Detection of Hypertension-Induced Changes in Erythrocytes by SERS Nanosensors

Surface-enhanced Raman spectroscopy (SERS) is a promising tool that can be used in the detection of molecular changes triggered by disease development. Cardiovascular diseases (CVDs) are caused by multiple pathologies originating at the cellular level. The identification of these deteriorations can provide a better understanding of CVD mechanisms, and the monitoring of the identified molecular changes can be employed in the development of novel biosensor tools for early diagnostics. We applied plasmonic SERS nanosensors to assess changes in the properties of erythrocytes under normotensive and hypertensive conditions in the animal model. We found that spontaneous hypertension in rats leads (i) to a decrease in the erythrocyte plasma membrane fluidity and (ii) to a decrease in the mobility of the heme of the membrane-bound hemoglobin. We identified SERS parameters that can be used to detect pathological changes in the plasma membrane and submembrane region of erythrocytes.

Cholesterol dynamics are essential for the growth and development of Plasmodium falciparum within the erythrocyte

Plasmodium spp. lack de novo cholesterol synthetic pathways and can only scavenge it from their host erythrocyte. Here we report that depletion of cholesterol from the erythrocyte plasma membrane by methyl-β-cyclodextrin (MBCD) has dramatic consequences. The removal of cholesterol results in invasion defects as well as inhibition of parasite development through the intra-erythrocytic cycle. These defects could be rescued by reconstitution with cholesterol and desmosterol but not with epicholesterol. By using live microscopy of fluorescently tagged trophozoite stage parasites, we detected rapid expulsion of the parasites from erythrocyte when exposed to MBCD for just 30 mins. Strikingly, the parasites transition from being intra-erythrocytic to extracellular within 10 seconds and do so without rupturing the erythrocyte membrane. These extruded parasites were still surrounded by the parasitophorous vacuolar membrane (PVM) and remained tethered to the erythrocyte. Electron microscopy revealed that although extracellular parasites retained their PVM, it was heavily compromised. Treatment with antimalarials that disrupt cholesterol homeostasis prior to MBCD exposure prevented the extrusion of trophozoites. These results reveal importance of cholesterol during the intra-erythrocytic development of P. falciparum and the dramatic consequences resulting from tampering with cholesterol content in the infected erythrocyte. These findings suggest dynamic nature of cholesterol within the infected erythrocyte that is critical for parasite survival.

Autoimmunity to phosphatidylserine and anemia in African Trypanosome infections

Anemia caused by trypanosome infection is poorly understood. Autoimmunity during Trypanosoma brucei infection was proposed to have a role during anemia, but the mechanisms involved during this pathology have not been elucidated. In mouse models and human patients infected with malaria parasites, atypical B-cells promote anemia through the secretion of autoimmune anti-phosphatidylserine (anti-PS) antibodies that bind to uninfected erythrocytes and facilitate their clearance. Using mouse models of two trypanosome infections, Trypanosoma brucei and Trypanosoma cruzi, we assessed levels of autoantibodies and anemia. Our results indicate that acute T. brucei infection, but not T. cruzi, leads to early increased levels of plasma autoantibodies against different auto antigens tested (PS, DNA and erythrocyte lysate) and expansion of atypical B cells (ABCs) that secrete these autoantibodies. In vitro studies confirmed that a lysate of T. brucei, but not T. cruzi, could directly promote the expansion of these ABCs. PS exposure on erythrocyte plasma membrane seems to be an important contributor to anemia by delaying erythrocyte recovery since treatment with an agent that prevents binding to it (Annexin V) ameliorated anemia in T. brucei-infected mice. Analysis of the plasma of patients with human African trypanosomiasis (HAT) revealed high levels of anti-PS antibodies that correlated with anemia. Altogether these results suggest a relation between autoimmunity against PS and anemia in both mice and patients infected with T. brucei.

