Detection of CD33 expression on monocyte surface is influenced by phagocytosis and temperature

CD33 is a myeloid-associated marker and belongs to the sialic acid-binding immunoglobulin (Ig)-like lectin (Siglec) family. Such types of receptors are highly expressed in acute myeloid leukemia, which could be used in its treatment. CD33 shows high variability in its expression levels with still unknown reasons. Here, we investigated the CD33 expression of monocytes in human blood samples processed at different temperatures and in dependence on their phagocytic activity against opsonized Escherichia coli. The samples were stained by fluorescently labelled anti-human CD14 to specify the monocyte population, anti-human CD33 antibodies to evaluate CD33 expression and analyzed by flow cytometry and confocal laser scanning microscopy. In blood samples kept at 37°C or first pre-chilled at 0°C with subsequent warming up to 37°C, the percentage of CD33-positive monocytes as well as their relative fluorescence intensity was up-regulated compared to samples kept constantly at 0°C. After exposure to E. coli the CD33 relative fluorescence intensity of the monocytes activated at 37°C was 3 to 4 times higher than that of those cells kept inactive at 0°C. Microscopic analysis showed internalisation of CD33 due to its enhanced expression on the surface followed by engulfment of E. coli

Improved upper limb function in non-ambulant children with SMA type 2 and 3 during nusinersen treatment: a prospective 3-years SMArtCARE registry study

Background The development and approval of disease modifying treatments have dramatically changed disease progression in patients with spinal muscular atrophy (SMA). Nusinersen was approved in Europe in 2017 for the treatment of SMA patients irrespective of age and disease severity. Most data on therapeutic efficacy are available for the infantile-onset SMA. For patients with SMA type 2 and type 3, there is still a lack of sufficient evidence and long-term experience for nusinersen treatment. Here, we report data from the SMArtCARE registry of non-ambulant children with SMA type 2 and typen 3 under nusinersen treatment with a follow-up period of up to 38 months. Methods SMArtCARE is a disease-specific registry with data on patients with SMA irrespective of age, treatment regime or disease severity. Data are collected during routine patient visits as real-world outcome data. This analysis included all non-ambulant patients with SMA type 2 or 3 below 18 years of age before initiation of treatment. Primary outcomes were changes in motor function evaluated with the Hammersmith Functional Motor Scale Expanded (HFMSE) and the Revised Upper Limb Module (RULM). Results Data from 256 non-ambulant, pediatric patients with SMA were included in the data analysis. Improvements in motor function were more prominent in upper limb: 32.4% of patients experienced clinically meaningful improvements in RULM and 24.6% in HFMSE. 8.6% of patients gained a new motor milestone, whereas no motor milestones were lost. Only 4.3% of patients showed a clinically meaningful worsening in HFMSE and 1.2% in RULM score. Conclusion Our results demonstrate clinically meaningful improvements or stabilization of disease progression in non-ambulant, pediatric patients with SMA under nusinersen treatment. Changes were most evident in upper limb function and were observed continuously over the follow-up period. Our data confirm clinical trial data, while providing longer follow-up, an increased number of treated patients, and a wider range of age and disease severity

