Lysis Of Human Erythrocytes Research Articles

Lysis of human erythrocytes due to Piezo1-dependent cytosolic calcium overload as a mechanism of circulatory removal

Hematopoietic stem cells surrender organelles during differentiation, leaving mature red blood cells (RBC) devoid of transcriptional machinery and mitochondria. The resultant absence of cellular repair capacity limits RBC circulatory longevity, and old cells are removed from circulation. The specific age-dependent alterations required for this apparently targeted removal of RBC, however, remain elusive. Here, we assessed the function of Piezo1, a stretch-activated transmembrane cation channel, within subpopulations of RBC isolated based on physical properties associated with aging. We subsequently investigated the potential role of Piezo1 in RBC removal, using pharmacological and mechanobiological approaches. Dense (old) RBC were separated from whole blood using differential density centrifugation. Tolerance of RBC to mechanical forces within the physiological range was assessed on single-cell and cell population levels. Expression and function of Piezo1 were investigated in separated RBC populations by monitoring accumulation of cytosolic Ca2+ and changes in cell morphology in response to pharmacological Piezo1 stimulation and in response to physical forces. Despite decreased Piezo1 activity with increasing cell age, tolerance to prolonged Piezo1 stimulation declined sharply in older RBC, precipitating lysis. Cell lysis was immediately preceded by an acute reversal of density. We propose a Piezo1-dependent mechanism by which RBC may be removed from circulation: Upon adherence of these RBC to other tissues, they are uniquely exposed to prolonged mechanical forces. The resultant sustained activation of Piezo1 leads to a net influx of Ca2+, overpowering the Ca2+-removal capacity of specifically old RBC, which leads to reversal of ion gradients, dysregulated cell hydration, and ultimately osmotic lysis.

New insight into the virulence and inflammatory response of Staphylococcus aureus strains isolated from diabetic foot ulcers.

Staphylococcus aureus strains isolated from diabetic foot ulcers (DFUs) have less virulence, but still cause severe infections. Furthermore, hypovirulent S. aureus strains appear to be localized in the deep tissues of diabetic foot osteomyelitis, indicating that the unique environment within DFUs affects the pathogenicity of S. aureus. In this study, the cell-free culture medium (CFCM) of S. aureus strains isolated from DFUs exhibited higher cytotoxicity to human erythrocytes than those isolated from non-diabetic patients with sepsis or wounds. Among these S. aureus strains isolated from DFUs, β-toxin negative strains have less virulence than β-toxin positive strains, but induced a higher expression of inflammatory cytokines. Our study and previous studies have shown that the synergistic effect of phenol-soluble modulin α and β-toxin contributes to the higher hemolytic activity of β-toxin positive strains. However, lysis of human erythrocytes by the CFCM of β-toxin negative strains was greatly inhibited by an autolysin inhibitor, sodium polyanethole sulfonate (SPS). A high level of glucose greatly reduced the hemolytic activity of S. aureus, but promoted the expression of interleukin-6 (IL-6) in human neutrophils. However, 5 mM glucose or glucose-6-phosphate (G6P) increased the hemolytic activity of SA118 (a β-toxin negative strain) isolated from DFUs. Additionally, patients with DFUs with growth of S. aureus had lower level of serum IL-6 than those with other bacteria, and the CFCM of S. aureus strains significantly reduced lipopolysaccharide-induced IL-6 expression in human neutrophils. Therefore, the virulence and inflammatory response of S. aureus strains isolated from DFUs are determined by the levels of glucose and its metabolites, which may explain why it is the predominant bacteria isolated from DFUs.

Synthesis of Fluorinated Hydrazinylthiazole Derivatives: A Virtual and Experimental Approach to Diabetes Management.

