Ana Torbidoni

Decoration of nanoparticles with specific molecules such as antibodies, peptides, and proteins that preserve their biological properties is essential for the recognition and internalization of their specific target cells. Inefficient preparation of such decorated nanoparticles leads to nonspecific interactions diverting them from their desired target. We report a simple two-step procedure for the preparation of biohybrid nanoparticles containing a core of hydrophobic quantum dots coated with a multilayer of human serum albumin. These nanoparticles were prepared by ultra-sonication, crosslinked using glutaraldehyde, and decorated with proteins such as human serum albumin or human transferrin in their native conformations. These nanoparticles were homogeneous in size (20–30 nm), retained the fluorescent properties of quantum dots, and did not show a “corona effect” in the presence of serum. The uptake of transferrin-decorated quantum dot nanoparticles was observed in A549 lung cancer ...

Trilateral retinoblastoma (TRb) presents a management challenge, since intracranial tumours are seldom times resectable and quickly disseminate. However, there are no risk factors to predict the final outcome in each patient.ObjectiveTo evaluate minimal disseminated disease (MDD) in the bone marrow (BM) and the cerebrospinal fluid (CSF) at diagnosis and during follow-up and reviewing its potential impact in the outcome of patients with TRb.Methods and analysisWe evaluated MDD in five patients with TRb, detecting the mRNA of CRX and/or GD2, in samples from BM and CSF, obtained at diagnosis, follow-up and relapse.ResultsTreatment involved intensive systemic chemotherapy in four patients, one did not receive this treatment and died of progression of the disease. Two patients underwent stem cell rescue. Three patients had leptomeningeal relapse and died. One patient remains disease-free for 84 months. RB1 mutations were identified in the five patients, all of them were null mutations. A...

Intravitreal injection of chemotherapy in retinoblastoma eyes with vitreous seeds may lead to a risk of extraocular tumour dissemination that has not been assessed so far. To develop a sensitive and clinically feasible technique to assess for potential retinoblastoma cell reflux after intravitreal injection of melphalan. Filter papers were cut in 6 mm diameter circles and sterilised before use. Eyes with retinoblastoma vitreous seeds (group D, International Classification) received weekly intravitreal melphalan injections (20 µg or 30 µg/dose) followed by cryotherapy as part of local treatment. Immediately after finishing the injection and cryotherapy, filter papers were placed on the injection site and on the cryoprobe tip to assess for the expression of the cone-rod homeobox gene (CRX) by real-time qPCR as a surrogate of retinoblastoma RNA. The assay was developed and validated to determine sensitivity, linearity, recovery, repeatability and reproducibility. The assay for quantita...

Fatal metastatic relapse may occur in children with retinoblastoma and high-risk pathologic features (HRPFs). Minimal dissemination (MD) may be an additional tool for risk estimation. The use of cone-rod homeobox (CRX) transcription factor messenger RNA for MD evaluation in metastatic retinoblastoma was previously reported, but no data in nonmetastatic cases with HRPFs are available. To evaluate whether MD is detectable in patients with nonmetastatic retinoblastoma and to assess its prognostic effect on disease-free survival (DFS). This single-institution cohort study of patients with nonmetastatic retinoblastoma and HRPFs used prospectively defined inclusion criteria and a sampling strategy to procure bone marrow (BM) and cerebrospinal fluid (CSF) samples from May 1, 2007, through October 31, 2013. Median follow-up was 38 months (range, 8-89 months). Survival analysis was closed in December 2015, and no further updates were made after that point. The study evaluated CRX messenger R...

