Low Molecular Weight Aggregates Research Articles

Structural Studies of Monounsaturated and ω-3 Polyunsaturated Free Fatty Acids in Solution with the Combined Use οf NMR and DFT Calculations-Comparison with the Liquid State.

Molecular structures, in chloroform and DMSO solution, of the free fatty acids (FFAs) caproleic acid, oleic acid, α-linolenic acid, eicosapentanoic acid (EPA) and docosahexaenoic acid (DHA) are reported with the combined use of NMR and DFT calculations. Variable temperature and concentration chemical shifts of the COOH protons, transient 1D NOE experiments and DFT calculations demonstrate the major contribution of low molecular weight aggregates of dimerized fatty acids through intermolecular hydrogen bond interactions of the carboxylic groups, with parallel and antiparallel interdigitated structures even at the low concentration of 20 mM in CDCl3. For the dimeric DHA, a structural model of an intermolecular hydrogen bond through carboxylic groups and an intermolecular hydrogen bond between the carboxylic group of one molecule and the ω-3 double bond of a second molecule is shown to play a role. In DMSO-d6 solution, NMR and DFT studies show that the carboxylic groups form strong intermolecular hydrogen bond interactions with a single discrete solvation molecule of DMSO. These solvation species form parallel and antiparallel interdigitated structures of low molecular weight, as in chloroform solution. This structural motif, therefore, is an intrinsic property of the FFAs, which is not strongly affected by the length and degree of unsaturation of the chain and the hydrogen bond ability of the solvent.

How Single Site Mutations Can Help Understanding Structure Formation of Amyloid β1-40.

Amyloid fibrils represent the structural endpoint on the energetic (mis)folding landscape of very many proteins. Physiologically, amyloid fibrils are observed as a characteristic hallmark in misfolding diseases often associated with degenerative and neurodegenerative disorders. In the beginning of the scientific discussion, the focus is laid on the fibrillar state, but over the time it becomes increasingly clear that low molecular weight and transient aggregates are of crucial importance for pathological mechanisms. Structural studies find different intra- and intermolecular contacts for the most well-studied peptide amyloid β (Aβ) depending on the stage of fibrillation. In particular, the contact between residues phenylalanine 19 (F19) and leucine 34 (L34) seems to be highly conserved, suggesting that it must be of particular significance for Aβ misfolding and possibly the pathological properties of the peptide. This review aims to highlight the rational and the usefulness of point mutations in Aβ peptides and their impact on the critical interstrand contact F19-L34 depending on the stage of fibrillation. While the amyloid structure of Aβ is very robust against quite a few modifications, the toxicity of mutated Aβ molecules highly depends on the F19-L34 contact.

Biological and methodological complexities of beta-amyloid peptide: Implications for Alzheimer's disease research.

Although controversial, the amyloid cascade hypothesis remains central to the Alzheimer's disease (AD) field and posits amyloid-beta (Aβ) as the central factor initiating disease onset. In recent years, there has been an increase in emphasis on studying the role of low molecular weight aggregates, such as oligomers, which are suggested to be more neurotoxic than fibrillary Aβ. Other Aβ isoforms, such as truncated Aβ, have also been implicated in disease. However, developing a clear understanding of AD pathogenesis has been hampered by the complexity of Aβ biochemistry in vitro and in vivo. This review explores factors contributing to the lack of consistency in experimental approaches taken to model Aβ aggregation and toxicity and provides an overview of the different techniques available to analyse Aβ, such as electron and atomic force microscopy, nuclear magnetic resonance spectroscopy, dye-based assays, size exclusion chromatography, mass spectrometry and SDS-PAGE. The review also explores how different types of Aβ can influence Aβ aggregation and toxicity, leading to variation in experimental outcomes, further highlighting the need for standardisation in Aβ preparations and methods used in current research.