A novel series of fluorophenyl-based thiazoles was synthesized following the Hanztsch method. All of the compounds were initially verified with physical parameters (color, melting point, retardation factor (R f)), which were further confirmed by several spectroscopic methods, including ultraviolet-visible (UV-visible), Fourier-transform infrared (FTIR), 1H, 13C, 19F NMR, and high-resolution mass spectrometry (HRMS). The binding interactions of all compounds were studied using a molecular docking simulation approach. Furthermore, each compound was evaluated for its alpha(α)-amylase, antiglycation, and antioxidant potentials. The biocompatibility of all compounds was checked with an in vitro hemolytic assay. All synthesized scaffolds were found biocompatible with minimal lysis of human erythrocytes as compared to the standard Triton X-100. Among the tested compounds, the analogue 3h (IC50 = 5.14 ± 0.03 μM) was found to be a highly potent candidate against α-amylase as compared to the standard (acarbose, IC50 = 5.55 ± 0.06 μM). The compounds 3d, 3f, 3i, and 3k exhibited excellent antiglycation inhibition potential with their IC50 values far less than the standard amino guanidine (IC50 = 0.403 ± 0.001 mg/mL). The antidiabetic potential was further supported by docking studies. Docking studies revealed that all synthesized compounds exhibited various interactions along enzyme active sites (pi-pi, H-bonding, van der Waals) with varied binding energies.

Soluble MAC is primarily released from MAC-resistant bacteria that potently convert complement component C5.

The membrane attack complex (MAC or C5b-9) is an important effector of the immune system to kill invading microbes. MAC formation is initiated when complement enzymes on the bacterial surface convert complement component C5 into C5b. Although the MAC is a membrane-inserted complex, soluble forms of MAC (sMAC), or terminal complement complex (TCC), are often detected in sera of patients suffering from infections. Consequently, sMAC has been proposed as a biomarker, but it remains unclear when and how it is formed during infections. Here, we studied mechanisms of MAC formation on different Gram-negative and Gram-positive bacteria and found that sMAC is primarily formed in human serum by bacteria resistant to MAC-dependent killing. Surprisingly, C5 was converted into C5b more potently by MAC-resistant compared to MAC-sensitive Escherichia coli strains. In addition, we found that MAC precursors are released from the surface of MAC-resistant bacteria during MAC assembly. Although release of MAC precursors from bacteria induced lysis of bystander human erythrocytes, serum regulators vitronectin (Vn) and clusterin (Clu) can prevent this. Combining size exclusion chromatography with mass spectrometry profiling, we show that sMAC released from bacteria in serum is a heterogeneous mixture of complexes composed of C5b-8, up to three copies of C9 and multiple copies of Vn and Clu. Altogether, our data provide molecular insight into how sMAC is generated during bacterial infections. This fundamental knowledge could form the basis for exploring the use of sMAC as biomarker.

Microfluidic device integrating single-cell extraction and electrical lysis for mass spectrometry detection of intracellular compounds.

Analysis of cellular composition and metabolism at a single-cell resolution allows gaining more information about complex relationships of cells within tissues or whole living organisms by resolving the variance stemming from the cellular heterogeneity. Mass spectrometry (MS) is a perfect analytical tool satisfying the demanding requirements of detecting and identifying compounds present in such ultralow-volume samples of high chemical complexity. However, the method of sampling and sample ionization is crucial in obtaining relevant information. In this work, we present a microfluidic sampling platform that integrates single-cell extraction from MS-incompatible media with electrical cell lysis and nanoESI-MS analysis of human erythrocytes. Hemoglobin alpha and beta chains (300amol/cell) were successfully identified in mass spectra of single-erythrocyte lysates.

Respirable stone particles differ in their ability to induce cytotoxicity and pro-inflammatory responses in cell models of the human airways