Hemoglobin-based oxygen carriers: in vitro hemocompatibility and functionality

Hemoglobin microparticles (HbMP) present a promising alternative for allogenic blood transfusions. The peanut shaped HbMP show an average size of about 400 nm x 700 nm. However, it is necessary to thoroughly characterize such artificial blood substitute for clinical approval. Physical properties of HbMP must be determined. The HbMP must be investigated in vitro regarding their function, pharmacokinetics and toxicity. Additionally, it is essential to evaluate the hemocompatibility of the particles. In this study, HbMP were fabricated by the co-precipitation, crosslinking and dissolution (CCD) technique using two different crosslinking agents. The functionality was determined by spectral extinction measurements and hemocompatibility was investigated. The CCD technique allows the fabrication of biopolymer particles with bioactive components. Relevant parameter – such as size, shape and entrapment efficiency – were optimized by varying the minerals used as templates. Particles prepared with MnCl2 and Na2CO3 exhibit a protein entrapment efficiency of nearly 100 %. Hyaluronic acid could not influence entrapment efficiency, morphology and size of HbMP. The procedure to investigate the functionality of HbMP is based on spectral extinction measurements between 300 nm and 850 nm. The optical setup features a white light source collimated to obtain a low divergence. In contrast to commercial devices, this setup allows rapid measurements within 10 s and the detection of collimated spectral transmittance under defined conditions. The observed wavelength range includes absorption bands for the important hemoglobin variants necessary to determine the functionality of the particles. Due to the differences of the three absorption bands of oxygenated and deoxygenated hemoglobin as well as methemoglobin, the quantities of these three variants are accessible at a known HbMP concentration. Hence, the functionality of the particles was determined. A modified CCD technique using oxidized dextran as crosslinker was applied to fabricate HbMP with a good hemocompatibility, which was investigated by testing hemolysis and platelet activation. An indirect phagocytosis test was established in order to determine the innate immunological response to HbMP.Hämoglobinmikropartikel (HbMP) stellen als künstliche Sauerstoffträger eine vielversprechende Alternative zur allogenen Bluttransfusion dar. Die hantelförmigen HbMP haben eine typische Größe von etwa 700 nm in der Längsachse und 400 nm in der Querachse. Für die Zulassung klinischer Studien ist es jedoch notwendig sowohl ihre physikalischen Eigenschaften als auch ihre Funktion, Pharmakokinetik und Toxizität zunächst in vitro zu charakterisieren. Von essenzieller Bedeutung für eine Anwendung der Suspensionen der HbMP als Blutersatzstoff sind überdies Untersuchungen zur Hämokompatibilität. Im Rahmen der Arbeit wurden durch Ko-Präzipitation, Vernetzung und Auflösung (co-precipitation-crosslinking-dissolution, CCD) HbMP mit unterschiedlichen Vernetzern hergestellt, ihre Funktionalität durch spektrale Extinktionsmessungen bestimmt und die Hämokompatibilität untersucht. Die CCD-Technik erlaubt die Herstellung von Biopolymerpartikeln mit bioaktiven Bestandteilen. Relevante Parameter wie Größe, Form und Einschlusseffizienz können durch die Variation der mineralischen Matrizen und Reagenzien optimiert werden. Nutzt man MnCl2 und Na2CO3 für die Präzipitation, weisen die Teilchen eine Hantelform auf und die Effizienz des Proteineinbaus erreicht nahezu 100 %. Hyaluronsäure hat keinen Einfluss auf die erreichte Einschlusseffizienz von Hämoglobin. Das Verfahren zur funktionellen Charakterisierung der HbMP basiert auf spektralen Extinktionsmessungen im Wellenlängenbereich von etwa 300 nm bis 850 nm. Der angegebene Wellenlängenbereich schließt die Absorptionsbanden der verschiedenen Hämoglobinvarianten von oxygeniertem bzw. deoxygeniertem Hämoglobin und Methämoglobin ein, so dass diese bei bekannter HbMP-Konzentration quantifiziert werden können. HbMP, die mit oxidiertem Dextran als Vernetzer hergestellt wurden, weisen eine gute Hämokompatibilität auf, so dass dieser Ansatz auch zukünftig verfolgt werden sollte. Als Methoden wurden dabei Tests zur Bestimmung der Hämolyse und der Plättchenaktivierung verwendet. Außerdem wurde ein indirekter Phagozytosetest entwickelt, um die Auswirkungen auf die angeborene Immunantwort zu ermitteln

Function of Hemoglobin-Based Oxygen Carriers: Determination of Methemoglobin Content by Spectral Extinction Measurements

Suspensions of hemoglobin microparticles (HbMPs) are promising tools as oxygen therapeutics. For the approval of clinical studies extensive characterization of these HbMPs with a size of about 750 nm is required regarding physical properties, function, pharmaco-kinetics and toxicology. The standard absorbance measurements in blood gas analyzers require dissolution of red blood cells which does not work for HbMP. Therefore, we have developed a robust and rapid optical method for the quality and functionality control of HbMPs. It allows simultaneous determination of the portion of the two states of hemoglobin oxygenated hemoglobin (oxyHb) and deoxygenated hemoglobin (deoxyHb) as well as the content of methemoglobin (metHb). Based on the measurement of collimated transmission spectra between 300 nm and 800 nm, the average extinction cross section of HbMPs is derived. A numerical method is applied to determine the composition of the HbMPs based on their wavelength-dependent refractive index (RI), which is a superposition of the three different states of Hb. Thus, light-scattering properties, including extinction cross sections can be simulated for different compositions and sizes. By comparison to measured spectra, the relative concentrations of oxyHb, deoxyHb, metHb are accessible. For validation of the optically determined composition of the HbMPs, we used X-ray fluorescence spectrometry for the ratio of Fe(II) (oxyHb/deoxyHb) and Fe(III) (metHb). High accuracy density measurements served to access heme-free proteins, size was determined by dynamic light scattering and analytical centrifugation and the shape of the HbMPs was visualized by electron and atomic force microscopy

