Josette Cadusseau

Slow CCL2-dependent translocation of biopersistent particles from muscle to brain

BackgroundLong-term biodistribution of nanomaterials used in medicine is largely unknown. This is the case for alum, the most widely used vaccine adjuvant, which is a nanocrystalline compound spontaneously forming micron/submicron-sized agglomerates. Although generally well tolerated, alum is occasionally detected within monocyte-lineage cells long after immunization in presumably susceptible individuals with systemic/neurologic manifestations or autoimmune (inflammatory) syndrome induced by adjuvants (ASIA).MethodsOn the grounds of preliminary investigations in 252 patients with alum-associated ASIA showing both a selective increase of circulating CCL2, the major monocyte chemoattractant, and a variation in the CCL2 gene, we designed mouse experiments to assess biodistribution of vaccine-derived aluminum and of alum-particle fluorescent surrogates injected in muscle. Aluminum was detected in tissues by Morin stain and particle induced X-ray emission) (PIXE) Both 500 nm fluorescent latex beads and vaccine alum agglomerates-sized nanohybrids (Al-Rho) were used.ResultsIntramuscular injection of alum-containing vaccine was associated with the appearance of aluminum deposits in distant organs, such as spleen and brain where they were still detected one year after injection. Both fluorescent materials injected into muscle translocated to draining lymph nodes (DLNs) and thereafter were detected associated with phagocytes in blood and spleen. Particles linearly accumulated in the brain up to the six-month endpoint; they were first found in perivascular CD11b+ cells and then in microglia and other neural cells. DLN ablation dramatically reduced the biodistribution. Cerebral translocation was not observed after direct intravenous injection, but significantly increased in mice with chronically altered blood-brain-barrier. Loss/gain-of-function experiments consistently implicated CCL2 in systemic diffusion of Al-Rho particles captured by monocyte-lineage cells and in their subsequent neurodelivery. Stereotactic particle injection pointed out brain retention as a factor of progressive particle accumulation.ConclusionNanomaterials can be transported by monocyte-lineage cells to DLNs, blood and spleen, and, similarly to HIV, may use CCL2-dependent mechanisms to penetrate the brain. This occurs at a very low rate in normal conditions explaining good overall tolerance of alum despite its strong neurotoxic potential. However, continuously escalating doses of this poorly biodegradable adjuvant in the population may become insidiously unsafe, especially in the case of overimmunization or immature/altered blood brain barrier or high constitutive CCL-2 production.

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

BackgroundTumor initiating cells (TICs) provide a new paradigm for developing original therapeutic strategies.MethodsWe 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.ResultsA 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.ConclusionsOur 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.

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.

Skilled forelimb use in the rat: amelioration of functional deficits resulting from neonatal damage to the frontal cortex by neonatal transplantation of fetal cortical tissue.

Limb preference and dexterity of right and left forelimbs were studied in a food reaching task in normal rats (control group), in rats that sustained a neonatal lesion of the left frontal cortex (lesion group) and in animals that received a transplant obtained from the frontal cortex of 16-day-old embryos immediately after the lesion (graft group). In addition, an anatomical study of host-transplant interconnections was performed in several transplanted animals. The results indicate that the lesion slightly increased the preference for the limb ipsilateral to the lesion and transplantation of fetal cortical tissue did not restore the preference for the contralateral limb. Furthermore, lesion of the motor cortex induced a deficit in the dexterity of limb use in the food-reaching task. This motor deficit was more pronounced when the limb contralateral to the lesion was used. Transplantation of embryonic cortical tissue led to a reduction of the motor deficit. Compared to the lesion group, the graft group had a higher success rate and a lower percentage of motor abnormalities, whichever forelimb was used, ipsi- or contralateral to the transplant. Nevertheless, larger improvement was noted with contralateral forelimb usage. Functional recovery was not complete since the control group still performed significantly better than the graft group, although almost complete sparing in skilled reaching was noted when the limb ipsilateral to the transplant was used. Analysis of host-transplant interconnections indicates that the transplants sent fibers to the host spinal cord, caudate-putamen, thalamus and homotopic contralateral cortex and received projections from the host thalamus and contralateral cortex. It is therefore suggested that neonatal transplantation of fetal cortical tissue promotes functional recovery from damage to the motor cortex occurring at birth.