Understanding the removal of V, Ni and S in crude oil atmospheric residue hydrodemetallization and hydrodesulfurization

This study describes the use of gel permeation chromatography inductively coupled plasma high-resolution mass spectrometry (GPC ICP HRMS) to examine and explain two important petroleum industry catalytic processes: hydrodemetallization (HDM) and hydrodesulfurization (HDS).The sulfur, nickel and vanadium species size distributions in atmospheric residue fractions were studied to track their evolution during both catalytic processes and examine their mechanisms, especially those mechanisms linked to changes in temperature and initial deactivation via coke laydown. Chromatogram shapes as well as peak areas were used to study the V, Ni and S aggregate types and concentrations in the feedstock as well as in the product after varying the operating temperature and residence time at a constant temperature. For the V and Ni compounds, the low and medium molecular weight (LMW and MMW, respectively) aggregates are easily hydrotreated under all conditions, while the high molecular weight (HMW) compounds are more refractory. For the S compounds, a different reactivity pattern was observed whereby the LMW, MMW and HMW aggregates were more similar in their reactivity, showing that the catalyst is less selective towards a certain aggregate size for the S compounds compared to the V or Ni compounds.

Influence of low-molecular-weight aggregates on aggregate growth kinetics and physical properties of solid-state proteins during storage

Low-molecular-weight (LMW) aggregate is a critical determinant of subsequent protein aggregation, but the aggregate growth kinetics of solid proteins have not been fully characterized. In this study, the high-molecular weight (HMW) aggregate formation process for solid-state proteins and the relationship between aggregation and physical properties of tablets were evaluated using proteins with various initial aggregate ratios. Quantitative changes in monomers, LMW aggregates, and HMW aggregates during storage were measured. The monomer amount decreased uniformly for all proteins. Proteins with low initial LMW aggregates showed remarkable increases in LMW aggregates but little increases in HMW aggregates during storage. Proteins with high initial LMW aggregates showed decreases in LMW aggregates but remarkable increases in HMW aggregates. A correlation analysis and logistic regression indicated that HMW aggregate formation depended on the initial quantity of LMW aggregates. Furthermore, the initial LMW aggregate ratio was related to the disintegratability of protein-containing tablets after storage. These results provide novel insight into solid-state protein aggregation and may guide the prediction of the long-term quality of solid protein-containing pharmaceuticals and foods without storage.

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy.

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with neurodegenerative conditions such as Alzheimer's and Parkinson's diseases. In particular low molecular weight aggregates, amyloid oligomers, have been shown to possess generic cytotoxic properties and are implicated as neurotoxins in many forms of dementia. We illustrate the use of methods based on atomic force microscopy (AFM) to address the challenging task of characterizing the morphological, structural and chemical properties of these aggregates, which are difficult to study using conventional structural methods or bulk biophysical methods because of their heterogeneity and transient nature. Scanning probe microscopy approaches are now capable of investigating the morphology of amyloid aggregates with sub-nanometer resolution. We show here that infrared (IR) nanospectroscopy (AFM-IR), which simultaneously exploits the high resolution of AFM and the chemical recognition power of IR spectroscopy, can go further and enable the characterization of the structural properties of individual protein aggregates, and thus offer insights into the aggregation mechanisms. Since the approach that we describe can be applied also to the investigations of the interactions of protein assemblies with small molecules and antibodies, it can deliver fundamental information to develop new therapeutic compounds to diagnose or treat neurodegenerative disorders.

Polymeric Micelles and Vesicles in Polyelectrolyte Multilayers: Introducing Hierarchy and Compartmentalization

The incorporation of functional colloidal building blocks into polyelectrolyte multilayers provides the films with a higher level of hierarchy and separate compartments. The simplicity of the layer‐by‐layer approach meets the complexity of supramolecular aggregates, such as micelles or vesicles from block copolymers, thereby opening a venue towards functional coatings with new functionality at the interface. The scope of this progress report is to highlight recent developments in the incorporation of polymeric micellar and vesicular aggregates into functional and hierarchically structured multilayer films. The stability and functionality of these films, especially in terms of responsiveness, are discussed on the basis of their physico‐chemical properties and compared to films containing low molecular weight aggregates. Particular emphasis is on coatings with a reversible response to external signals, rendering these films potential surface‐mediated drug delivery systems with the possibility of refilling hydrophobic and/or hydrophilic reservoirs. Finally, prospects of future developments are given based on recent advances in synthetic polymer chemistry.

