Persistent Aggregates Research Articles

Aggregates in apheresis-derived platelet concentrates: A 5-year single-centre experience.

The phenomenon of aggregates in apheresis-derived platelet concentrates (APCs) has not yet been fully elucidated. Initially, visible aggregates (IVA) usually dissolve within 24 h after collection, but some persist till the end of the shelf life (persistent aggregates, PA). A study conducted at the Croatian Institute of Transfusion Medicine aimed to identify factors that influence the aggregate occurrence in APCs. We conducted a cross-sectional study for the 2018-2022 period and collected data on APCs with IVA. We analysed APCs discarded due to PA separately for two apheresis technologies and compared them to the control group. Significantly more donations were discarded in the IVA group compared with the control group and total number of discarded APCs. A total of 205 APCs were discarded due to PA (14.7% of IVA APCs and 1.27% of all APCs collected). Amicus APCs with PA had a significantly lower platelet count and mean platelet volume. They were obtained by procedures with less anticoagulant used. In contrast to Amicus APCs, Haemonetics APCs with PA had a significantly higher platelet count. None of the donor-related factors examined was predictive of PA. APCs with IVA are more often discarded, not only due to aggregates, but also for impairment of other quality control parameters. Type of apheresis technology, being one of the most common risk factors for IVA, was not confirmed as the main risk factor for PA. There seem to be some donor-related causal factors.

Growth-driven displacement of protein aggregates along the cell length ensures partitioning to both daughter cells in Caulobacter crescentus.

SummaryAll living cells must cope with protein aggregation, which occurs as a result of experiencing stress. In previously studied bacteria, aggregated protein is collected at the cell poles and is retained throughout consecutive cell divisions only in old pole‐inheriting daughter cells, resulting in aggregation‐free progeny within a few generations. In this study, we describe the in vivo kinetics of aggregate formation and elimination following heat and antibiotic stress in the asymmetrically dividing bacterium Caulobacter crescentus. Unexpectedly, in this bacterium, protein aggregates form as multiple distributed foci located throughout the cell volume. Time‐lapse microscopy revealed that under moderate stress, the majority of these protein aggregates are short‐lived and rapidly dissolved by the major chaperone DnaK and the disaggregase ClpB. Severe stress or genetic perturbation of the protein quality control machinery induces the formation of long‐lived aggregates. Importantly, the majority of persistent aggregates neither collect at the cell poles nor are they partitioned to only one daughter cell type. Instead, we show that aggregates are distributed to both daughter cells in the same ratio at each division, which is driven by the continuous elongation of the growing mother cell. Therefore, our study has revealed a new pattern of protein aggregate inheritance in bacteria.

Inverse Thermoreversible Mechanical Stiffening and Birefringence in a Methylcellulose/Cellulose Nanocrystal Hydrogel.

We show that composite hydrogels comprising methyl cellulose (MC) and cellulose nanocrystal (CNC) colloidal rods display a reversible and enhanced rheological storage modulus and optical birefringence upon heating, i.e., inverse thermoreversibility. Dynamic rheology, quantitative polarized optical microscopy, isothermal titration calorimetry (ITC), circular dichroism (CD), and scanning and transmission electron microscopy (SEM and TEM) were used for characterization. The concentration of CNCs in aqueous media was varied up to 3.5 wt % (i.e, keeping the concentration below the critical aq concentration) while maintaining the MC aq concentration at 1.0 wt %. At 20 °C, MC/CNC underwent gelation upon passing the CNC concentration of 1.5 wt %. At this point, the storage modulus (G′) reached a plateau, and the birefringence underwent a stepwise increase, thus suggesting a percolative phenomenon. The storage modulus (G′) of the composite gels was an order of magnitude higher at 60 °C compared to that at 20 °C. ITC results suggested that, at 60 °C, the CNC rods were entropically driven to interact with MC chains, which according to recent studies collapse at this temperature into ring-like, colloidal-scale persistent fibrils with hollow cross-sections. Consequently, the tendency of the MC to form more persistent aggregates promotes the interactions between the CNC chiral aggregates towards enhanced storage modulus and birefringence. At room temperature, ITC shows enthalpic binding between CNCs and MC with the latter comprising aqueous, molecularly dispersed polymer chains that lead to looser and less birefringent material. TEM, SEM, and CD indicate CNC chiral fragments within a MC/CNC composite gel. Thus, MC/CNC hybrid networks offer materials with tunable rheological properties and access to liquid crystalline properties at low CNC concentrations.