BackgroundRespirable stone- and mineral particles may be a major constituent in occupational and ambient air pollution and represent a possible health hazard. However, with exception of quartz and asbestos, little is known about the toxic properties of mineral particles. In the present study, the pro-inflammatory and cytotoxic responses to six stone particle samples of different composition and with diameter below 10 μm were assessed in human bronchial epithelial cells (HBEC3-KT), THP-1 macrophages and a HBEC3-KT/THP-1 co-culture. Moreover, particle-induced lysis of human erythrocytes was assessed to determine the ability of the particles to lyse biological membranes. Finally, the role of the NLRP3 inflammasome was assessed using a NLRP3-specific inhibitor and detection of ASC oligomers and cleaved caspase-1 and IL-1β. A reference sample of pure α-quartz was included for comparison.ResultsSeveral stone particle samples induced a concentration-dependent increase in cytotoxicity and secretion of the pro-inflammatory cytokines CXCL8, IL-1α, IL-1β and TNFα. In HBEC3-KT, quartzite and anorthosite were the most cytotoxic stone particle samples and induced the highest levels of cytokines. Quartzite and anorthosite were also the most cytotoxic samples in THP-1 macrophages, while anorthosite and hornfels induced the highest cytokine responses. In comparison, few significant differences between particle samples were detected in the co-culture. Adjusting responses for differences in surface area concentrations did not fully account for the differences between particle samples. Moreover, the stone particles had low hemolytic potential, indicating that the effects were not driven by membrane lysis. Pre-incubation with a NLRP3-specific inhibitor reduced stone particle-induced cytokine responses in THP-1 macrophages, but not in HBEC3-KT cells, suggesting that the effects are mediated through different mechanisms in epithelial cells and macrophages. Particle exposure also induced an increase in ASC oligomers and cleaved caspase-1 and IL-1β in THP-1 macrophages, confirming the involvement of the NLRP3 inflammasome.ConclusionsThe present study indicates that stone particles induce cytotoxicity and pro-inflammatory responses in human bronchial epithelial cells and macrophages, acting through NLRP3-independent and -dependent mechanisms, respectively. Moreover, some particle samples induced cytotoxicity and cytokine release to a similar or greater extent than α-quartz. Thus, these minerals warrant further attention in future research.

Antibacterial functionalization of nylon monofilament surgical sutures through in situ deposition of biogenic silver nanoparticles

Antibacterial coating of nylon monofilament sutures presents difficulties unlike non-absorbable braided sutures due to the nature of nylon material and absence of interstices. To solve these problems, a new technique using in situ deposition of biogenic silver nanoparticles (AgNPs) has been developed to coat nylon surgical sutures. The effect of AgNPs deposition on the surface roughness was observed, with significant reduction in average roughness (9.41 ± 0.96 nm), compared with uncoated sutures (12.35 ± 0.20 nm) (P < 0.05). The ICP-OES analysis of coated sutures revealed total Ag+ concentration of 11.25 ± 0.14 μg/mL, with an AgNPs release of 11.25, 11.27, 19.94, and 24.03% at 1, 3, 5, and 7 days, respectively. The sutures demonstrated bactericidal effects on Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae) wound pathogens with more than 99.9% reduction. The coated sutures indicated stable antibacterial activity against representative Gram-positive and Gram-negative isolates after 12-week storage. Haemolytic evaluation of coated sutures showed less than 1% lysis of human erythrocytes. In addition, coated sutures inhibited lipopolysaccharide-induced nitric oxide generation by 15.46 μM reduction. Cytocompatibility test on L929 fibroblast cells revealed approximately 81% cell viability and promoted cell migration by 51% after 24 h treatment. The study revealed that in situ deposition of AgNPs on nylon monofilament sutures did not alter the mechanical properties of the sutures, but conferred antibacterial and wound healing properties.

Searching for the toxic potential of Loxosceles amazonica and Loxosceles willianilsoni spiders’ venoms

The Loxosceles genus belongs to the Sicariidae family and it comprises species whose venom can cause accidents with potentially fatal consequences. We have previously shown that SMase D is the enzyme responsible for the main pathological effects of Loxosceles venom. Despite the severity of accidents with Loxosceles, few species are considered to be of medical importance. Little is known about the venom of non-synanthropic species that live in natural environments. To contribute to a better understanding about the venom's toxicity of Loxosceles genus, the aim of this study was to (i) characterize the toxic properties of Loxosceles amazonica from two different localities and a recent described cave species Loxosceles willianilsoni and (ii) compare these venoms with that from Loxosceles laeta, which is among the most toxic ones. We show here that both L. amazonica venoms (from the two studied locations) and L. willianilsoni presented SMase D activity similar to that exhibited by L. laeta venom. Although L. amazonica and L. willianilsoni venoms were able to induce complement dependent human erythrocytes lysis, they were not able to induce cell death of human keratinocytes, as promoted by L. laeta venom, in the concentrations tested. These results indicate that other species of Loxosceles, in addition to those classified as medically important, have toxic potential to cause accidents in humans, despite interspecific variations that denote possible less toxicity.

