Development of a neural rosette formation assay (RoFA) to identify neurodevelopmental toxicants and to characterize their transcriptome disturbances.

The first in vitro tests for developmental toxicity made use of rodent cells. Newer teratology tests, e.g. developed during the ESNATS project, use human cells and measure mechanistic endpoints (such as transcriptome changes). However, the toxicological implications of mechanistic parameters are hard to judge, without functional/morphological endpoints. To address this issue, we developed a new version of the human stem cell-based test STOP-tox(UKN). For this purpose, the capacity of the cells to self-organize to neural rosettes was assessed as functional endpoint: pluripotent stem cells were allowed to differentiate into neuroepithelial cells for 6 days in the presence or absence of toxicants. Then, both transcriptome changes were measured (standard STOP-tox(UKN)) and cells were allowed to form rosettes. After optimization of staining methods, an imaging algorithm for rosette quantification was implemented and used for an automated rosette formation assay (RoFA). Neural tube toxicants (like valproic acid), which are known to disturb human development at stages when rosette-forming cells are present, were used as positive controls. Established toxicants led to distinctly different tissue organization and differentiation stages. RoFA outcome and transcript changes largely correlated concerning (1) the concentration-dependence, (2) the time dependence, and (3) the set of positive hits identified amongst 24 potential toxicants. Using such comparative data, a prediction model for the RoFA was developed. The comparative analysis was also used to identify gene dysregulations that are particularly predictive for disturbed rosette formation. This 'RoFA predictor gene set' may be used for a simplified and less costly setup of the STOP-tox(UKN) assay.

Malignant Transformation of a Rosette-Forming Glioneuronal Tumor to Glioblastoma.

BACKGROUND: A rosette-forming glioneuronal tumor (RGNT), a rare brain tumor, presents as a benign feature with a favorable outcome. To date, a few cases with aggressive behaviors, such as recurrence or dissemination, but none with malignant transformation, have been reported. We describe 1 case that recurred as glioblastoma after complete resection of the benign RGNT. CASE DESCRIPTION: A man aged 58 years presented with headache and dizziness without neurologic symptoms. Magnetic resonance imaging showed a 4 × 2.5 cm, well-demarcated mass in the left cerebellar hemisphere. The patient underwent gross total resection of the tumor and a diagnosis of RGNT was made. There was no evidence of recurrence on serial follow-up. However, a recurrent heterogeneous enhancing mass in the previous surgical cavity was observed on a 7-year postoperative magnetic resonance imaging scan. Reoperation was performed and a histopathological study revealed a glioblastoma. CONCLUSIONS: To the best of our knowledge, this is the first case of spontaneous malignant transformation of an RGNT. Our case may be helpful in better understanding the biological behavior and clinical outcome of RGNT. We emphasize the malignant potential of this rare tumor and the necessity of future large-scaled research for most appropriate therapeutic strategies.

Rosette-forming and papillary glioneuronal tumors - A clinicopathological and molecular analysis.

INTRODUCTION: Rosette-forming glioneuronal tumors (RGNT) and papillary glioneuronal tumors (PGNT) account for < 1% of brain tumors. Genetic data regarding RGNT and PGNT is still evolving. We aimed to perform a detailed clinicopathological analysis on rosette-forming and papillary glioneuronal tumors and to evaluate these for common, known genetic mutations. MATERIALS AND METHODS: Our cohort consisted of 6 cases of these rare glioneuronal tumors diagnosed over a period of 5 years. IDH1, ATRX, p53, and BRAF V600E mutations were evaluated on immunohistochemistry, and cases of RGNT were screened for the mutations in PIK3CA gene at hotspots exon 4, 9, and 20. RESULTS AND CONCLUSIONS: Our findings confirm the presence of PIK3CA gene mutations in RGNT along with two novel mutations in PIK3CA gene, of which one is proposed to be of prognostic significance.
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Lateral attachment of kinetochores to microtubules is enriched in prometaphase rosette and facilitates chromosome alignment and bi-orientation establishment.