Size-exclusion chromatography-mass spectrometry with m-nitrobenzyl alcohol as post-column additive for direct characterization of size variants of monoclonal antibodies.

Size-exclusion chromatography (SEC) is commonly used to monitor low molecular weight fragments and aggregates present in recombinant monoclonal antibody (mAb) biopharmaceuticals. It has been previously demonstrated that SEC could be coupled with mass spectrometry (MS) to directly measure the molecular weights of these protein species to aid in their identification. However, the use of certain mobile phase modifiers led to compromised sensitivity in MS detection. In this work, we demonstrate that the use of m-nitrobenzyl alcohol (m-NBA) as a post-column additive in an SEC-MS method is able to improve the ionization of antibody light chain and heavy chain approximately 7-fold and 2-fold, respectively, and thus allows the MS detection of low-abundance size variants present in mAb biopharmaceuticals. Application of the 15-min reducing SEC-UV/MS method enabled the direct identification of size variants present in an IgG1 mAb sample. One high molecular weight species observed under reducing conditions was identified to be a thioether-linked heterodimer of light chain and heavy chain. Multiple lower molecular weight species were found to result from cleavage of the heavy chain at a number of sites throughout the conserved sequence. The reducing SEC-UV/MS method provides a straightforward approach for identification of size variants present in mAb and may be applicable generally to all types of mAb biopharmaceuticals.

Analyses of Protease Resistance and Aggregation State of Abnormal Prion Protein across the Spectrum of Human Prions

Prion diseases are characterized by tissue accumulation of a misfolded, β-sheet-enriched isoform (scrapie prion protein (PrP(Sc))) of the cellular prion protein (PrP(C)). At variance with PrP(C), PrP(Sc) shows a partial resistance to protease digestion and forms highly aggregated and detergent-insoluble polymers, two properties that have been consistently used to distinguish the two proteins. In recent years, however, the idea that PrP(Sc) itself comprises heterogeneous species has grown. Most importantly, a putative proteinase K (PK)-sensitive form of PrP(Sc) (sPrP(Sc)) is being increasingly investigated for its possible role in prion infectivity, neurotoxicity, and strain variability. The study of sPrP(Sc), however, remains technically challenging because of the need of separating it from PrP(C) without using proteases. In this study, we have systematically analyzed both PK resistance and the aggregation state of purified PrP(Sc) across the whole spectrum of the currently characterized human prion strains. The results show that PrP(Sc) isolates manifest significant strain-specific differences in their PK digestion profile that are only partially explained by differences in the size of aggregates, suggesting that other factors, likely acting on PrP(Sc) aggregate stability, determine its resistance to proteolysis. Fully protease-sensitive low molecular weight aggregates were detected in all isolates but in a limited proportion of the overall PrP(Sc) (i.e. <10%), arguing against a significant role of slowly sedimenting PK-sensitive PrP(Sc) in the biogenesis of prion strains. Finally, we highlight the limitations of current operational definitions of sPrP(Sc) and of the quantitative analytical measurements that are not based on the isolation of a fully PK-sensitive PrP(Sc) form.