Multiscale organization of thermoplastic elastomers with varying content of hard segments

Thermoplastic elastomers (TPEs) based on segmented block-copolymers containing poly(tetrahydrofuran) (pTHF) and terephthalate-based diamide groups (T4T) were synthesized via polycondensation. While pTHF is known to be flexible and amorphous at rest, the more rigid T4T crystallized in different ways depending on both chain composition and sample preparation conditions. Increasing the content of hard-segments (HS) from 5 to 20% in weight leads to a substantial increase of the melting point Tm by more than 60 °C. We have systematically investigated the multiscale (1 Å - 50 nm) organization of the HSs (of fractions from 5% to 20%) by means of DSC, WAXS and (ultra) small angle X-ray scattering (U)SAXS. By increasing HS content, hence the rigidity of the chain, scattering experiments unambiguously show the formation of bigger and better defined ribbon-like crystallites, as well as the densification of the network they form. We propose a scenario for rationalizing the local T4T-HSs packing and the crystallites anisotropy at the mesoscale (1–10 nm) for HS fraction above 5%. Moreover, following in-situ the crystallization of TPEs with large HS fraction (20%), we highlight the presence of “persistent aggregates” present at T > Tm and study the ribbon-like crystallites growth mechanism during cooling from the melt state.

Persistent aggregates in apheresis platelet concentrates are commonly collected from donors with a history of aggregate donation.

Platelet apheresis sometimes causes persistent aggregates (PA). This study (n = 211) shows that changing the apheresis settings to reach fixed product volumes instead of yields does not influence PA incidence, even though PA products on average contain more platelets than controls. Furthermore, logistic regression was used to model if PA can be predicted on the basis of certain predonation parameters. PA donation history was the only parameter retained, proving a strong determinant of predictability [AUC = 0.735 (SE = 0.022)]. Consequently, donations from a donor with previous PA history are 7.8 times more likely to contain PA than from a donor without preceding history.

The contribution of von Willebrand factor-GPIbα interactions to persistent aggregate formation in apheresis platelet concentrates.

Apheresis platelet concentrates sometimes contain persistent aggregates (PA). Because apheresis involves extracorporeal circulation, we hypothesized that interactions between GPIbα and von Willebrand factor (VWF) underlie their origin. Platelets in donations with PA were compared to aggregate-free (AF) controls. Flow cytometry was used to determine platelet bound VWF. Degranulation was measured using P-selectin expression in flow cytometry and cytokine release using immunosorbent assays. Platelet adhesion to VWF was assessed in hydrodynamic flow and real-time video microscopy. Platelets in PA concentrates had significantly more (P = 0·009, n ≥ 8) bound VWF compared to AF platelets, but differences in VWF concentration, VWF collagen binding, activated VWF or GPIbα expression were not found. Degranulation was higher (P = 0·030, n = 7) in PA than AF concentrates on day 1 of storage, but adhesion to immobilized VWF under hydrodynamic flow conditions was normal at that moment. On day 6, however, significantly less VWF adhesion (P = 0·009, n ≥ 6) was found for PA platelets compared to AF, indicating accelerated storage lesion in PA products. In a model that mimicks PA formation by chemically induced binding of VWF to platelets, we found that degranulation, phosphatidylserine expression and metabolism did not differ with paired controls at any time during subsequent storage. Accelerated storage lesion is found in concentrates with PA, but this cannot be explained solely by increased platelet bound VWF following apheresis. Therefore, additional stressors are probably responsible for the increases observed in platelet degranulation and storage lesion in products with PA.

Persistent aggregates in apheresis platelet concentrates.

Aggregates often appear during apheresis. Sometimes, these persist throughout storage, causing product wastage. This study assessed product quality of apheresis concentrates containing persistent aggregates (PA) and aimed to identify the factors that contribute to their formation. Donation (n=180) and platelet indices (n≥10) from apheresis concentrates with PA were compared with aggregate-free products. The proportion of donors with at least one previous PA donation was twofold higher in the PA group (P<0·0001) indicating a donor dependence. Significantly higher donor whole blood platelet counts (286±50 vs. 266±49×10(3) /μl, P<0·0001) and higher apheresis yields (6·0±1·6 vs. 5·4±1·5×10(11) , P<0·0001) were noted in the PA group. Haematocrit was also slightly higher, but age, gender and body mass were similar. The pH of PA products on day six postdonation was significantly lower (P<0·001), in line with higher lactic acid concentrations. Flow cytometry showed no differences in GPIbα levels or phosphatidylserine exposure. However, there was slightly more integrin activation as well as increased degranulation measured by P-selectin expression. Cytokine concentrations were also significantly higher in PA concentrates. Aggregation was normal in response to SFLLRN peptide and collagen stimulation, but agglutination at low-dose ristocetin was significantly higher (P=0.01) in PA products. Finally, PA were disintegrated by plasmin-mediated thrombolysis but not by integrin αIIb β3 inhibition. Products with PA have acceptable quality parameters, but additional functional studies are warranted. Furthermore, PA are more likely to recur in certain donors who have higher platelet counts.