Faithful chromosome segregation is ensured by the establishment of bi-orientation; the attachment of sister kinetochores to the end of microtubules extending from opposite spindle poles. In addition, kinetochores can also attach to lateral surfaces of microtubules; called lateral attachment, which plays a role in chromosome capture and transport. However, molecular basis and biological significance of lateral attachment are not fully understood. We have addressed these questions by focusing on the prometaphase rosette, a typical chromosome configuration in early prometaphase. We found that kinetochores form uniform lateral attachments in the prometaphase rosette. Many transient kinetochore components are maximally enriched, in an Aurora B activity-dependent manner, when the prometaphase rosette is formed. We revealed that rosette formation is driven by rapid poleward motion of dynein, but can occur even in its absence, through slow kinetochore movements caused by microtubule depolymerization that is supposedly dependent on kinetochore tethering at microtubule ends by CENP-E. We also found that chromosome connection to microtubules is extensively lost when lateral attachment is perturbed in cells defective in end-on attachment. Our findings demonstrate that lateral attachment is an important intermediate in bi-orientation establishment and chromosome alignment, playing a crucial role in incorporating chromosomes into the nascent spindle.

Rosette-forming glioneuronal tumour of the fourth ventricle: case report and review of the literature.

Rosette-forming glioneuronal tumour (RGNT) of the fourth ventricle is one of the newly described primary tumours of the central nervous system. These tumours have two components of both neurocytic and glial areas but usually the glial component of the tumour predominates. They have biphasic cytoarchitecture with two elements; neurocytic rosettes resembling Homer-Wright rosettes, and astrocytic component resembling a pilocytic astrocytoma. They are low-grade tumours with lack of histopathological signs of malignancy. Here, clinical, magnetic resonance, computed tomography (CT) and pathological features of rosette-forming glioneuronal tumour of posterior fossa are presented. A 29-year-man was admitted with an acute neurological deterioration. A three ventricular hydrocephalus and a hypo-density around vermis in the posterior fossa were seen in his CT scans. He did well after an emergency external ventricular drainage. He had an elective operation and a mass that was reported to be a rosette-forming glioneuronal tumour of the fourth ventricle was excised.

Thalamic rosette-forming a glioneuronal tumor in an elderly patient: Case report and literature review.

The rosette-forming glioneuronal tumor (RGNT) is a novel type of brain tumor recently listed in the WHO 2007 classification of central nervous system (CNS) tumors. We report the case of a 75-year-old woman harboring a thalamic RGNT with third ventricle dissemination. Age and location make the present case exceptional and which has never previously been reported. A review of the clinical, pathological and radiological features is presented along with the relevant literature.

Negative and positive separation techniques for the isolation of antigen-specific CD8(+) T cells from blood and tumor tissue.

Adoptive transfer of tumor-reactive cytotoxic T cells (CTLs) is a promising therapeutic approach for the treatment of solid tumors. To develop these strategies, it is necessary to isolate specific leukocyte subpopulations from peripheral blood or tumor tissue (referred to as tumor-infiltrating lymphocytes (TIL)) that will be reinfused into the patient after expansion in vitro. The ideal cell isolation approach should be performed rapidly, thereby maximizing the recovery and viability of the purified cells. Here, we describe the negative or the positive separation procedures to isolate CD8(+) T cells from whole blood, from peripheral blood mononuclear cells (PBMCs), or from cancer tissue. Purified CD8(+) cells will be used for different downstream applications such as protein and gene expression profile analysis in order to assess their intrinsic cytotoxic ability.

Glycophorin C (CD236R) mediates vivax malaria parasite rosetting to normocytes.