Small Misfolded Tau Species Are Internalized via Bulk Endocytosis and Anterogradely and Retrogradely Transported in Neurons

The accumulation of Tau into aggregates is associated with key pathological events in frontotemporal lobe degeneration (FTD-Tau) and Alzheimer disease (AD). Recent data have shown that misfolded Tau can be internalized by cells in vitro (Frost, B., Jacks, R. L., and Diamond, M. I. (2009) J. Biol. Chem. 284, 12845-12852) and propagate pathology in vivo (Clavaguera, F., Bolmont, T., Crowther, R. A., Abramowski, D., Frank, S., Probst, A., Fraser, G., Stalder, A. K., Beibel, M., Staufenbiel, M., Jucker, M., Goedert, M., and Tolnay, M. (2009) Nat. Cell Biol. 11, 909-913; Lasagna-Reeves, C. A., Castillo-Carranza, D. L., Sengupta, U., Guerrero-Munoz, M. J., Kiritoshi, T., Neugebauer, V., Jackson, G. R., and Kayed, R. (2012) Sci. Rep. 2, 700). Here we show that recombinant Tau misfolds into low molecular weight (LMW) aggregates prior to assembly into fibrils, and both extracellular LMW Tau aggregates and short fibrils, but not monomers, long fibrils, nor long filaments purified from brain extract are taken up by neurons. Remarkably, misfolded Tau can be internalized at the somatodendritic compartment, or the axon terminals and it can be transported anterogradely, retrogradely, and can enhance tauopathy in vivo. The internalized Tau aggregates co-localize with dextran, a bulk-endocytosis marker, and with the endolysosomal compartments. Our findings demonstrate that exogenous Tau can be taken up by cells, uptake depends on both the conformation and size of the Tau aggregates and once inside cells, Tau can be transported. These data provide support for observations that tauopathy can spread trans-synaptically in vivo, via cell-to-cell transfer.

APP mutations in the Aβ coding region are associated with abundant cerebral deposition of Aβ38

Aβ is the main component of amyloid deposits in Alzheimer disease (AD) and its aggregation into oligomers, protofibrils and fibrils is considered a seminal event in the pathogenesis of AD. Aβ with C-terminus at residue 42 is the most abundant species in parenchymal deposits, whereas Aβ with C-terminus at residue 40 predominates in the amyloid of the walls of large vessels. Aβ peptides with other C-termini have not yet been thoroughly investigated. We analysed Aβ38 in the brains of patients with Aβ deposition linked to sporadic and familial AD, hereditary cerebral haemorrhage with amyloidosis, or Down syndrome. Immunohistochemistry, confocal microscopy, immunoelectron microscopy, immunoprecipitation and the electrophoresis separation of low molecular weight aggregates revealed that Aβ38 accumulates consistently in the brains of patients carrying APP mutations in the Aβ coding region, but was not detected in the patients with APP mutations outside the Aβ domain, in the patients with presenilin mutations or in subjects with Down syndrome. In the patients with sporadic AD, Aβ38 was absent in the senile plaques, but it was detected only in the vessel walls of a small subset of patients with severe cerebral amyloid angiopathy. Our results suggest that APP mutations in the Aβ coding region favour Aβ38 accumulation in the brain and that the molecular mechanisms of Aβ deposition in these patients may be different from those active in patients with familial AD associated with other genetic defects and sporadic AD.

The Coarse-Grained OPEP Force Field for Non-Amyloid and Amyloid Proteins

Coarse-grained protein models with various levels of granularity and degrees of freedom offer the possibility to explore many phenomena including folding, assembly, and recognition in terms of dynamics and thermodynamics that are inaccessible to all-atom representations in explicit aqueous solution. Here, we present a refined version of the coarse-grained optimized potential for efficient protein structure prediction (OPEP) based on a six-bead representation. The OPEP version 4.0 parameter set, which uses a new analytical formulation for the nonbonded interactions and adds specific side-chain-side-chain interactions for α-helix, is subjected to three tests. First, we show that molecular dynamics simulations at 300 K preserve the experimental rigid conformations of 17 proteins with 37-152 amino acids within a root-mean-square deviation (RMSD) of 3.1 Å after 30 ns. Extending the simulation time to 100 ns for five proteins does not change the RMSDs. Second, replica exchange molecular dynamics (REMD) simulations recover the NMR structures of three prototypical β-hairpin and α-helix peptides and the NMR three-stranded β-sheet topology of a 37-residue WW domain, starting from randomly chosen states. Third, REMD simulations on the ccβ peptide show a temperature transition from a three-stranded coiled coil to amyloid-like aggregates consistent with experiments, while simulations on low molecular weight aggregates of the prion protein helix 1 do not. Overall, these studies indicate the effectiveness of our OPEP4 coarse-grained model for protein folding and aggregation, and report two future directions for improvement.