Вплив сумісної дії проникального і непроникального компонентів середовища на розвиток постгіпертонічного лізису еритроцитів людини

In this research, a post-hypertonic lysis of human erythrocytes has been studied by varying the medium composition at dehydration stage. The osmolality of combined media, containing different concentrations of glycerol and NaCl was ~ 2370 mOsm/L. The level of post-hypertonic lysis of erythrocytes was shown as determined not by the total osmolality of dehydration medium, but glycerol concentration within the combined media. It was found that the nature of post-hypertonic lysis development of erythrocytes if using glycerol within the combined media at dehydration stage did not depend on preliminary cell saturation with cryoprotectant. A comparative study of post-hypertonic shock of erythrocytes at 37 and 0°C showed that in the latter case the cell hemolysis developed if using glycerol in lower concentration (10%) within the combined media (at 0°C the level of erythrocyte post-hypertonic lysis was 2.7 times higher). The observed effect may be due to a significant difference in the rates of transport for water and cryoprotectant through erythrocyte membrane at low temperatures. Probl Cryobiol Cryomed 2020; 30(3): 236–246

Vitex negundo L. leaf extract inhibits IL-6 and TNF-α secretion and phagocytosis in human leukocytes

Vitex negundo L. is used as an anti-inflammatory agent in Ayurveda and Sri Lankan traditional medicine. Here, the anti-inflammatory effects of an aqueous leaf extract (ALE) of V. negundo L. was investigated. The inhibitory effects of freeze-dried ALE (15.7–500 μg/mL) against interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumour necrosis factor-α (TNFα) secretion by human mononuclear cells, phagocytic activity of human neutrophils, and reactive oxygen species (ROS) and nitric oxide (NO) production by human leukocytes were tested in vitro. The inhibitory effects were dose dependent, and the strongest effect was observed at 500 μg/mL (P < 0.05). ALE caused 58.0 ± 1.0 % and 26.1 ± 2.0 % inhibition of IL-6 and TNFα secretion, respectively, whereas the positive control, Andrographis paniculata, caused complete inhibition. Phagocytic activity and ROS production were inhibited by 55 ± 4.5 % and 58.6 ± 8.6 %, respectively, whereas the positive controls, Ixora coccinea and diphenyleneiodonium chloride, caused 100 ± 4.4 % and 80 ± 6.4 % inhibition, respectively. ALE markedly inhibited NO production (87.5 ± 17.7 %), and the effect was comparable to that of the positive control, N-monomethyl-l-arginine (95.0 ± 1.0 %). The membrane-stabilizing activity of ALE (0.01–100 μg/mL) was assessed using heat-induced lysis of human erythrocytes. The highest membrane stabilizing activity (91 %) was observed at 0.01 μg/mL and was comparable with that of aspirin (positive control; 86.6 ± 0.51 %). These inhibitory effects may be the mechanisms through which V. negundo L. elicits its anti-inflammatory activities and support the traditional use of V. negundo L. as an anti-inflammatory agent.

The Design and Anticancer Activity of a Citropin1.1 Hybrid Peptide with Selective Activity Against Highly Invasive Metastatic Cell Lines