Rosetting phenomenon has been linked to malaria pathogenesis. Although rosetting occurs in all causes of human malaria, most data on this subject has been derived from Plasmodium falciparum. Here, we investigate the function and factors affecting rosette formation in Plasmodium vivax. To achieve this, we used a range of novel ex vivo protocols to study fresh and cryopreserved P vivax (n = 135) and P falciparum (n = 77) isolates from Thailand. Rosetting is more common in vivax than falciparum malaria, both in terms of incidence in patient samples and percentage of infected erythrocytes forming rosettes. Rosetting to P vivax asexual and sexual stages was evident 20 hours postreticulocyte invasion, reaching a plateau after 30 hours. Host ABO blood group, reticulocyte count, and parasitemia were not correlated with P vivax rosetting. Importantly, mature erythrocytes (normocytes), rather than reticulocytes, preferentially form rosetting complexes, indicating that this process is unlikely to directly facilitate merozoite invasion. Although antibodies against host erythrocyte receptors CD235a and CD35 had no effect, Ag-binding fragment against the BRIC 4 region of CD236R significantly inhibited rosette formation. Rosetting assays using CD236R knockdown normocytes derived from hematopoietic stem cells further supports the role of glycophorin C as a receptor in P vivax rosette formation.

Rosetting Plasmodium falciparum-infected erythrocytes bind to human brain microvascular endothelial cells in vitro, demonstrating a dual adhesion phenotype mediated by distinct P. falciparum erythrocyte membrane protein 1 domains.

Adhesion interactions between Plasmodium falciparum-infected erythrocytes (IE) and human cells underlie the pathology of severe malaria. IE cytoadhere to microvascular endothelium or form rosettes with uninfected erythrocytes to survive in vivo by sequestering IE in the microvasculature and avoiding splenic clearance mechanisms. Both rosetting and cytoadherence are mediated by the parasite-derived IE surface protein family Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). Rosetting and cytoadherence have been widely studied as separate entities; however, the ability of rosetting P. falciparum strains to cytoadhere has received little attention. Here, we show that IE of the IT/R29 strain expressing a rosette-mediating PfEMP1 variant (IT4var09) cytoadhere in vitro to a human brain microvascular endothelial cell line (HBEC-5i). Cytoadherence was inhibited by heparin and by treatment of HBEC-5i with heparinase III, suggesting that the endothelial receptors for IE binding are heparan sulfate proteoglycans. Antibodies to the N-terminal regions of the IT4var09 PfEMP1 variant (NTS-DBL1α and DBL2γ domains) specifically inhibited and reversed cytoadherence down to low concentrations (<10 µg/ml of total IgG). Surface plasmon resonance experiments showed that the NTS-DBLα and DBL2γ domains bind strongly to heparin, with half-maximal binding at a concentration of ∼0.5 µM in both cases. Therefore, cytoadherence of IT/R29 IE is distinct from rosetting, which is primarily mediated by NTS-DBL1α interactions with complement receptor 1. These data show that IT4var09-expressing parasites are capable of dual interactions with both endothelial cells and uninfected erythrocytes via distinct receptor-ligand interactions.

Onset of rosette formation during spontaneous neural differentiation of hESC and hiPSC colonies.

In vitro neural differentiation of human embryonic stem cells (hESCs) is an advantageous system for studying early neural development. The process of early neural differentiation in hESCs begins by initiation of primitive neuroectoderm, which is manifested by rosette formation, with consecutive differentiation into neural progenitors and early glial-like cells. In this study, we examined the involvement of early neural markers - OTX2, PAX6, Sox1, Nestin, NR2F1, NR2F2, and IRX2 - in the onset of rosette formation, during spontaneous neural differentiation of hESC and human induced pluripotent stem cell (hiPSC) colonies. This is in contrast to the conventional way of studying rosette formation, which involves induction of neuronal differentiation and the utilization of embryoid bodies. Here we show that OTX2 is highly expressed at the onset of rosette formation, when rosettes comprise no more than 3-5 cells, and that its expression precedes that of established markers of early neuronal differentiation. Importantly, the rise of OTX2 expression in these cells coincides with the down-regulation of the pluripotency marker OCT4. Lastly, we show that cells derived from rosettes that emerge during spontaneous differentiation of hESCs or hiPSCs are capable of differentiating into dopaminergic neurons in vitro, and into mature-appearing pyramidal and serotonergic neurons weeks after being injected into the motor cortex of NOD-SCID mice.