Specific Soluble Oligomers of Amyloid-β Peptide Undergo Replication and Form Non-fibrillar Aggregates in Interfacial Environments

Aggregates of amyloid-β (Aβ) peptides have been implicated in the etiology of Alzheimer disease. Among the different forms of Aβ aggregates, low molecular weight species ranging between ~2- and 50-mers, also called "soluble oligomers," have emerged as the species responsible for early synaptic dysfunction and neuronal loss. Emerging evidence suggests that the neurotoxic oligomers need not be formed along the obligatory nucleation-dependant fibril formation pathway. In our earlier work, we reported the isolation of one such "off-pathway" 12-18-mer species of Aβ42 generated from fatty acids called large fatty acid-derived oligomers (LFAOs) (Kumar, A., Bullard, R. L., Patel, P., Paslay, L. C., Singh, D., Bienkiewicz, E. A., Morgan, S. E., and Rangachari, V. (2011) PLoS One 6, e18759). Here, we report the physiochemical aspects of LFAO-monomer interactions as well as LFAO-LFAO associations in the presence of interfaces. We discovered that LFAOs are a replicating strain of oligomers that recruit Aβ42 monomers and quantitatively convert them into LFAO assemblies at the expense of fibrils, a mechanism similar to prion propagation. We also found that in the presence of hexane-buffer or chloroform-buffer interfaces LFAOs are able to associate with themselves to form larger but non-fibrillar aggregates. These results further support the hypothesis that low molecular weight oligomers can be generated via non-fibril formation pathways. Furthermore, the unique replicating property of off-pathway oligomers may hold profound significance for Alzheimer disease pathology.

Development of a GMP Phase III purification process for VB4-845, an immunotoxin expressed in E. coli using high cell density fermentation

VB4-845 is a recombinant immunotoxin comprised of an anti-epithelial cell adhesion molecule (EpCAM) scFv fused to a truncated form of the bacterial toxin, Pseudomonas exotoxin A. VB4-845, purified from TB fed-batch fermentation, showed clinical efficacy when administered locally to treat non-muscle invasive bladder cancer (NMIBC) and squamous cell carcinomas of the head and neck (SCCHN). Here, we describe the implementation of an Escherichia coli high cell density (HCD) cultivation and purification process for VB4-845. HCD cultivation was a prerequisite for achieving higher yields necessary for Phase III clinical trials and commercialization. Using this process, the VB4-845 titer in the supernatant was increased by 30-fold over the original TB fed-batch cultivation. To obtain clinical grade material, a process involving a five-step column purification procedure was implemented and led to an overall recovery of ∼40%. VB4-845 purity of >97% was achieved after the first three columns following the removal of low-molecular weight product-related impurities and aggregates. Endotoxins were effectively separated from VB4-845 on the Q-columns and by washing the Ni-column with a detergent buffer while host cell proteins were removed using ceramic hydroxyapatite. Comparability studies demonstrated that the purified product from the Phase III process was identical to the Phase II reference standard produced using TB fed-batch fermentation.