Citropin 1.1 is an amphipathic alpha-helical cationic peptide that exhibits potent anticancer activity in vitro. Citropin 1.1 was found to be active against 60 cancer cell lines, and this activity was mainly attributed to its ability to bind and lyse membranes of cancer cells. One of the major drawbacks of developing Citropin 1.1 as an anticancer agent is its lack of apparent selectivity toward cancer cells and its ability to cause significant lysis of normal human erythrocytes and mammalian cells at high concentrations. This low selectivity index places severe restraints on the development of Citropin 1.1 as a novel anticancer agent. In this study, we have designed a Citropin 1.1 analog named Citropin A that retained the biological activity of the parent peptide. Citropin A was fused to an anionic fragment in order to neutralize the positive charge carried on the parent peptide rendering it inactive. The resultant hybrid peptide named Citropin-MMP was designed to contain a Matrix metalloproteinase (MMP) cleavable consensus sequence that would be cleaved to release the active Citropin A once it encounters highly metastatic MMP producing cancer cells. Citropin-MMP was found to be completely inactive against non-MMP producing cancer cells and normal mammalian cells. However, when Citropin-MMP was administered to MMP producing cells, its antiproliferative activity was regained, and the peptide displayed exclusive activity against MMP producing cancer cell lines. The data of our study indicate that this enzyme-based cleavage strategy could prove to be successful for the development of Citropin-MMP as a novel therapeutic agent for the purpose of inhibiting the proliferation and invasion of highly metastatic invasive cancer cells.

Interaction of Magnesium-Based Materials with Human Blood Cells and Culture of Human Diploid Cells In Vitro.

We studied morphofunctional properties of human blood cells and diploid culture cells exposed to different types of magnesium materials: pure magnesium (Mg), magnesium-yttrium-neodymium-zirconium alloy (Mg-Y-Nd-Zr) and magnesium-zinc-calcium alloy (Mg-Zn-Ca). The materials were incubated with donor blood and mesenchymal multipotent stromal cells over 3 days. The studied materials did not induce massive lysis of human erythrocytes and leukocytes in vitro, but gradually impaired their structural integrity. In all cases, spontaneous platelet aggregation was observed in 6 h. In the presence of pure Mg and Mg-Zn-Ca alloy, this was accompanied by a decrease in the number of platelets with granules. In 24 h, substantial platelet degranulation occurred in all cases and in 72 h, the platelets did not contain granules. In parallel, the formation of large aggregates (60 μ) was observed. In the culture of stromal cells, all Mg-based materials reduced structural integrity of cells in 24 h, but did not significantly inhibit cell proliferation. Structural integrity of stromal cells partially recovered by day 3 in culture. The studied materials (Mg, Mg-Y-Nd-Zr, and Mg-Zn-Ca) seemed to be low-toxic for human cells during short-term contact, but could stimulate platelet aggregation and spontaneous degranulation and reduced the viability of diploid cells in vitro.

Development and Characterization of Monoclonal Antibodies Against Glycophorin A Applicable for Blood Sample Processing.

The separation of plasma from blood cells is critical for the accuracy of blood tests because cellular fractions can create discrepancies in analysis. The most common method to separate blood cells from the liquid part of the blood is centrifugation, which is not always applicable in resource-constrained areas and countries. In this study, we describe the generation of monoclonal antibodies (mAbs) against glycophorin A (GPA) of human erythrocytes. BALB/c mice were immunized with human erythrocytes followed by purified GPA. The splenocytes of the immunized mice were fused with Sp2/0 myeloma cells by hybridoma technique. Hybridoma clones were screened by hemagglutination assay and enzyme-linked immunosorbent assay (ELISA). Six hybridoma clones were obtained and subcloned. The characterization of the purified mAbs demonstrates that they are able to bind and retain erythrocytes in hemagglutination assay. Furthermore, one of the mAbs 1A9 recognizes purified GPA in ELISA, whereas the other mAb 1G7 is able to immunoprecipitate GPA from human erythrocyte lysates, and a band of 38 kDa is detected. In conclusion, the anti-GPA mAbs are useful tools in developing a quick and easy way to separate blood plasma from whole blood for clinical tests, and in developing bi-specific antibodies for other clinical applications.