Kinetic tracking of Plasmodium falciparum antigen presentation reveals determinants of protein export and membrane insertion

Intracellular malaria parasites export many proteins into their host cell, inserting several into the erythrocyte plasma membrane to enable interactions with their external environment. While static techniques have identified some surface-exposed proteins, other candidates have eluded definitive localization and membrane topology determination. Moreover, both export kinetics and the mechanisms of membrane insertion remain largely unexplored. We introduce Reporter of Insertion and Surface Exposure (RISE), a method for continuous nondestructive tracking of antigen exposure on infected cells. RISE utilizes a small 11 aa NanoLuc fragment inserted into a target protein and detects surface exposure through high-affinity complementation. We tracked insertion of CLAG3, a malaria parasite protein linked to nutrient uptake, throughout the P. falciparum cycle in human erythrocytes. Our approach also revealed key determinants of trafficking and surface exposure. Removal of a C-terminal transmembrane domain aborted export. Unexpectedly, certain increases in the exposed reporter size improved surface exposure by up to 50-fold, revealing that both size and charge of the extracellular epitope influence membrane insertion. Insertion of parasite proteins at the host cell surface and antigen accessibility is regulated by multiple factors, enabling intracellular parasite survival and immune evasion under a broad range of conditions.

Changes in Human Erythrocyte Membrane Exposed to Aqueous and Ethanolic Extracts from Uncaria tomentosa

Uncaria tomentosa (Willd.) DC is a woody climber species originating from South and Central America that has been used in the therapy of asthma, rheumatism, hypertension, and blood purification. Our previous study showed that U. tomentosa extracts altered human erythrocyte shape, which could be due to incorporation of the compounds contained in extracts into the erythrocyte membrane. The aim of the present study was to determine how the compounds contained in U. tomentosa extracts incorporate into the human erythrocyte membrane. The study has assessed the effect of aqueous and ethanolic extracts from leaves and bark of U. tomentosa on the osmotic resistance of the human erythrocyte, the viscosity of erythrocyte interior, and the fluidity of erythrocyte plasma membrane. Human erythrocytes were incubated with the studied extracts in the concentrations of 100, 250, and 500 µg/mL for 2, 5, and 24 h. All extracts tested caused a decrease in erythrocyte membrane fluidity and increased erythrocyte osmotic sensitivity. The ethanolic extracts from the bark and leaves increased viscosity of the erythrocytes. The largest changes in the studied parameters were observed in the cells incubated with bark ethanolic extract. We consider that the compounds from U. tomentosa extracts mainly build into the outer, hydrophilic monolayer of the erythrocyte membrane, thus protecting the erythrocytes against the adverse effects of oxidative stress.

RELATION OF DISTURBANCES IN THE ERYTHROCYTES ELECTRICAL ACTIVITY WITH DYSLIPIDEMIA IN THE METABOLIC SYNDROME

Aim. To study the erythrocyte electric charge (EEC) in patients with metabolic syndrome (MS).Material and methods. 112 patients (mean age 61.4±7.2 years) with MS (average duration of MS 8.7±5.2 years) were examined. Control group consisted of healthy volunteers (n=25) of similar age. The level of EEC was detected by the method of adsorption of a positive cationic dye (cationic blue O) on the surface of the erythrocyte plasma membrane up to the complete neutralization of their negative charge, followed by photometry of the solution and calculation of the number of charges on the erythrocyte cell surface. The results of the study were processed by Statistica 10.0 software.Results. Hypertension was found in 73% of patients with MS, hyperglycemia – in 39%, diabetes – in 36%, dyslipidemia – in 80% of patients. The EEC in MS patients (1.59±0.05×107) was lower than this in the control group (1.65±0.03×107; p<0.05). With MS duration more than 5 years, the EEC was significantly lower in comparison with control group (p<0.05). In patients with obesity II-III degrees as well as dyslipidemia the EEC values were significantly lower vs control group (p<0.05). In patients with MS, a significant negative correlation of EEC with total cholesterol (r=-0.51, p<0.05) and triglycerides (r=-0.51, p<0.05) levels were revealed.Conclusion. In patients with MS with increase in the MS duration, degree of obesity and lipid metabolism disorders, significantly lower values of EEC were observed.