Structural and Dynamics Characteristics of Acylphosphatase from Sulfolobus solfataricus in the Monomeric State and in the Initial Native-like Aggregates

It has previously been shown that the acylphosphatase from Sulfolobus solfataricus is capable of forming amyloid-like aggregates under conditions in which the native structure is maintained and via the transient formation of native-like aggregates. Based on the previously determined NMR structure of the native protein, showing a ferredoxin-like fold and the peculiar presence of an unstructured N-terminal segment, we show here, at a molecular level using NMR spectroscopy, that indeed S. solfataricus acylphosphatase remains in a native-like conformation when placed in aggregating conditions and that such a native-like structure persists when the protein forms the initial aggregates, at least within the low molecular weight species. The analysis carried out under different solution conditions, based on the measurement of the combined (1)H and (15)N chemical shifts and hydrogen/deuterium exchange rates, enabled the most significant conformational changes to be monitored upon transfer of the monomeric state into aggregating conditions and upon formation of the initial native-like aggregates. Important increases of the hydrogen/deuterium exchange rates throughout the native protein, accompanied by small and localized structural changes, in the monomeric protein were observed. The results also allow the identification of the intermolecular interaction regions within the native-like aggregates, that involve, in particular, the N-terminal unstructured segment, the apical region including strands S4 and S5 with the connecting loop, and the opposite active site.

Aβ Oligomers Induce Neuronal Cell Cycle Events in Alzheimer's Disease

Neurons subject to degeneration in Alzheimer's disease (AD) exhibit evidence of re-entry into a mitotic cell cycle even before the development of substantial AD brain pathology. In efforts to identify the initiating factors underlying these cell cycle events (CCEs), we have characterized the appearance of the neuronal CCEs in the genomic-based R1.40 transgenic mouse model of AD. Notably, R1.40 mice exhibit neuronal CCEs in a reproducible temporal and spatial pattern that recapitulates the neuronal vulnerability seen in human AD. Neuronal CCEs first appear at 6 months in the frontal cortex layers II/III. This is 6-8 months before detectable amyloid beta (Abeta) deposition, suggesting that specific amyloid precursor protein (APP) processing products are responsible for the induction of neuronal CCEs. Furthermore, a reduction in the levels of Abeta (achieved by shifting the genetic background from C57BL/6 to the DBA/2 mouse strain) dramatically delays the appearance of neuronal CCEs. More significantly, elimination of beta-secretase activity blocks the appearance of CCEs, providing direct genetic evidence that the amyloidogenic processing of APP is required for the induction of CCEs. Finally, in vitro preparations of oligomeric, but not monomeric, Abeta induce DNA synthesis in dissociated cortical neurons, and this response is blocked by antioligomer specific antibodies. Together, our data suggest that low molecular weight aggregates of Abeta induce neuronal cell cycle re-entry in mouse models of Alzheimer's disease.

Characterization of protein aggregation: The case of a therapeutic immunoglobulin

In this paper, a therapeutic immunoglobulin (Antibody A) has been characterized in two solutions: (1) 0.1% acetic acid containing 50 mM magnesium chloride, a solution in which the immunoglobulin is stable, and (2) 10 mM sodium phosphate buffer pH ∼ 7. The protein solutions were characterized by microscopy, asymmetrical flow field-flow fractionation (FFF), light scattering, circular dichroism, fluorescence and fluorescence lifetime spectroscopy. The results show that Antibody A dissolved in 0.1% acetic acid containing 50 mM magnesium chloride exists as 88% monomer, 2% low molecular weight aggregates and 10% high molecular weight aggregates (> 1 million Dalton). In phosphate buffer, Antibody A formed micrometre-sized aggregates that were best characterized by fluorescence microscopy. The aggregation of Antibody A in phosphate buffer was shown to be concomitant with conformational changes in amino acid residue side chains. The aggregates formed in phosphate buffer were easily disrupted during FFF analysis, indicating that they are formed by weak interactions. The combination of microscopy, asymmetrical flow field-flow fractionation (FFF) and spectroscopy allowed a reliable assessment of protein self association and aggregation.