ВПЛИВ ФЕНІЛГІДРАЗИНУ НА ГІПЕРТОНІЧНИЙ СТРЕС ЕРИТРОЦИТІВ ССАВЦІВ І АНТИГЕМОЛІТИЧНУ АКТИВНІСТЬ АМФІФІЛЬНИХ СПОЛУК

In the research work the initial level of erythrocyte damage of the investigated mammals has been shown to be significantly differed under hypertonic conditions. The level of human erythrocyte lysis in 4.0 mol/l NaCl at 37 °C made 90 %, for bovine cells it was 80 % and 60 % for equine ones. During the medium temperature decrease down to 0 °C the resistance of human, bovine and equine erythrocytes did not rise significantly. It has been demonstrated that sensitivity of mammalian erythrocytes to hypertonic stress at 37 °C after treatment with phenylhydrazine depended on the species of cells. Herewith the sensitivity of modified bovine and human erythrocytes was reduced and the equine ones increased. Sensitivity of rabbit erythrocytes after modification did not change statistically and significantly. Under hypertonic stress at temperature of 0 °C the rise in the damage rate of modified erythrocytes of all the investigated mammal species was observed. Trifluoperazine and dodecyl-b,D-maltoside has been established to manifest a high protective effect under hypertonic stress of erythrocytes for all the studied mammal species, excluding the rabbit erythrocytes. Trifluoperazine has a somewhat higher anithemolytic activity (about 80–90 %) if compared with dodecyl-b,D-maltoside (70 %). When decreasing in experiment the temperature down to 0 °C the antihemolytic activity of trifluoperazine and dodecyl-b,D-maltoside was strongly decreased. It has been shown that under hypertonic stress antihemolytic activity of amphiphilic compounds after modification of mammalian erythrocytes with phenylhydrazine was strongly decreased.

Hemolysis in human erythrocytes by Clostridium perfringens epsilon toxin requires activation of P2 receptors

ABSTRACT Epsilon-toxin (ETX) is produced by types B and D strains of Clostridium perfringens, which cause fatal enterotoxaemia in sheep, goats and cattle. Previous studies showed that only a restricted number of cell lines are sensitive to ETX and ETX-induced hemolysis has not previously been reported. In this study, the hemolytic ability of ETX was examined using erythrocytes from 10 species including murine, rabbit, sheep, monkey and human. We found that ETX caused hemolysis in human erythrocytes (HC50 = 0.2 μM) but not erythrocytes from the other test species. Moreover, the mechanism of ETX-induced hemolysis was further explored. Recent studies showed that some bacterial toxins induce hemolysis through purinergic receptor (P2) activation. Hence, the function of purinergic receptors in ETX-induced hemolysis was tested, and we found that the non-selective P2 receptor antagonists PPADS inhibited ETX-induced lysis of human erythrocytes in a concentration-dependent manner, indicating that ETX-induced hemolysis requires activation of purinergic receptors. P2 receptors comprise seven P2X (P2X1–7) and eight P2Y (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11–P2Y14) receptor subtypes. The pattern of responsiveness to more selective P2-antagonists implies that both P2Y13 and P2X7 receptors are involved in ETX-induced hemolysis in human species. Furthermore, we demonstrated that extracellular ATP is likely not involved in ETX-induced hemolysis and the activation of P2 receptors. These findings clarified the mechanism of ETX-induced hemolysis and provided new insight into the activities and ETX mode of action.

THE IMPORTANCE OF A PHOTOSENSITIZER BLEACHING REGISTRATION FOR THE EVALUATION OF MECHANISM OF PREPARATION ACTION ON THE PHOTO-INDUCED HEMOLYSIS

The influence of three pharmaceutical preparations on photo-induced lysis (irradiation with light-emitting diode of 653 nm) of human erythrocytes in the presence of photosensitizer radachlorin and also on the quantum yield of radachlorin photobleaching was studied. Both processes are mediated by singlet oxygen generation and therefore can become a target for antioxidant or prooxidant regulation. The pyridine preparation mexidol (action principle – 3-hydroxy-6-methyl-2-ethylpyridine succinate) in concentration of 152 µM used as an antioxidant and a membrane protector and aminazine (action principle – chlorpromazine) in concentration of 70 µM many times accelerated manyfold the photo-induced hemolysis. However only mexidol increased the quantum yield of radachlorin photobleaching in many times. The obtained data showed the direct prooxidant action of mexidol, but not aminazine that accelerated a photo-induced hemolysis due to other mechanism. A pyridine preparation nicotinic acid tested in equimolar to mexidol concentration, failed to influence both the velocity of the photo-induced hemolysis and the quantum yield of radachlorin photobleaching. Our study shows the possible use of a photobleaching tests for detection of the direct prooxidant action.