Efecto in vitro de una solución de Passiflora incarnata L. sobre la respuesta de linfocitos humanos de donantes sanos y de enfermos con inmunodeficiencia celular / In vitro effect of a Passiflora incarnata L. solution on the response of human lymphocytes from healthy donors and from patients with cellular immunodeficiency

La Passiflora incarnata L. es una especie que se ha utilizado por el hombre con diversos fines. Se estudió el efecto in vitro de un extracto fluido de esta planta sobre los linfocitos de 20 donantes voluntarios de sangre y de 20 enfermos con diagnóstico de inmunodeficiencia celular, mediante la técnica de formación de roseta activa, roseta espontánea y el ultramicrométodo inmunocitoquímico, así como en la función fagocítica de los neutrófilos. No se hallaron diferencias estadísticamente significativas entre las condiciones experimentales sin pasiflora y con pasiflora, en las técnicas de formación de rosetas ni en la expresión de los marcadores de linfocitos CD2 y CD3. Similares resultados se hallaron con la función fagocítica de los neutrófilos en la misma dilución

Passiflora incarnata L. is a species that has been used by man for various purposes. It was studied the effect in vitro of a fluid extract of this plant on lymphocytes from 20 blood donors and 20 patients with cellular immunodeficiency diagnosis, using the technique of active rosette formation, and spontaneous rosette immunocytochemical ultramicromethod and in the phagocytic function of neutrophils. We found no statistically relevant differences between experimental conditions with and without Passiflora, neither in the rosette formation techniques or the expression of lymphocyte markers CD2 and CD3. Similar results were found with the phagocytic function of neutrophils in the same dilution

Evaluación inmunológica de pacientes con parotiditis recurrente / Immunological assessment of patients with recurrent parotiditis

La parotiditis recurrente se define como una inflamación parotídea, generalmente asociada a una sialectasia no obstructiva glandular. Se realizó un estudio en 74 niños menores de 15 años con diagnñstico de parotiditis recurrente en el período de 2000 a 2007. A cada paciente se le realizó interrogatorio, examen físico y estudio inmunológico mediante cuantificación de inmunoglobulinas séricas M y G, rosetas espontánea y activa e índice opsonofagocítico. La enfermedad afectó de forma similar a los 2 sexos. La edad de presentación de la primera crisis fue alrededor de los 3 años, con un promedio de 7 crisis por niño y una duración de 6 d. El 95,9 por ciento de los pacientes presentó alguna alteración de la respuesta inmune, 41,8 por ciento de células T, 12,2 por ciento de células fagocíticas, y 41,8 por ciento combinadas

Recurrent parotiditis is defined as parotic inflammation that is generally associated to non-obstructive glandular sialectasia. Seventy four children under 15 years of age, diagnosed with recurrent parotiditis from 2000 to 2007, were studied. Each patient was questioned and they also underwent physical exam and immunological study through quantification of serum M and G immunoglobulins, the spontaneous and active rosettes and the opsonocytophagic index. The disease affected males and females in a similar way. The age of onset of the first crisis was 3 years, with an average of 7 crises per child and 6 days of duration. Of these patients, 95.9 percent presented with some disorder in the immune response, that is, 41.8 percent in T-cells, 12.2 percent in phagocytic cells and 41.8 percent combined

Efecto in vitro de una solución de Hibiscus elatus SW (Majagua) sobre la respuesta de linfocitos y neutrófilos humanos de donantes sanos y enfermos con inmunodeficiencia celular / In vitro effects of a Hibiscus elatus SW solution (Majagua) on the response of human lymphocytes and neutrophils from healthy donors and ills with cellular immunodeficiency

El Hibiscus elatus SW (majagua) es una especie que se ha utilizado por el hombre con diversos fines. Se estudió el efecto in vitro de una solución acuosa de las flores de esta planta sobre los linfocitos y neutrófilos de 20 donantes de sangre sanos y de 20 enfermos con diagnóstico de inmunodeficiencia celular, mediante la técnica de roseta activa y espontánea, el ultramicrométodo inmunocitoquímico (UMICIQ) y la prueba de función fagocítica. No se hallaron diferencias estadísticamente significativas entre las condiciones experimentales sin Hibiscus elatus SW y con esta planta (dilución 1:2), en los parámetros estudiados

Hibiscus elatus SW (majagua) is a species used by man due to its diverse ends. Authors studied the in vitro effect of a aqueous solution of flowers from this plant on lymphocytes and neutrophils of 20 healthy blood donors and from 20 ills diagnosed with cellular immunodeficiency using active and spontaneous rosette technique, the immunocytochemical ultramicromethod (UMICIQ) and the phagocytic function test. There weren & rsquo;t significant statistically differences among experimental conditions without Hibiscus elatus SW and with this plant (dilution 1:2) in study parameters

Cerebrospinal fluid B lymphocyte identification for diagnosis and follow-up in human African trypanosomiasis in the field.