Astrocytes Reverted to a Neural Progenitor‐like State with Transforming Growth Factor Alpha Are Sensitized to Cancerous Transformation

Gliomas, 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)‐α, 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‐α is sufficient to trigger their reversion to a neural progenitor‐like state. To determine whether TGF‐α 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‐α 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‐α 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. STEM CELLS 2009;27:2373–2382

Biopersistence and brain translocation of aluminum adjuvants of vaccines.

Aluminum oxyhydroxide (alum) is a crystalline compound widely used as an immunological 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 phagocytosed 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.

Clinical features in patients with long-lasting macrophagic myofasciitis

Macrophagic myofasciitis (MMF) is an emerging condition characterized by specific muscle lesions assessing abnormal long-term persistence of aluminum hydroxide within macrophages at the site of previous immunization. Affected patients usually are middle-aged adults, mainly presenting with diffuse arthromyalgias, chronic fatigue, and marked cognitive deficits, not related to pain, fatigue, or depression. Clinical features usually correspond to that observed in chronic fatigue syndrome/myalgic encephalomyelitis. Representative features of MMF-associated cognitive dysfunction include dysexecutive syndrome, visual memory impairment, and left ear extinction at dichotic listening test. Most patients fulfill criteria for non-amnestic/dysexecutive mild cognitive impairment, even if some cognitive deficits appear unusually severe. Cognitive dysfunction seems stable over time despite marked fluctuations. Evoked potentials may show abnormalities in keeping with central nervous system involvement, with a neurophysiological pattern suggestive of demyelination. Brain perfusion SPECT shows a pattern of diffuse cortical and subcortical abnormalities, with hypoperfusions correlating with cognitive deficiencies. The combination of musculoskeletal pain, chronic fatigue, and cognitive disturbance generates chronic disability with possible social exclusion. Classical therapeutic approaches are usually unsatisfactory making patient care difficult.

Two temporal stages of oligodendroglial response to excitotoxic lesion in the gray matter of the adult rat brain

Excitotoxic lesions in the gray matter induce profuse demyelination of passage and afferent fibers in areas of neuronal loss, independent of Wallerian degeneration. The time course of this phenomenon, which extends over weeks after the excitotoxin injection, suggests that demyelination is not related only to a direct effect of the toxin. In order to define mechanisms at work, a parallel study of myelin and oligodendrocytes was carried out following kainate injections into the adult rat thalamus. Within the 1st day postlesion, myelin alteration appeared throughout the area exhibiting neuronal loss, while the number of oligodendrocytes fell by 45%. No apoptotic oligodendrocytes were identified at that time. Over the following 2 days, there was no further loss of myelin and oligodendrocytes, but there was an increase in the number of oligodendrocytes displaying typical signs of apoptosis as revealed with TUNEL-end-labeled nuclei, Hoechst-labeled condensed chromatin bodies, or bax immunoreactivity. This resulted in a second, progressive loss of both myelin and oligodendrocytes leading to their almost complete disappearance 2 weeks postlesion. These results demonstrate two temporal stages of oligodendroglial cell death. The excitotoxin injection resulted in the rapid destruction of a first oligodendroglial population, most probably by necrosis. A second population died in a delayed manner from apoptosis. This second wave of death coincided with an activated microglia/macrophage invasion of the lesion, suggesting that delayed oligodendroglial death results from toxic microglia/macrophage effects. In addition, the longest surviving oligodendrocytes were located next to reactive astrocytes, suggesting the existence of trophic interactions between these two glial populations.

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

BackgroundAluminum 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.MethodsWe 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.ResultsIn 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.ConclusionsThe fluorescent nanodiamond technology is able to overcome the limitations of previously used organic fluorophores, thus appearing as a choice methodology for studying distribution, persistence and long-term neurotoxicity of alum adjuvants and beyond of other types of nanoparticles.

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

Several 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.