Generation of dimeric single-chain antibodies neutralizing the cytolytic activity of vaginolysin

Background: Gardnerella vaginalis is a bacterial vaginosis-associated vaginal bacterium, which produces toxin vaginolysin (VLY). VLY is a pore-forming toxin suggested to be the main virulence factor of G. vaginalis. The high recurrence rate of BV and emergence of antibiotic-resistant bacterial species demonstrate the need for development of recombinant antibodies as novel therapeutic agents for disease treatment. Single-chain variable fragments (scFvs) generated against VLY exhibited reduced efficacy to neutralize VLY activity compared to the respective full-length antibodies. To improve properties of scFvs the monospecific dimeric scFvs were generated by a genetic fusion of two anti-VLY scFv molecules connected with the alpha-helix-forming peptide linker. Results: N-terminally hexahistidine-tagged d imeric scFvs were constructed, produced in Escherichia coli and purified using metal-chelate affinity chromatography. Inhibition of VLY-mediated human erythrocyte lysis by dimeric and monomeric scFvs was detected by the in vitro hemolytic assay. Circulating half-life of purified scFvs in blood plasma of mice was accessed using ELISA. Dimeric anti-VLY scFvs showed higher neutralizing potency and extended circulating half-life in comparison with parental monomeric scFv. Conclusions: The protein obtained by a genetic fusion of two anti-VLY scFvs into dimeric molecule exhibited increased properties in comparison with monomeric scFv. This new recombinant antibody might implement new possibilities for the prophylaxis and treatment of the diseases caused by bacteria G. vaginalis . Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:Calibri,sans-serif; mso-bidi-font-family:Times New Roman;}

Loop Diuretics Diminish Hemolysis Induced by α-Hemolysin from Escherichia coli.

Uropathogenic Escherichia coli often produce the virulence factor α-hemolysin (HlyA), and the more severe the infection, the likelier it is to isolate HlyA-producing E. coli from patients. HlyA forms pores upon receptor-independent insertion of the toxin into biological membranes and it has been substantiated that HlyA-induced hemolysis is amplified by toxin-induced ATP release and activation of P2X receptors. Thus, hemolysis inflicted by HlyA is a protracted process involving signal transduction. It consists of early, marked cell shrinkage followed by swelling and eventually lysis. The initially shrinkage is a consequence of a substantial Ca2+-influx and activation of Ca2+-sensitive K+ and Cl- channels (KCa3.1/TMEM16A). The shrinkage is followed by gradual cell swelling, which ultimately lyses the cells. These findings clearly show that the HlyA pore provides a substantial volume challenge for the cells, and the fate of the given cell is co-determined by intrinsic erythrocytal volume regulation. We therefore speculated that other mechanisms involved in erythrocyte volume regulation may influence the hemolytic process inflicted by HlyA. Strikingly, HlyA-induced hemolysis is markedly reduced in erythrocytes isolated from NKCC1-deficient (NKCC1-/-) mice compared to controls. The NKCC1 inhibitors furosemide and bumetanide concentration-dependently inhibit HlyA-induced lysis of human and murine erythrocytes. However, in high concentrations bumetanide further reduced hemolysis in erythrocytes from NKCC1-/- mice and, thus, also exhibit indirect effects on hemolysis. The effect of loop diuretics on the hemolysis is not unique to HlyA but is similarly seen in LtxA- and α-toxin-induced hemolysis. Bumetanide clearly potentiates HlyA-induced volume reduction and delays the following erythrocyte swelling. This allows increased phagocytosis of damaged erythrocytes by THP-1 cell as a result of prolonged cell shrinkage. These data suggest that erythrocyte susceptibility to cytolysins is modified by NKCC1 and signifies intrinsic volume regulators as important determinants of cellular outcome of pore-forming toxins.