OBJECTIVES: In human African trypanosomiasis (HAT, sleeping sickness), staging of disease and treatment follow-up relies on white cell count in the cerebrospinal fluid (CSF). As B lymphocytes (CD19 positive cells) are not found in the CSF of healthy individuals but occur in neurological disorders such as multiple sclerosis, B lymphocyte count may be useful for field diagnosis/staging and therapeutic follow-up in HAT. METHODS: Seventy-one HAT patients were diagnosed and 50 were followed-up 6-24 months after treatment. White cell counts were used for conventional staging (stage 1, < or =5 cells/microl CSF, n = 42; stage 2, > or =20 cells/microl, n = 16) and intermediate stage (6-19 cells/microl, n = 13). Slides containing 1 microl of CSF mixed with Dynabeads CD19 pan B were examined microscopically to detect B cell rosettes (bound to at least four beads). RESULTS: Stage 1 patients exhibited zero (n = 37) or one CSF rosette/microl (n = 5), contrary to most stage 2 patients (14/16: > or =2 rosettes/microl). Intermediate stage patients expressed 0 (n = 9), 1 (n = 3) or 2 (n = 1) rosettes/microl of CSF. During follow-up, rosette counts correlated with white cell count staging but were much easier to read. CONCLUSION: B cell rosettes being easily detected in the CSF in field conditions may be proposed to replace white cell count for defining HAT stages 1 and 2 and limit uncertainty in treatment decision in patients with intermediate stage.

Relapsing fever Borrelia binds to neolacto glycans and mediates rosetting of human erythrocytes.

A hallmark of acute relapsing fever borreliosis is severe bacteremia. Some Borrelia species, such as B. duttonii and B. crocidurae, associate with erythrocytes and induce aggregation recognized as erythrocyte rosetting. Erythrocyte rosettes contribute to disease severity by increased tissue invasiveness (such as invasion of CNS and encephalitis), hemorrhaging, and reduced blood flow in affected microcapillaries. Here we report that relapsing fever Borrelia binds to neolacto (Galbeta4GlcNAcbeta3Galbeta4Glcbeta1)-carrying glycoconjugates that are present on human erythrocytes. This interaction is of low affinity but is compensated for by the multivalency of neo-lacto-oligosaccharides on the erythrocyte cell surface. Hence, the protein-carbohydrate interaction is dependent on multivalent neolacto-glycans to mediate binding.

An in vivo and in vitro model of Plasmodium falciparum rosetting and autoagglutination mediated by varO, a group A var gene encoding a frequent serotype.

In the Saimiri sciureus monkey, erythrocytes infected with the varO antigenic variant of the Plasmodium falciparum Palo Alto 89F5 clone bind uninfected red blood cells (rosetting), form autoagglutinates, and have a high multiplication rate, three phenotypic characteristics that are associated with severe malaria in human patients. We report here that varO parasites express a var gene having the characteristics of group A var genes, and we show that the varO Duffy binding-like 1alpha(1) (DBL1alpha(1)) domain is implicated in the rosetting of both S. sciureus and human erythrocytes. The soluble varO N-terminal sequence (NTS)-DBL1alpha(1) recombinant domain, produced in a baculovirus-insect cell system, induced high titers of antibodies that reacted with varO-infected red blood cells and disrupted varO rosettes. varO parasites were culture adapted in vitro using human erythrocytes. They formed rosettes and autoagglutinates, and they had the same surface serotype and expressed the same varO gene as the monkey-propagated parasites. To develop an in vitro model with highly homogeneous varO parasites, rosette purification was combined with positive selection by panning with a varO NTS-DBL1alpha(1)-specific mouse monoclonal antibody. The single-variant, clonal parasites were used to analyze seroprevalence for varO at the village level in a setting where malaria is holoendemic (Dielmo, Senegal). We found 93.6% (95% confidence interval, 89.7 to 96.4%) seroprevalence for varO surface-reacting antibodies and 86.7% (95% confidence interval, 82.8 to 91.6%) seroprevalence for the recombinant NTS-DBL1alpha(1) domain, and virtually all permanent residents had seroconverted by the age of 5 years. These data imply that the varO model is a relevant in vivo and in vitro model for rosetting and autoagglutination that can be used for rational development of vaccine candidates and therapeutic strategies aimed at preventing malaria pathology.