Disseminated retinoblastoma is usually fatal. Identification of small amounts (minimal dissemination [MD]) of tumor cells in extraocular sites might be a tool for designing appropriate treatments. To test cone-rod homeobox (CRX) transcription factor as a lineage-specific molecular marker for metastatic retinoblastoma and for evaluation of MD. In a prospective cohort design study, we evaluated CRX messenger RNA (mRNA) by retrotranscription followed by real-time polymerase chain reaction as a diagnostic test in samples obtained from bone marrow, peripheral blood, and cerebrospinal fluid (CSF) at diagnosis, after induction chemotherapy, and during follow-up. The study was conducted from June 30, 2008, to June 30, 2014. Seventeen retinoblastoma primary tumors, 2 retinoblastoma cell lines, and 47 samples of bone marrow from other cancers (controls) were studied. Seventeen patients with metastatic retinoblastoma (9 at diagnosis, 8 at relapse; age range: 18-41 months) were included. Detection of CRX mRNA as a marker for metastatic retinoblastoma and MD in bone marrow and CSF and its correlation with clinical findings. Cone-rod homeobox mRNA was expressed in all tumors (relative expression levels range, 8.1 × 10-5 to 5.6) and cell lines. In control samples, there was no amplification of CRX; only the housekeeping gene (GAPDH) demonstrated amplification. Bone marrow metastatic cells showed expression of CRX mRNA in all 9 children presenting with metastasis at the diagnosis (relative expression levels, 6.0 × 10-5 to 0.67). After induction chemotherapy, no evidence of MD of tumor cells was seen in any of the 8 responding children since only GAPDH showed amplification. In the CSF of children who had a metastatic relapse, CRX mRNA detection was positive in 2 patients in whom no conclusive results were reached by immunocytology for disialoganglioside GD2. Minimal dissemination in the CSF was associated with a clinical relapse in 2 cases. No concomitant MD was evident in the bone marrow in any case. These data suggest that CRX mRNA is a novel marker for retinoblastoma at extraocular sites. In this study among patients with bone marrow metastasis, there was a quick, complete, and sustained molecular response after induction chemotherapy. In all patients with secondary metastasis, CSF relapse occurred independently from the bone marrow, suggesting a sanctuary site.

To characterize melphalan pharmacokinetics after superselective ophthalmic artery infusion (SSOAI) in animals and children with retinoblastoma. Vitreous and plasma samples of five Landrace pigs were obtained over a 4-hour period after SSOAI of melphalan (7 mg). Melphalan cytotoxicity was evaluated in retinoblastoma cell lines with and without topotecan. Plasma samples were obtained from 17 retinoblastoma patients after SSOAI of 3 to 6 mg of melphalan to one (n=14) or two eyes (n=3). Correlation between plasma pharmacokinetics and age, dosage, and systemic toxicity was studied in patients. In animals, melphalan peak vitreous levels were greater than its IC50 and resulted in 3-fold vitreous-to-plasma exposure. In patients, a large variability in pharmacokinetic parameters was observed and it was explained mainly by body weight (P<0.05). A significantly higher systemic area under the curve was obtained in children receiving more than 0.48 mg/kg for bilateral tandem infusions (P<0.05). These children had 50% probability of grades 3-4 neutropenia. Plasma concentrations after 2 and 4 hours of SSOAI were significantly higher in these children (P<0.05). A synergistic cytotoxic effect of melphalan and topotecan was evident in cell lines. Potentially active levels of melphalan after SSOAI were achieved in the vitreous of animals. Low systemic exposure was found in animals and children. Doses greater than 0.48 mg/kg, given for bilateral tandem infusions, were associated with significantly higher plasma levels and increased risk of neutropenia. Synergistic in vitro cytotoxicity between melphalan and topotecan favors combination treatment.