Enzymatic kinetics regarding reversible metabolism of CS-0777, a sphingosine 1-phosphate receptor modulator, via phosphorylation and dephosphorylation in humans

1. CS-0777, a candidate compound for autoimmune diseases, becomes phosphorylated active metabolite, M1, by fructosamine 3-kinase (FN3K), FN3K-related protein (FN3K-RP); and M1 is reverted back to CS-0777 by alkaline phosphatase (ALP) in the body. We performed enzyme kinetic analysis of phosphorylation of CS-0777 by FN3K, FN3K-RP, human erythrocytes and human platelets; and dephosphorylation of M1 by various ALP isozymes and human liver, kidney, lung and small intestine microsomes.2. The Michaelis constants of human FN3K, FN3K-RP and erythrocytes for CS-0777 phosphorylation were in the range from 498 μM to 1060 μM. FN3K inhibitor, 1-deoxy-1-morpholinofructose, suppressed only about 20% of CS-0777 phosphorylation activity in human erythrocyte lysate. Immunodepletion of FN3K and FN3K-RP decreased M1 formation activity by about 25% and 50%, respectively, in human erythrocyte lysate.3. The Michaelis constants of four human ALPs and microsomes were in the range from 10.9 μM to 32.1 μM. The ALP inhibitor, levamisole, suppressed over 50% of M1 dephosphorylation activity in liver, kidney and lung microsomes.4. FN3K-RP is expected to take a prominent role in the phosphorylation of CS-0777 in human erythrocytes; dephosphorylation of M1 was observed in all ALPs and human tissue microsomes examined, with a similar affinity towards M1 among them.

Toxicity screening of 13 Gambierdiscus strains using neuro-2a and erythrocyte lysis bioassays

Species in the epi-benthic dinoflagellate genus Gambierdiscus produce ciguatoxins (CTXs) and maitotoxins (MTXs), which are among the most potent marine toxins known. Consumption of fish contaminated with sufficient quantities of CTXs causes Ciguatera Fish Poisoning (CFP), the largest cause of non-bacterial food poisoning worldwide. Maitotoxins, which can be found in the digestive system of fish, could also contribute to CFP if such tissues are consumed. Recently, an increasing number of Gambierdiscus species have been identified; yet, little is known about the variation in toxicity among Gambierdiscus strains or species.This study is the first assessment of relative CTX- and MTX-toxicity of Gambierdiscus species from areas as widespread as the North-Eastern Atlantic Ocean, Pacific Ocean and the Mediterranean Sea. A total of 13 strains were screened: (i) seven Pacific strains of G. australes, G. balechii, G. caribaeus, G. carpenteri, G. pacificus, G. scabrosus and one strain of an undetermined species (Gambierdiscus sp. Viet Nam), (ii) five strains from the North-Eastern Atlantic Ocean (two G. australes, a single G. excentricus and two G. silvae strains), and (iii) one G. carolinianus strain from the Mediterranean Sea. Cell pellets of Gambierdiscus were extracted with methanol and the crude extracts partitioned into a CTX-containing dichloromethane fraction and a MTX-containing aqueous methanol fraction. CTX-toxicity was estimated using the neuro-2a cytoxicity assay, and MTX-toxicity via a human erythrocyte lysis assay.Different species were grouped into different ratios of CTX- and MTX-toxicity, however, the ratio was not related to the geographical origin of species (Atlantic, Mediterranean, Pacific). All strains showed MTX-toxicity, ranging from 1.5 to 86pg MTX equivalents (eq) cell−1. All but one of the strains showed relatively low CTX-toxicity ranging from 0.6 to 50 fg CTX3C eq cell−1. The exception was the highly toxic G. excentricus strain from the Canary Islands, which produced 1426 fg CTX3C eq cell−1. As was true for CTX, the highest MTX-toxicity was also found in G. excentricus. Thus, the present study confirmed that at least one species from the Atlantic Ocean demonstrates similar toxicity as the most toxic strains from the Pacific, even if the metabolites in fish have so far been shown to be more toxic in the Pacific Ocean.