HAT-field: a very cheap, robust and quantitative point-of-care serological test for Covid-19.

We have recently described a very simple and cheap serological test called HAT to detect antibodies directed against the RBD of the SARS-Cov-2 virus. HAT is based on hemagglutination, triggered by a single reagent (IH4-RBD) comprised of the viral RBD domain fused to a nanobody specific for glycophorin, which is expressed at very high levels at the surface of human red blood cells (RBCs). One of the main initial goals of this study was to devise a test protocol that would be sensitive and reliable, yet require no specialized laboratory equipment such as adjustable pipets, so that it could be performed in the most remote corners of the world by people with minimal levels of training. Because antibody levels against the viral RBD have been found to correlate closely with sero-neutralisation titers, and thus with protection against reinfection, it has become obvious during the course of this study that making this test reliably quantitative would be a further significant advantage. We have found that, in PBN, a buffer which contains BSA and sodium azide, IH4-RBD is stable for over 6 months at room temperature, and that PBN also improves HAT performance compared to using straight PBS. We also show that performing HAT at either 4°C, room temperature or 37°C has minimal influence on the results, and that quantitative evaluation of the levels of antibodies directed against the SARS-CoV-2 RBD can be achieved in a single step using titration of the IH4-RBD reagent. The HAT-field protocol described here requires only very simple disposable equipment and a few microliters of whole blood, such as can be obtained by finger prick. Because it is based on a single soluble reagent, the test can be adapted very simply and rapidly to detect antibodies against variants of the SARS-CoV-2, or conceivably against different pathogens. HAT-field appears well suited to provide quantitative assessments of the serological protection of populations as well as individuals, and given its very low cost, the stability of the IH4-RBD reagent in the adapted buffer, and the simplicity of the procedure, could be deployed pretty much anywhere, including in the poorest countries and the most remote corners of the globe.

Human red blood cells express the RNA sensor TLR7 and bind viral RNA

Red blood cells (RBCs) express the nucleic acid-sensing toll-like receptor 9 (TLR9) and bind CpG-containing DNA. However, whether human RBCs express other nucleic acid-sensing TLRs and bind RNA is unknown. Here we show that human RBCs express the RNA sensor, TLR7. TLR7 is present on the red cell membrane and associates with the RBC membrane protein Band 3. RBCs bind synthetic single-stranded RNA and RNA from pathogenic single-stranded RNA viruses. RNA acquisition by RBCs is attenuated by recombinant TLR7 and inhibitory oligonucleotides. Thus, RBCs may represent a previously unrecognized reservoir for RNA, although how RNA-binding by RBCs modulates the immune response has yet to be elucidated. These findings add to the growing list of non-gas exchanging RBC immune functions.

Facilitating trehalose entry into hRBCs at 4 ºC by alkylated ε-poly(L-lysine) for glycerol-free cryopreservation

Currently, glycerol is a conventional cryoprotectant of human red blood cells (hRBCs), but the time-consuming thawing and deglycerolization processes are essential before transfusion. Much of the research up to now...

Morphometry and Stiffness of Red Blood Cells—Signatures of Neurodegenerative Diseases and Aging

Human red blood cells (RBCs) are unique cells with the remarkable ability to deform, which is crucial for their oxygen transport function, and which can be significantly altered under pathophysiological conditions. Here we performed ultrastructural analysis of RBCs as a peripheral cell model, looking for specific signatures of the neurodegenerative pathologies (NDDs)—Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS) and Alzheimer’s disease (AD), utilizing atomic force (AFM) and conventional optical (OM) microscopy. We found significant differences in the morphology and stiffness of RBCs isolated from patients with the selected NDDs and those from healthy individuals. Neurodegenerative pathologies' RBCs are characterized by a reduced abundance of biconcave discoid shape, lower surface roughness and a higher Young’s modulus, compared to healthy cells. Although reduced, the biconcave is still the predominant shape in ALS and AD cells, while the morphology of PD is dominated by crenate cells. The features of RBCs underwent a marked aging-induced transformation, which followed different aging pathways for NDDs and normal healthy states. It was found that the diameter, height and volume of the different cell shape types have different values for NDDs and healthy cells. Common and specific morphological signatures of the NDDs were identified.

Blood compatibility evaluations of two-dimensional Ti3C2Tx nanosheets

Two-dimensional (2D) nanomaterial Ti3C2Tx is a novel biomaterial used for medical apparatus. For its application, biosafety serves as a prerequisite for their use in vivo. So far, no research has systematically reported how Ti3C2Tx interacts with various components in the blood. In this work, we evaluated the hemocompatibility of Ti3C2Tx nanosheets which we prepared by HF etching. Effects of the concentration and size of Ti3C2Tx on the morphology and hemolysis rate of human red blood cells (RBCs), the structure and conformation of plasma proteins, the complement activation, as well as in vitro blood coagulation were studied. In general, Ti3C2Tx takes on good blood compatibility, but in the case of high concentration (>30 μg/mL) and “Small size” (about 100 nm), it led to the rupture of RBCs membrane and a higher rate of hemolysis. Meanwhile, platelets and complement were inclined to be activated with the increased concentration, accompanying the changed configuration of plasma proteins dependent on concentration. Surprisingly, the presence of Ti3C2Tx did not significantly disrupt the coagulation. In vitro cell culture, the results prove that when the Ti3C2Tx concentration is as high as 60μg/mL and still has good biological safety. By establishing a fuzzy mathematical model, it was proved that the hemocompatibility of Ti3C2Tx is more concentration-dependent than size-dependent, and the hemolysis rate is the most sensitive to the size and concentration of the Ti3C2Tx. These findings provide insight into the potential use of Ti3C2Tx as biofriendly nanocontainers for biomaterials in vivo.