Transthyretin oligomers induce calcium influx via voltage‐gated calcium channels

The deposition of transthyretin (TTR) amyloid in the PNS is a major pathological feature of familial amyloidotic polyneuropathy. The aim of the present study was to examine whether TTR could disrupt cytoplasmic Ca(2+) homeostasis and to determine the role of TTR aggregation in this process. The aggregation of amyloidogenic TTR was examined by solution turbidity, dynamic light scattering and atomic force microscopy. A nucleation-dependent polymerization process was observed in which TTR formed low molecular weight aggregates (oligomers < 100 nm in diameter) before the appearance of mature fibrils. TTR rapidly induced an increase in the concentration of intracellular Ca(2+) ([Ca(2+)](i)) when applied to SH-SY5Y human neuroblastoma cells. The greatest effect on [Ca(2+)](i) was induced by a preparation that contained the highest concentration of TTR oligomers. The TTR-induced increase in [Ca(2+)](i) was due to an influx of extracellular Ca(2+), mainly via L- and N-type voltage-gated calcium channels (VGCCs). These results suggest that increasing [Ca(2+)](i) via VGCCs may be an important early event which contributes to TTR-induced cytotoxicity, and that TTR oligomers, rather than mature fibrils, may be the major cytotoxic form of TTR.

Antigen valency as a determinant of the responsiveness of IgE-sensitised rat basophil leukemia cells.

Rat basophil leukemia (RBL) cells were sensitised with varying proportions of monoclonal IgE anti-ovalbumin (OVA) and anti-DNP antibodies, and serotonin release was measured after challenge with aggregated OVA or dinitrophenylated human serum albumin (DNP-HSA). Highly aggregated OVA was shown to provoke the degranulation of RBL cells that had been sensitised with an IgE preparation containing 2% IgE anti-OVA antibodies. Highly substituted DNP32-HSA induced degranulation of RBL cells sensitised with just 0.5% antigen-specific IgE. When cells were sensitised with high percentages of specific IgE, maximum degranulation was seen at concentrations of 2 micrograms/ml (aggregated OVA) and 50 ng/ml (DNP-HSA), while moderate degranulation was still seen at antigen concentrations as low as 50 and 2 ng/ml, respectively. Low-molecular weight aggregates of OVA and low-valency DNP4-HSA only stimulated degranulation when high percentages of RBL Fc epsilon receptor were occupied by antigen-specific IgE. The sensitising abilities of two anti-DNP monoclonal antibodies of differing affinities were compared. When challenged with low-valency antigen, only cells sensitised with the higher-affinity monoclonal antibody exhibited moderate levels of degranulation. Degranulation required exposure to high antigen challenge doses (5 micrograms/ml). Cells sensitised with either monoclonal antibody responded strongly when challenged with a wide range of concentrations (1-250 ng/ml) of high-valency DNP32-HSA, although greater sensitivity was always seen with the higher-affinity antibody. These results suggest that antigen valency is a critical parameter for mast cell function, and that low-affinity antibody may be capable of sensitising mast cells to high-valency antigen.

The Involvement of Kupffer Cells in the Clearance of High Molecular Weight Rat IgA Aggregates in Rats

In the present study the clearance kinetics and tissue distribution of aggregated 125I-labelled monoclonal rat IgA [( 125I] AIgA) of different sizes were studied in rats. Soluble [125I]AIgA disappeared from the circulation in a biphasic manner with an initial rapid distribution half-life (T1) and a second slower half-life (T2). T2 was directly related to the size of the aggregates. High molecular weight [125I]AIgA, containing 10-12 IgA molecules per aggregate [( IgA]10-12), was cleared much faster than low molecular weight aggregates. The main site of clearance was the liver. The larger the size of the AIgA, the more degradation products were found in the circulation. After injection of [IgA]10-12, non-parenchymal cells (NPC) contained three times more radioactivity than parenchymal cells (PC) (NPC:PC ratio 3.06 +/- 0.96). Ratios of 0.82 +/- 0.03 and 0.62 +/- 0.12 were observed when [IgA]5-6 and [IgA]2 were injected respectively, suggesting a greater role for Kupffer cells in the clearance of large-sized IgA aggregates. Kupffer cells were shown to be the main cells for localization of large-sized AIgA established by immunohistochemical staining on liver cryostat sections.