The plasma protein fibrinogen stabilizes clusters of red blood cells in microcapillary flows

The supply of oxygen and nutrients and the disposal of metabolic waste in the organs depend strongly on how blood, especially red blood cells, flow through the microvascular network. Macromolecular plasma proteins such as fibrinogen cause red blood cells to form large aggregates, called rouleaux, which are usually assumed to be disaggregated in the circulation due to the shear forces present in bulk flow. This leads to the assumption that rouleaux formation is only relevant in the venule network and in arterioles at low shear rates or stasis. Thanks to an excellent agreement between combined experimental and numerical approaches, we show that despite the large shear rates present in microcapillaries, the presence of either fibrinogen or the synthetic polymer dextran leads to an enhanced formation of robust clusters of red blood cells, even at haematocrits as low as 1%. Robust aggregates are shown to exist in microcapillaries even for fibrinogen concentrations within the healthy physiological range. These persistent aggregates should strongly affect cell distribution and blood perfusion in the microvasculature, with putative implications for blood disorders even within apparently asymptomatic subjects.

Hemorheological implications of perfluorocarbon based oxygen carrier interaction with colloid plasma expanders and blood

Perfluorocarbon (PFC) emulsions used as artificial oxygen carriers lack colloid osmotic pressure (COP) and must be administered with colloid-based plasma expanders (PEs). Although PFC emulsions have been widely studied, there is limited information about PFC emulsion interaction with PEs and blood. Their interaction forms aggregates due to electrostatic and rheological phenomena, and change blood rheology and blood flow. This study analyzes the effects of the interaction between PFC emulsions with blood in the presence of clinically-used PEs. The rheological behavior of the mixtures was analyzed in vitro in parallel with in vivo analysis of blood flow in the microcirculation using intravital microscopy, when PEs were administered in a clinically relevant scenario. The interaction between the PFC emulsion and PE with blood produced PFC droplets and red blood cell (RBCs) aggregation and increased blood viscosity in a shear dependent fashion. The PFC droplets formed aggregates when mixed with PEs containing electrolytes, and the aggregation increased with the electrolyte concentration. Mixtures of PFC with PEs that produced PFC aggregates also induced RCBs aggregation when mixed with blood, increasing blood viscosity at low shear rates. The more viscous suspension at low shear rates produced a blunted blood flow velocity profile in vivo compared to nonaggregating mixtures of PFC and PEs. For the PEs evaluated, human serum albumin produced minimal to undetectable aggregation. PFC and PEs interaction with blood can affect sections of the microcirculation with low shear rates (e.g., arterioles, venules, and pulmonary circulation) when used in a clinical setting, because persistent aggregates could cause capillary occlusion, decreased perfusion, pulmonary emboli or focal ischemia.

Soil biotransformation of thiodiglycol, the hydrolysis product of mustard gas: understanding the factors governing remediation of mustard gas contaminated soil

Thiodiglycol (TDG) is both the precursor for chemical synthesis of mustard gas and the product of mustard gas hydrolysis. TDG can also react with intermediates of mustard gas degradation to form more toxic and/or persistent aggregates, or reverse the pathway of mustard gas degradation. The persistence of TDG have been observed in soils and in the groundwater at sites contaminated by mustard gas 60 years ago. The biotransformation of TDG has been demonstrated in three soils not previously exposed to the chemical. TDG biotransformation occurred via the oxidative pathway with an optimum rate at pH 8.25. In contrast with bacteria isolated from historically contaminated soil, which could degrade TDG individually, a consortium of three bacterial strains isolated from the soil never contaminated by mustard gas was able to grow on TDG in minimal medium and in hydrolysate derived from an historical mustard gas bomb. Exposure to TDG had little impacts on the soil microbial physiology or on community structure. Therefore, the persistency of TDG in soils historically contaminated by mustard gas might be attributed to the toxicity of mustard gas to microorganisms and the impact to soil chemistry during the hydrolysis. TDG biodegradation may form part of a remediation strategy for mustard gas contaminated sites, and may be enhanced by pH adjustment and aeration.