Synthesis, Molecular Docking and Antiplasmodial Activities of New Tetrahydro-β-Carbolines

Malaria is still one of the most dangerous infectious diseases and the emergence of drug resistant parasites only worsens the situation. A series of new tetrahydro-β-carbolines were designed, synthesized by the Pictet–Spengler reaction, and characterized. Further, the compounds were screened for their in vitro antiplasmodial activity against chloroquine-sensitive (D10) and chloroquine-resistant (W2) strains of Plasmodium falciparum. Moreover, molecular modeling studies were performed to assess the potential action of the designed molecules and toxicity assays were conducted on the human microvascular endothelial (HMEC-1) cell line and human red blood cells. Our studies identified N-(3,3-dimethylbutyl)-1-octyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b] indole-3-carboxamide (7) (a mixture of diastereomers) as the most promising compound endowed with the highest antiplasmodial activity, highest selectivity, and lack of cytotoxicity. In silico simulations carried out for (1S,3R)-7 provided useful insights into its possible interactions with enzymes essential for parasite metabolism. Further studies are underway to develop the optimal nanosized lipid-based delivery system for this compound and to determine its precise mechanism of action.

Type II Activation of Monocytes in Multiple Sclerosis

<p><b>Multiple sclerosis (MS) is an incurable autoimmune disease of the CNS. Although its cause is not known, immune cells are involved in the disease progression. Among these cells, type I monocytes are first to arrive to the brain and initiate inflammation; however, if monocytes are type II activated, they can inhibit inflammation. Previous research has shown that immune responses can be modulated by treatments, such as glatiramer acetate (GA) and immune complexes (IC). Therefore, we aimed to determine whether GA and IC can induce type II activation of monocytes in MS.</b></p> <p>Human blood monocytes from healthy volunteers and MS patients were stimulated in vitro with bacterial lipopolysaccharide (classical activation) in the presence or absence of GA and immune complexes (IC) composed of IVIG and human red blood cells (type II activation). Flow cytometry, ELISA and cytometric bead array were used to assess levels of marker expression and cytokine production in order to define the activation of monocytes.</p> <p>Interestingly, while both GA and IC induced type II activation of monocytes, the characteristics of these type II monocytes were distinct. We have found that monocytes from both healthy people and MS patients have significantly lower levels of inflammatory marker CD40 and higher levels of the anti-inflammatory cytokine IL-10 after treatment with IC. In contrast, GA treatment reduced the levels of CD40, CD86 and the inflammatory cytokine IL- 12. Moreover, the combined addition of GA and IC appeared to be more effective in type II activating monocytes than either agent alone. We also found that both CD14++CD16- and CD14+CD16+ monocyte subsets can be type II activated by the treatments; however, an interaction between the subsets impaired their response to the treatments.</p> <p>Our study suggests that treatments with GA and IC, especially in combination, are effective in type II activation of human monocytes and can be beneficial therapeutic approaches for multiple sclerosis.</p>

Type II Activation of Monocytes in Multiple Sclerosis

<p><b>Multiple sclerosis (MS) is an incurable autoimmune disease of the CNS. Although its cause is not known, immune cells are involved in the disease progression. Among these cells, type I monocytes are first to arrive to the brain and initiate inflammation; however, if monocytes are type II activated, they can inhibit inflammation. Previous research has shown that immune responses can be modulated by treatments, such as glatiramer acetate (GA) and immune complexes (IC). Therefore, we aimed to determine whether GA and IC can induce type II activation of monocytes in MS.</b></p> <p>Human blood monocytes from healthy volunteers and MS patients were stimulated in vitro with bacterial lipopolysaccharide (classical activation) in the presence or absence of GA and immune complexes (IC) composed of IVIG and human red blood cells (type II activation). Flow cytometry, ELISA and cytometric bead array were used to assess levels of marker expression and cytokine production in order to define the activation of monocytes.</p> <p>Interestingly, while both GA and IC induced type II activation of monocytes, the characteristics of these type II monocytes were distinct. We have found that monocytes from both healthy people and MS patients have significantly lower levels of inflammatory marker CD40 and higher levels of the anti-inflammatory cytokine IL-10 after treatment with IC. In contrast, GA treatment reduced the levels of CD40, CD86 and the inflammatory cytokine IL- 12. Moreover, the combined addition of GA and IC appeared to be more effective in type II activating monocytes than either agent alone. We also found that both CD14++CD16- and CD14+CD16+ monocyte subsets can be type II activated by the treatments; however, an interaction between the subsets impaired their response to the treatments.</p> <p>Our study suggests that treatments with GA and IC, especially in combination, are effective in type II activation of human monocytes and can be beneficial therapeutic approaches for multiple sclerosis.</p>