Methods</p> <p>We screened for TICs in 47 human adult brain malignant tumors. Cells forming floating spheres in culture, and endowed with all of the features expected from tumor cells with stem-like properties were obtained from glioblastomas, medulloblastoma but not oligodendrogliomas.</p> <p>Results</p> <p>A long-term self-renewal capacity was particularly observed for cells of malignant glio-neuronal tumors (MGNTs). Cell sorting, karyotyping and proteomic analysis demonstrated cell stability throughout prolonged passages. Xenografts of fewer than 500 cells in Nude mouse brains induced a progressively growing tumor. CD133, CD15/LeX/Ssea-1, CD34 expressions, or exclusion of Hoechst dye occurred in subsets of cells forming spheres, but was not predictive of their capacity to form secondary spheres or tumors, or to resist high doses of temozolomide.</p> <p>Conclusions</p> <p>Our results further highlight the specificity of a subset of high-grade gliomas, MGNT. TICs derived from these tumors represent a new tool to screen for innovative therapies.</p

Selective elevation of circulating CCL2/MCP1 levels in patients with longstanding post-vaccinal macrophagic myofasciitis and ASIA.

International audienceSeveral medical conditions sharing similar signs and symptoms may be related to immune adjuvants. These conditions described as ASIA (Autoimmune/inflammatory Syndrome Induced by Adjuvants), include a condition characterized by macrophagic myofasciitis (MMF) assessing long-term persistence of vaccine derived-alum adjuvants into macrophages at sites of previous immunization. Despite increasing data describing clinical manifestations of ASIA have been reported, biological markers are particularly lacking for their characterization and follow up. We report an extensive cytokine screening performed in serum from 44 MMF patients compared both to sex and age matched healthy controls and to patients with various types of inflammatory neuromuscular diseases. Thirty cytokines were quantified using combination of Luminex® technology and ELISA. There was significant mean increase of serum levels of the monocytechemoattractant protein 1 (CCL2/MCP-1) in MMF patients compared to healthy subjects. MMF patients showed no elevation of other cytokines. This contrasted with inflammatory patients in whom CCL2/MCP-1 serum levels were unchanged, whereas several other inflammatory cytokines were elevated (IL1β, IL5 and CCL3/MIP1α). These results suggest that CCL2 may represent a biological marker relevant to the pathophysiology of MMF rather than a non specific inflammatory marker and that it should be checked in the other syndromes constitutive of ASIA

Transforming growth factor α (TGF α) expression in degenerating motoneurons of the murine mutant wobbler: a neuronal signal for astrogliosis?

International audienceThe enhanced expression of the trophic factor transforming growth factor alpha (TGF alpha) in reactive astrocytes following CNS injury suggests that TGF alpha has a role in the development of astrogliosis. We explored this hypothesis in the murine mutant wobbler, which presents a progressive motoneuronal degeneration associated with an astrogliosis. Evolution of astrogliosis, and expression of TGF alpha precursor (pro-TGF alpha) and of its receptor were examined over the course of the disease, using genetically diagnosed animals and immunocytochemical techniques. We report here that degenerating motoneurons of the cervical spinal cord and a subset of astrocytes express pro-TGF alpha, prior to the onset of astrogliosis, when the first clinical manifestations of the disease are observed at 4 weeks of age. TGF alpha expression appeared strongly correlated with motoneuronal degeneration. All pro-TGF alpha-immunoreactive neurons exhibited a degenerative morphology, and the number of pro-TGF alpha-immunoreactive neurons increased with the progression of the disease. At the glial level, we observed that astrogliosis was a transitory phenomenon in the wobbler mice, developing in coordination with the motoneuronal expression of pro-TGF alpha. Astrogliosis became evident in 6-week-old wobbler mice, when the number of pro-TGF alpha-immunoreactive motoneurons was maximal, and regressed in older mutant mice in correlation with the disappearance of pro-TGF alpha-immunoreactive motoneurons. Furthermore, TGF alpha/EGF receptor immunoreactivity was exclusively localized in a subset of reactive astrocytes, its expression following closely the course of the astrogliosis. These data show that TGF alpha synthesis by the affected motoneurons is an early event in the course of the wobbler disease, and suggest a role for TGF alpha as a neuronal inducer of astrocytic reactivity