SOCIETE FRANCAISE DE NEUROLOGIE (SFN)

Unstable biopolymer solutions inevitably lead to mis-characterization of macromolecular properties and irreproducible results. Even under stable or quasi-stable conditions, persistent aggregates can hamper reliable characterization, especially using light scattering methods. Various pitfalls in characterizing biopolymers were worked through, including determination of solution stability zones, dissolution kinetics, estimation of fraction of aggregate populations, and the relationship between batch and fractionation methods. Chitosans, polyampholytic biopolymers with isoelectric point around pH = 6.0, with varying degrees of carboxymethylation were studied. Instability was determined vs. pH and ionic strength using a high throughput screening method, simultaneous multiple sample light scattering (SMSLS). With stable solution conditions determined, equilibrium batch and multi-detector GPC characterization of molecular weight, intrinsic viscosity, and polyelectrolyte properties was made. Finally, a first attempt at continuous online monitoring of the modification reaction itself was made and compared to FTIR analysis of carboxymethylation on discrete aliquots. Given the range of possible characterization problems, multiple approaches with independent instruments may be required for reliable natural product characterization. Online monitoring of modification reactions may lead to rapid advances in understanding and preparation of natural products.

Characterization of stability, aggregation, and equilibrium properties of modified natural products; The case of carboxymethylated chitosans

Unstable biopolymer solutions inevitably lead to mis-characterization of macromolecular properties and irreproducible results. Even under stable or quasi-stable conditions, persistent aggregates can hamper reliable characterization, especially using light scattering methods. Various pitfalls in characterizing biopolymers were worked through, including determination of solution stability zones, dissolution kinetics, estimation of fraction of aggregate populations, and the relationship between batch and fractionation methods. Chitosans, polyampholytic biopolymers with isoelectric point around pH = 6.0, with varying degrees of carboxymethylation were studied. Instability was determined vs. pH and ionic strength using a high throughput screening method, simultaneous multiple sample light scattering (SMSLS). With stable solution conditions determined, equilibrium batch and multi-detector GPC characterization of molecular weight, intrinsic viscosity, and polyelectrolyte properties was made. Finally, a first attempt at continuous online monitoring of the modification reaction itself was made and compared to FTIR analysis of carboxymethylation on discrete aliquots. Given the range of possible characterization problems, multiple approaches with independent instruments may be required for reliable natural product characterization. Online monitoring of modification reactions may lead to rapid advances in understanding and preparation of natural products.

Nucleoside analogs in treatment of chronic lymphocytic leukemia.

The nucleoside analogs fludarabine monophosphate, 2-chlorodeoxyadenosine, and 2-deoxycoformycin (pentostatin) all have activity in chronic lymphocytic leukemia. The most widely studied drug is fludarabine which is able to obtain complete or partial responses in more than 50% of previously treated patients. The response rate is 44% for 2-CDA and approximately 25% for pentostatin. Fludarabine has also been used to treat patients as initial therapy, and has resulted in overall response rate of 79% with 75% of the patients achieving complete remission. The NCI and International Working Group for CLL criteria for complete remission allow for persistent nodules or lymphoid infiltrates in the bone marrow biopsy. Studies have now demonstrated persistent lymphoid aggregates are associated with a shorter time to progression for responders but no survival disadvantage. There is a strong association of documented refractoriness to alkylating agents with probability of response to fludarabine and also survival. The major morbidity associated with the use of these drugs are infections, which, in some circumstances, are associated with neutropenia but in other circumstances are probably related to the hypogammaglobulinemia and T-cell immunodeficiency which are part of the disease. The T-cell immunodeficiency is aggravated by the nucleoside analogs. Even after discontinuation of therapy the immunodeficiency as measured by CD4 cell number is sustained for 12 to 24 months. Opportunistic organisms such as herpes simplex, herpes zoster, Listeria monocytogenes, and pneumocystis carinii are being noted in patients treated with these agents. The potency of these drugs and low incidence of toxicities to other organs suggests that they will be effectively combined with other agents.(ABSTRACT TRUNCATED AT 250 WORDS)

The ultrastructural cytology and taxonomy of Duboscqia sidaeJírovec, 1942 (Microspora, Duboscqiidae), with establishment of the new genus Agglomerata gen. nov.

Summary The cytology of the microsporidium Duboscqia sidae (Microspora, Duboscqiidae), a parasite of Holopedium gibberum in a Canadian Shield Lake, was investigated using light and electron microscopical methods. Persistent aggregates of spores, generally 16 in number, were produced in polysporous sporophorous vesicles. The integrity of aggregates was maintained by a dense reticulum of tubules which were modified from characteristic inclusions observed in vesicles with sporoblasts and immature spores. The ultrastructure of mature spores was unique, characterized by a five-layered exospore, a slightly anisofilar polar filament and a polaroplast with three structurally distinct regions. The species is compared with other microsporidia reported from Cladocera and its systematic position is examined. As the ultrastructural cytology is specialised and the developmental attributes of the species differ from those of the type species of Duboscqia and all other genera with polysporoblastic sporogony in sporophorous vesicles, a new genus, Agglomerata, is created for D. sidae.