Albumin Submicron Particles with Entrapped Riboflavin—Fabrication and Characterization

Although riboflavin (RF) belongs to the water-soluble vitamins of group B, its solubility is low. Therefore, the application of micro-formulations may help to overcome this limiting factor for the delivery of RF. In this study we immobilized RF in newly developed albumin submicron particles prepared using the Co-precipitation Crosslinking Dissolution technique (CCD-technique) of manganese chloride and sodium carbonate in the presence of human serum albumin (HSA) and RF. The resulting RF containing HSA particles (RF-HSA-MPs) showed a narrow size distribution in the range of 0.9 to 1 μm, uniform peanut-like morphology, and a zeta-potential of −15 mV. In vitro release studies represented biphasic release profiles of RF in a phosphate buffered saline (PBS) pH 7.4 and a cell culture medium (RPMI) 1640 medium over a prolonged period. Hemolysis, platelet activation, and phagocytosis assays revealed a good hemocompatibility of RF-HSA-MPs

Nonvasoconstrictive Hemoglobin Particles as Oxygen Carriers

Artificial oxygen carriers, favorably hemoglobin-based oxygen carriers (HBOCs), are being investigated intensively during the last 30 years with the aim to develop a universal blood substitute. However, serious side effects mainly caused by vasoconstriction triggered by nitric oxide (NO) scavenging due to penetration of nanosized HBOCs through the endothelial gaps of the capillary walls and/or oxygen oversupply in the precapillary arterioles due to their low oxygen affinity led to failure of clinical trials and FDA disapproval. To avoid these effects, HBOCs with a size between 100 and 1000 nm and high oxygen affinity are needed. Here we present for the first time unique hemoglobin particles (HbPs) of around 700 nm with high oxygen affinity and low immunogenicity using a novel, highly effective, and simple technique. The fabrication procedure provides particles with a narrow size distribution and nearly uniform morphology. The content of hemoglobin (Hb) in the particles corresponded to 80% of the Hb content in native erythrocytes. Furthermore, we demonstrate a successful perfusion of isolated mouse glomeruli with concentrated HbP suspensions <i>in vitro</i>. A normal, nonvasoconstrictive behavior of the afferent arterioles is observed, suggesting no oxygen oversupply and limited NO scavenging by these particles, making them a highly promising blood substitute

Nonvasoconstrictive Hemoglobin Particles as Oxygen Carriers

Artificial oxygen carriers, favorably hemoglobin-based oxygen carriers (HBOCs), are being investigated intensively during the last 30 years with the aim to develop a universal blood substitute. However, serious side effects mainly caused by vasoconstriction triggered by nitric oxide (NO) scavenging due to penetration of nanosized HBOCs through the endothelial gaps of the capillary walls and/or oxygen oversupply in the precapillary arterioles due to their low oxygen affinity led to failure of clinical trials and FDA disapproval. To avoid these effects, HBOCs with a size between 100 and 1000 nm and high oxygen affinity are needed. Here we present for the first time unique hemoglobin particles (HbPs) of around 700 nm with high oxygen affinity and low immunogenicity using a novel, highly effective, and simple technique. The fabrication procedure provides particles with a narrow size distribution and nearly uniform morphology. The content of hemoglobin (Hb) in the particles corresponded to 80% of the Hb content in native erythrocytes. Furthermore, we demonstrate a successful perfusion of isolated mouse glomeruli with concentrated HbP suspensions <i>in vitro</i>. A normal, nonvasoconstrictive behavior of the afferent arterioles is observed, suggesting no oxygen oversupply and limited NO scavenging by these particles, making them a highly promising blood substitute