Treatment of intraocular retinoblastoma with vitreous seeding is a challenge. Different routes of chemotherapy administration have been explored in order to attaining pharmacological concentrations into the posterior chamber. Intravitreal drug injection is a promissing route for maximum bioavailability to the vitreous but it requires a well defined dose for achieving tumor control while limited toxicity to the retina. Topotecan proved to be a promising agent for retinoblastoma treatment due to its pharmacological activity and limited toxicity. High and prolonged concentrations were achieved in the rabbit vitreous after 5 μg of intravitreal topotecan. However, whether a lower dose could achieve potentially therapeutic levels remained to be determined. Thus, we here study the pharmacokinetics of topotecan after 0.5 μg and the toxicity profile of intravitreal topotecan in the rabbit eye as a potential treatment of retinoblastoma. A cohort of rabbits was used to study topotecan disposition in the vitreous after a single dose of 0.5 μg of intravitreal topotecan. In addition, an independent cohort of non-tumor bearing rabbits was employed to evaluate the clinical and retinal toxicity after four weekly injections of two different doses of intravitreal topotecan (Group A, 5 μg/dose; Group B, 0.5 μg/dose) to the right eye of each animal. The same volume (0.1 ml) of normal saline was administered to the left eye as control. A third group of rabbits (Group C) served as double control (both eyes injected with normal saline). Animals were weekly evaluated for clinical and hematologic values and ocular evaluations were performed with an inverse ophthalmoscope to establish potential topotecan toxicity. Weekly controls included topotecan quantitation in plasma of all rabbits. Electroretinograms (ERGs) were recorded before and after topotecan doses. One week after the last injection, topotecan concentrations were measured in vitreous of all eyes and samples for retinal histology were obtained. Our results indicate that topotecan shows non linear pharmacokinetics after a single intravitreal dose in the range of 0.5-5 μg in the rabbit. Vitreous concentration of lactone topotecan was close to the concentration assumed to be therapeutically active after 5 h of 0.5 μg intravitreal administration. Eyes injected with four weekly doses of topotecan (0.5 or 5 μg/dose) showed no significant differences in their ERG wave amplitudes and implicit times in comparison with control (p > 0.05). Animals showed no weight, hair loss or significant changes in hematologic values during the study period. There were no significant histologic damage of the retinas exposed to topotecan treatments. After intravitreal administration no topotecan could be detected in plasma during the follow-up period nor in the vitreous of treated and control animals after 1 week of the last injection. The present data shows that four weekly intravitreal injection of 5 μg of topotecan is safe for the rabbit eye. Despite multiple injections of 0.5 μg of topotecan are also safe to the rabbit eye, lactone topotecan vitreous concentrations were potentially active only after 5 h of the administration. We postulate promising translation to clinics for retinoblastoma treatment.

The synapse between olivocochlear (OC) neurons and cochlear mechanosensory hair cells is cholinergic, fast, and inhibitory. The inhibitory sign of this cholinergic synapse is accounted for by the activation of Ca(2+)-permeable postsynaptic α9α10 nicotinic receptors coupled to the opening of hyperpolarizing Ca(2+)-activated small-conductance type 2 (SK2)K(+) channels. Acetylcholine (ACh) release at this synapse is supported by both P/Q- and N-type voltage-gated calcium channels (VGCCs). Although the OC synapse is cholinergic, an abundant OC GABA innervation is present along the mammalian cochlea. The role of this neurotransmitter at the OC efferent innervation, however, is for the most part unknown. We show that GABA fails to evoke fast postsynaptic inhibitory currents in apical developing inner and outer hair cells. However, electrical stimulation of OC efferent fibers activates presynaptic GABA(B(1a,2)) receptors [GABA(B(1a,2))Rs] that downregulate the amount of ACh released at the OC-hair cell synapse, by inhibiting P/Q-type VGCCs. We confirmed the expression of GABA(B)Rs at OC terminals contacting the hair cells by coimmunostaining for GFP and synaptophysin in transgenic mice expressing GABA(B1)-GFP fusion proteins. Moreover, coimmunostaining with antibodies against the GABA synthetic enzyme glutamic acid decarboxylase and synaptophysin support the idea that GABA is directly synthesized at OC terminals contacting the hair cells during development. Thus, we demonstrate for the first time a physiological role for GABA in cochlear synaptic function. In addition, our data suggest that the GABA(B1a) isoform selectively inhibits release at efferent cholinergic synapses.