Clinical implications of the infratentorial rosette-forming glioneuronal tumor: case report.

OBJECTIVE: This article describes our experience with two patients who presented with unusual tumors in the cerebellar vermis and cerebral aqueduct. Although sparing the fourth ventricle proper, both tumors had histological features consistent with the rare diagnosis of a rosette-forming glioneuronal tumor of the fourth ventricle, of which only 19 cases have been reported previously. A review of the clinical features and courses of all 21 cases is presented and management recommendations are given. CLINICAL PRESENTATION: Patient 1 was a 42-year-old man who presented with a headache of 1 day's duration and no neurological signs, in whom magnetic resonance imaging disclosed a nonenhancing mass lesion occupying the proximal cerebral aqueduct. Patient 2 was a 38-year-old woman with a long history of intermittent giddiness, no neurological signs, and a magnetic resonance imaging scan that demonstrated a nonenhancing and subtle abnormality in the cerebellar vermis. INTERVENTION: Biopsy was performed on both lesions, the first endoscopically and the second via craniotomy. The only postoperative complication was short-lived double vision and poor upgaze in Patient 1. CONCLUSION: These cases demonstrate that the rosette-forming glioneuronal tumor may be more accurately categorized as an infratentorial tumor rather than a tumor of the fourth ventricle. Because the literature indicates that this is a tumor with little potential for malignant behavior and considerable morbidity can accompany attempts at resection, a conservative management approach would seem well advised. If this tumor is to be managed conservatively, because of the paucity of extended follow-up data, long-term radiological and clinical surveillance is strongly recommended.

Anandamide and Delta9-tetrahydrocannabinol directly inhibit cells of the immune system via CB2 receptors.

This study shows that two cannabinoids, Delta(9)-tetrahydrocannabinol (THC) and anandamide, induce dose-related immunosuppression in both the primary and secondary in vitro plaque-forming cell assays of antibody formation. The immunosuppression induced by both compounds could be blocked by SR144528, an antagonist specific for the CB(2) receptor, but not by SR141716, a CB(1) antagonist. These studies are novel in that they show that both anandamide and THC are active in the nanomolar to picomolar (for anandamide) range in these assays of immune function, and that both mediate their effects directly on cells of the immune system through the CB(2) receptor.

Involvement of dopamine D1 and D2 receptors in the rat nucleus accumbens in immunostimulation.

Analysis of the nature of changes in the immune response in operated Wistar rats showed that electrolytic lesioning of the nucleus accumbens, the site of the greatest density of dopamine D1 and D2 receptors, led to suppression of the immune response in animals immunized with sheep erythrocytes. Administration of SKF 38393 (20 mg/kg) and quinpirol (1 mg/kg), selective agonists of dopamine D1 and D2 receptors respectively, to sham-operated rats induced significant increases in immune responses. However, no immunostimulation was seen on administration of the selective dopamine D2 agonist quinpirol to animals with lesions to the nucleus accumbens as compared with controls. At the same time, treatment of animals with nucleus accumbens lesions using the dopamine D1 receptor agonist SKF 38393 had no effect on the immune response as compared with that in sham-operated animals given the D1 receptor agonist. These data provide evidence that dopamine D2 receptors in the nucleus accumbens have a role in the mechanisms of immunostimulation, though D2 receptors in other brain structures may also make some contribution to this process; D1 receptors in the nucleus accumbens make no significant contribution to controlling the immune response.