Biopersistence and Brain Translocation of Aluminum Adjuvants of Vaccines

Aluminum oxyhydroxide (alum) is a crystaline compound widely used as an immunologic adjuvant of vaccines. Concerns linked to the use of alum particles emerged following recognition of their causative role in the so-called macrophagic myofasciitis (MMF) lesion detected in patients with myalgic encephalomyelitis/chronic fatigue/syndrome. MMF revealed an unexpectedly long-lasting biopersistence of alum within immune cells in presumably susceptible individuals, stressing the previous fundamental misconception of its biodisposition. We previously showed that poorly biodegradable aluminum-coated particles injected into muscle are promptly phagocytozed in muscle and the draining lymph nodes, and can disseminate within phagocytic cells throughout the body and slowly accumulate in brain. This strongly suggests that long-term adjuvant biopersistence within phagocytic cells is a prerequisite for slow brain translocation and delayed neurotoxicity. The understanding of basic mechanisms of particle biopersistence and brain translocation represents a major health challenge, since it could help to define susceptibility factors to develop chronic neurotoxic damage. Biopersistence of alum may be linked to its lysosome-destabilizing effect, which is likely due to direct crystal-induced rupture of phagolysosomal membranes. Macrophages that continuously perceive foreign particles in their cytosol will likely reiterate, with variable interindividual efficiency, a dedicated form of autophagy (xenophagy) until they dispose of alien materials. Successful compartmentalization of particles within double membrane autophagosomes and subsequent fusion with repaired and re-acidified lysosomes will expose alum to lysosomal acidic pH, the sole factor that can solubilize alum particles. Brain translocation of alum particles is linked to a Trojan horse mechanism previously described for infectious particles (HIV, HCV), that obeys to CCL2 signaling the major inflammatory monocyte chemoattractant

Low concentrations of aluminum hydroxide adjuvant, forming limited size aggregates, selectively induce cerebral aluminum increase and long-term neurotoxicity in mouse

International audienceBackgroundAluminium hydroxide (alum) has long been added as an adjuvant of vaccines. It consists of nanoparticles forming aggregates. Unexpectedly long-lasting biopersistence of alum aggregates were found within immune cells of patients with chronic fatigue, cognitive dysfunction, myalgias and dysimmunity [1], [2]. We documented in mice slow translocation of alum aggregates captured by monocyte-lineage cells from the injected muscle to brain [3], [4], [5]. Herein, brain function and aluminium (Al) concentration were examined long after injections.MethodsAlhydrogel® was injected in TA muscle in adult female CD1 mice at 3 doses ranging from 133 to 800 μg Al/kg. Eight validated tests were used to evaluate cognitive and motor performances 180 days after injection. Brains were collected for Al level determination and Iba-1 immunohistochemistry.ResultsA most unusual neuro-toxicological pattern limited to lower doses of alum was observed. Neurobehavioral changes, including decreased activity levels and altered anxiety-like behaviour, were documented in animals exposed to the two lowest doses (133 and 200 μg Al/kg) but not at the highest dose (800 μg Al/kg), compared to controls. Consistently, cerebral Al levels were increased in animals exposed to the lowest doses. Microglial cell increase was found in amygdala of the 200 μg Al/kg group. Interestingly, the injected suspensions corresponding to the two lowest doses contained much smaller aggregates (1.50–1.75 μm) compared to the highest dose (4.70 μm).ConclusionAlum particles injected in muscle may induce neurotoxic effects and Al cerebral accumulation six months after injection in mice. Neurotoxic effects are restricted to low concentration suspensions forming small particle aggregates. Such bacteria-sized aggregates are known to be selectively captured by monocyte-lineage cells. This study strongly suggests that, in contrast to “the dose makes the poison” paradigm of classical toxicology, alum toxicology obeys the specific rules of small particle toxicology, thus deserving in depth revaluation. (This study was supported by ANSM)

Assessment of the neurotoxic effects of aluminum hydroxide vaccine adjuvant injections in mice

50th Congress of the European-Societies-of-Toxicology, Edinburgh, SCOTLAND, SEP 07-10, 2014International audienceno abstrac

Fluorescent nanodiamonds as a relevant tag for the assessment of alum adjuvant particle biodisposition

International audienceAluminum oxyhydroxide (alum) is a crystalline compound widely used as an immunologic adjuvant of vaccines. Concerns linked to alum particles have emerged following recognition of their causative role in the so-called macrophagic myofasciitis (MMF) lesion in patients with myalgic encephalomyelitis, revealing an unexpectedly long-lasting biopersistence of alum within immune cells and a fundamental misconception of its biodisposition. Evidence that aluminum-coated particles phagocytozed in the injected muscle and its draining lymph nodes can disseminate within phagocytes throughout the body and slowly accumulate in the brain further suggested that alum safety should be evaluated in the long term. However, lack of specific staining makes difficult the assessment of low quantities of bona fide alum adjuvant particles in tissues. Methods: We explored the feasibility of using fluorescent functionalized nanodiamonds (mfNDs) as a permanent label of alum (Alhydrogel ®). mfNDs have a specific and perfectly photostable fluorescence based on the presence within the diamond lattice of nitrogen-vacancy centers (NV centers). As the NV center does not bleach, it allows the microspectrometric detection of mfNDs at very low levels and in the long-term. We thus developed fluorescent nanodiamonds functionalized by hyperbranched polyglycerol (mfNDs) allowing good coupling and stability of alum:mfNDs (AluDia) complexes. Specificities of AluDia complexes were comparable to the whole reference vaccine (anti-hepatitis B vaccine) in terms of particle size and zeta potential. Results: In vivo, AluDia injection was followed by prompt phagocytosis and AluDia particles remained easily detectable by the specific signal of the fND particles in the injected muscle, draining lymph nodes, spleen, liver and brain. In vitro, mfNDs had low toxicity on THP-1 cells and AluDia showed cell toxicity similar to alum alone. Expectedly, AluDia elicited autophagy, and allowed highly specific detection of small amounts of alum in autophagosomes