Differential Internalization of Amphotericin B - Conjugated Nanoparticles in Human Cells and the Expression of Heat Shock Protein 70

Although a variety of nanoparticles (NPs) functionalized with amphotericin B, an antifungal agent widely used in the clinic, have been studied in the last years their cytotoxicity profile remains elusive. Here we show that human endothelial cells take up high amounts of silica nanoparticles (SNPs) conjugated with amphotericin B (AmB) (SNP-AmB) (65.4 12.4 pg of Si per cell) through macropinocytosis while human fibroblasts internalize relatively low amounts (2.3 0.4 pg of Si per cell) because of their low capacity for macropinocytosis. We further show that concentrations of SNP-AmB and SNP up to 400 mg/mL do not substantially affect fibroblasts. In contrast, endothelial cells are sensitive to low concentrations of NPs (above 10 mg/mL), in particular to SNP-AmB. This is because of their capacity to internalize high concentration of NPs and high sensitivity of their membrane to the effects of AmB. Low-moderate concentrations of SNP-AmB (up to 100 mg/mL) induce the production of reactive oxygen species (ROS), LDH release, high expression of pro-inflammatory cytokines and chemokines (IL-8, IL-6, G-CSF, CCL4, IL-1b and CSF2) and high expression of heat shock proteins (HSPs) at gene and protein levels. High concentrations of SNP-AmB (above 100 ug/mL) disturb membrane integrity and kill rapidly human cells(60% after 5 h). This effect is higher in SNP-AmB than in SNP.

Analysis of Synaptic Proteins in the Cerebrospinal Fluid as a New Tool in the Study of Inborn Errors of Neurotransmission

Abstract In a few rare diseases, specialised studies in cerebrospinal fluid (CSF) are required to identify the underlying metabolic disorder. We aimed to explore the possibility of detecting key synaptic proteins in the CSF, in particular dopaminergic and gabaergic, as new procedures that could be useful for both pathophysiological and diagnostic purposes in investigation of inherited disorders of neurotransmission. Dopamine receptor type 2 (D2R), dopamine transporter (DAT) and vesicular monoamine transporter type 2 (VMAT2) were analysed in CSF samplesfrom 30 healthy controls (11 days to 17 years) by western blot analysis. Because VMAT2 was the only protein with intracellular localisation, and in order to compare results, GABA vesicular transporter, which is another intracellular protein, was also studied. Spearman’s correlation and Student’s t tests were applied to compare optical density signals between different proteins. All these synaptic proteins could be easily detected and quantified in the CSF. DAT, D2R and GABA VT expression decrease with age, particularly in the first months of life, reflecting the expected intense synaptic activity and neuronal circuitry formation. A statistically significant relationship was found between D2R and DAT expression, reinforcing the previous evidence of DAT regulation by D2R. To our knowledge, there are no previous studies on human CSF reporting a reliable analysis of these proteins. These kinds of studies could help elucidate new causes of disturbed dopaminergic and gabaergic transmission as well as understanding different responses to L-dopa in inherited disorders affecting dopamine metabolism. Moreover, this approach to synaptic activity in vivo can be extended to different groups of proteins and diseases.

Cerebrospinal Fluid Synaptic Proteins as Useful Biomarkers in Tyrosine Hydroxylase Deficiency

Tyrosine hydroxylase (TH) deficiency is an inborn error of dopamine biosynthesis and a cause of early parkinsonism. Two clinical phenotypes have been described. Type “B”: early onset severe encephalopathy; type “A”: later onset, less severe and better response to L-dopa. We aimed to study the expression of several key dopaminergic and gabaergic synaptic proteins in the cerebrospinal fluid (CSF) of a series of patients with TH deficiency and their possible relation with the clinical phenotype and response to L-DOPA. Dopamine transporter (DAT), D2-receptor and vesicularmonoamine transporter (VMAT2)weremeasured in the CSF of 10 subjectswith THdeficiency byWestern blot analysis. In 3 patients, data of pre- and post-treatmentwith L-DOPA were available, and in one of them, GABA vesicular transporter was determined. Results were compared to an age-matched control population. The concentration of D2-receptors in CSFwas significantly higher in patients with TH deficiency than in controls. Similarly, DAT and vesicular monoamine transporter type 2 were up-regulated. Studies performed before LDOPA, and on L-DOPA therapy showed a paradoxical response with D2 receptor expression increase as L-Dopa doses and homovanillic concentration gradually raised in a B phenotype patient. The opposite results were found in two patients with A phenotype. However, this is a very small sample, and further studies are needed to conclude robust differences between phenotypes. Synaptic proteins are detectable in the CSF and their quantification can be useful for understanding the pathophysiology of neurotransmitter defects and potentially to adjust and personalize treatments in the future.

Transthyretin Amyloidosis: Chaperone Concentration Changes and Increased Proteolysis in the Pathway to Disease

Transthyretin amyloidosis is a conformational pathology characterized by the extracellular formation of amyloid deposits and the progressive impairment of the peripheral nervous system. Point mutations in this tetrameric plasma protein decrease its stability and are linked to disease onset and progression. Since non-mutated transthyretin also forms amyloid in systemic senile amyloidosis and some mutation bearers are asymptomatic throughout their lives, non-genetic factors must also be involved in transthyretin amyloidosis. We discovered, using a differential proteomics approach, that extracellular chaperones such as fibrinogen, clusterin, haptoglobin, alpha-1-anti-trypsin and 2-macroglobulin are overrepresented in transthyretin amyloidosis. Our data shows that a complex network of extracellular chaperones are over represented in human plasma and we speculate that they act synergistically to cope with amyloid prone proteins. Proteostasis may thus be as important as point mutations in transthyretin amyloidosis.

Identification of de Novo Germline Mutations in the HRPT2 Gene in Two Apparently Sporadic Cases with Challenging Parathyroid Tumor Diagnoses

The diagnosis of parathyroid carcinomas is often difficult. HRPT2 mutations have been identified in familial [hyperparathyroidism-jaw tumor (HPT-JT) syndrome] and sporadic parathyroid carcinomas, supporting that HRPT2 mutations may confer a malignant potential to parathyroid tumors. In this study, we report the clinical, histopathological, and genetic investigation of two unrelated cases, whom had apparently sporadic malignant parathyroid tumors, initially diagnosed as adenomas. In one case, the differential diagnosis was complicated by cervical seeding of parathyroid tumor cells. Genetic studies identified de novo HRPT2 germline mutations in cases 1 (c.518_521delTGTC [p.Ser174LysfsX27]) and 2 (c.226 C > T [p.Arg76X]), unveiling the hereditary HPT-JT syndrome in both patients. Furthermore, the identification of somatic mutations in the patients‟ parathyroid tumors provided evidence for complete inactivation of the HRPT2 gene, which was consistent with the tumor malignant features. The sensitivity of parafibromin immunostaining to detect HRPT2 mutations was limited. The present data suggests that patients with apparently sporadic parathyroid carcinomas, or parathyroid tumors with atypical histological features, should undergo molecular genetic testing, as it may detect germline HRPT2 mutations. Establishing the diagnosis of hereditary HPT-JT syndrome is relevant for clinical counseling and management of the carriers and their relatives.