Understanding the nanoparticle-protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles

Due to their small size, nanoparticles have distinct properties compared with the bulk form of the same materials. These properties are rapidly revolutionizing many areas of medicine and technology. Despite the remarkable speed of development of nanoscience, relatively little is known about the interaction of nanoscale objects with living systems. In a biological fluid, proteins associate with nanoparticles, and the amount and presentation of the proteins on the surface of the particles leads to an in vivo response. Proteins compete for the nanoparticle "surface," leading to a protein "corona" that largely defines the biological identity of the particle. Thus, knowledge of rates, affinities, and stoichiometries of protein association with, and dissociation from, nanoparticles is important for understanding the nature of the particle surface seen by the functional machinery of cells. Here we develop approaches to study these parameters and apply them to plasma and simple model systems, albumin and fibrinogen. A series of copolymer nanoparticles are used with variation of size and composition (hydrophobicity). We show that isothermal titration calorimetry is suitable for studying the affinity and stoichiometry of protein binding to nanoparticles. We determine the rates of protein association and dissociation using surface plasmon resonance technology with nanoparticles that are thiol-linked to gold, and through size exclusion chromatography of protein-nanoparticle mixtures. This method is less perturbing than centrifugation, and is developed into a systematic methodology to isolate nanoparticle-associated proteins. The kinetic and equilibrium binding properties depend on protein identity as well as particle surface characteristics and size.     

Sulfonation and sialylation of gonadotropins in women during the menstrual cycle, after menopause, and with polycystic ovarian syndrome and in men

CONTEXT: More basic isoforms of LH and FSH appear in blood at midcycle and more acidic after menopause. The LH isoforms are more basic in women with polycystic ovarian syndrome (PCOS). These charge alterations may reflect differences in the number of two negatively charged residues on the gonadotropins: sialic acid and sulfonated N-acetylgalactosamine, residues that modulate the half-life of the gonadotropins in blood. OBJECTIVE: The objective of the study was to determine the contributions of sialic acid and sulfonated N-acetylgalactosamine and sialic acid on LH and FSH to the observed alterations in charge. DESIGN/PARTICIPANTS: Serum samples were obtained from 59 young women with regular cycles, nine postmenopausal women, 12 women with PCOS, and 40 young men. MAIN OUTCOME MEASURES: The number of sulfonated N-acetylgalactosamine and sialic acid residues per LH and FSH molecule in serum and the distributions of molecules with 0-1-2-3-4 sulfonated residues were determined by electrophoretic analyses before and after removal of sialic acid. RESULTS: Considerably decreased sulfonation of LH was found at midcycle and in women with PCOS concomitant with slightly increased sialylation. The sulfonation of LH increased in the luteal phase, and the sialylation was highest after menopause for both hormones. The frequencies of sulfonated LH and FSH isoforms were directly related (P < 0.01) to body mass index in women with PCOS. CONCLUSION: The observed variations in sialic acid and sulfonated residues on serum gonadotropins are suggested to reflect alterations in the isoform composition of the hormones secreted by the pituitary, resulting in modulations of their biological properties, such as half-life in blood.     

Cellular injury and neuroinflammation in children with chronic intractable epilepsy

Objective  

To elucidate the presence and potential involvement of brain inflammation and cell death in neurological morbidity and intractable seizures in childhood epilepsy, we quantified cell death, astrocyte proliferation, microglial activation and cytokine release in brain tissue from patients who underwent epilepsy surgery.     

Some microbiological, histopathological and immunohistochemical characteristics of progressive periodontal disease

Abstract The aim of the present investigation was to study the local nature of human periodontal disease by assessing the microbiota and the composition of the tissue lesions at sites with progressive attachment loss in periodontitis susceptible subjects. 300 subjects with periodontal disease were monitored for 2 years without treatment. 8 subjects lost >2 mm of attachment at 3 sites during both the first and the second 12 month interval. These 8 subjects (progressive disease group; PD)were recalled for a microbiological and histopathological examination. A group of age- and sex-matched subjects were identified who during the 2 years of monitoring exhibited gingivitis and deep pockets, but no further attachment loss. This group of 11 subjects (non-progressive disease group; NPD) served as controls. From the 8 active disease subjects, 1 interproximal site which had displayed disease activity (progressive disease active; PDA) and 1 contralateral site without disease progression (progressive disease inactive; PDI) were sampled. From the 11 control subjects, 1 site/subject was sampled (NPD). The total number of viable micro-organisms (TVC) in the subgingival microbiota was estimated and a series of bacterial species were identified and enumerated. The gingival tissue of the sampling site was excised and the soft tissue prepared for morphometrical and immunohistochemical analyses. No differences were observed in the subgingival microbiota of the sample sites in the subjects who exhibited disease progression (PD) when compared with the subjects with periodontally diseased but stable conditions (NPD). Furthermore, no marked difference could be noted between progressive (PDA) and non-progressive (PDI) sites in the PD group of subjects. The results from the morphometric determinations revealed that the lesions from the PD and NPD subjects on the average were of similar size, but the PD lesions were comprised of a larger relative volume of plasma cells, a higher % number of plasma cells and monocytes/macrophages and a lower numerical density of lymphocytes than the corresponding sites in the NPD group. Both T cell markers (CDS and CD4) and B cell markers (CD22) examined were significantly elevated in the PDA compared to the PDI lesions. The CD4/CD8 ratios calculated from assessments made in the PD and NPD tissue samples were 2.4 and 2, while the corresponding ratios for PDA and PDI lesions were 3 and 2.1 (p<0.05) respectively. The present data indicate that differences exist between disease active and inactive subjects and sites and it is suggested that the human model described may be used to study disease progression using shorter time intervals between examinations and additional parameters.     

Epithelial remnants in the crestal periodontium of the deciduous and permanent dentition of beagle dogs

Abstract. The purpose of this study was to document and characterize epithelial remnants (EPRs) of the crestal periodontium of the deciduous dentition of a diphyodont and compare them with EPR units found in the corresponding area of the permanent dentition. 7 beagle dogs were used. At the age of 10 weeks (deciduous dentition) and 15 months (permanent dentition), respectively, a 6-week plaque control period was initiated. At the end of each plaque control period, biopsies were obtained from the mandibular ₀₂P, ₀₃P (deciduous dentition) and P₃, P₄ (permanent dentition) premolar regions and prepared for histologic analysis. 2 regions, (1) the supracrestal region and (2) the periodontal ligament region, were identified. The supracrestal region was divided into 4 compartments of equal height. The histologic parameters studied included the (i) EPR frequency: number of EPRs/mm of root length, (ii) EPR size, (iii) EPR-root distance, (iv) EPR-bone distance and (v) cell area. No differences were observed between the 2 dentitions with respect to the number, size and relative location of EPR units in the supracrestal regions or the periodontal ligament regions. Epithelial remnants of the supracrestal region in both dentitions tended to be more frequent, larger and positioned further from the root surface than the EPRs of the periodontal ligament region. EPR units of the periodontal ligament region were located significantly further from the bone in the deciduous dentition than in the permanent dentition. The cell area of EPRs did not differ between the 2 dentitions. It was concluded that EPRs are a normal component of the crestal periodontal tissues of the deciduous dentitions of the diphyodont beagle dog and they appear to be similar to those found in the permanent dentition of young dogs.     

Some anatomical features of the periodontium of the deciduous and permanent dentition in the beagle dog

The present study describes some anatomical characteristics of teeth and periodontal tissues in the deciduous and permanent dentition of the beagle dog. Five animals were used. At the age of 10 weeks (Period A) and 15 months (Period B), respectively, a plaque control period was initiated. At the end of each plaque control period, clinical examinations were performed. Biopsies were obtained from the 02P, 03P (Period A) and P3, P4 (Period B) tooth regions and were examined with the light microscope. Histometric and morphometric measurements were made. The macroscopic and microscopic measurements revealed that marked differences exist between the teeth and the periodontium of the deciduous and the permanent dentition. The permanent premolar erupting into the position of the deciduous premolar was found to be significantly wider and higher than its precursor. Also the shape of the crown of the permanent premolar differed from that of the deciduous premolar. The sinuous contour of the buccal gingival margin was more accentuated in the deciduous than in the permanent dentition. The free gingiva was shorter and the periodontal ligament space was wider in the deciduous than in the permanent dentition. The free gingival unit in the deciduous dentition consisted of a larger volume of epithelium and a smaller volume of connective tissue than the corresponding unit in the permanent dentition. The connective tissue of the deciduous gingiva contained a larger proportion of fibroblasts and a lower proportion of collagen fibres than the corresponding tissue of the permanent gingiva.     

Processing of the lipocalin alpha(1)-microglobulin by hemoglobin induces heme-binding and heme-degradation properties

Alpha(1)-microglobulin is a 26-kd protein, widespread in plasma and tissues and well-conserved among vertebrates. Alpha(1)-microglobulin belongs to the lipocalins, a protein superfamily with highly conserved 3-dimensional structures, forming an internal ligand binding pocket. The protein, isolated from urine, has a heterogeneous yellow-brown chromophore bound covalently to amino acid side groups around the entrance of the lipocalin pocket. Alpha(1)-microglobulin is found in blood both in free form and complex-bound to immunoglobulin A (IgA) via a half-cystine residue at position 34. It is shown here that an alpha(1)-microglobulin species, which we name t-alpha(1)-microglobulin (t = truncated), with a free Cys34 thiol group, lacking its C-terminal tetrapeptide, LIPR, and with a more polar environment around the entrance of the lipocalin pocket, is released from IgA-alpha(1)-microglobulin as well as from free alpha(1)-microglobulin when exposed to the cytosolic side of erythrocyte membranes or to purified oxyhemoglobin. The processed t-alpha(1)-microglobulin binds heme and the alpha(1)-microglobulin-heme complex shows a time-dependent spectral rearrangement, suggestive of degradation of heme concomitantly with formation of a heterogeneous chromophore associated with the protein. The processed t-alpha(1)-microglobulin is found in normal and pathologic human urine, indicating that the cleavage process occurs in vivo. The results suggest that alpha(1)-microglobulin is involved in extracellular heme catabolism.