Lymphocyte Membrane Research Articles

A CD6 homolog of Nile tilapia (Oreochromis niloticus) conserved binding bacteria involved in the regulation of Streptococcus agalactiae induced inflammation

As a lymphocyte-specific surface receptor belonging to the cysteine-rich superfamily of scavenger receptors, CD6 acts as a pattern recognition receptor for microbial components and is involved in the regulation of inflammatory responses. However, the characteristics and functions of CD6 molecules in lower vertebrates represented by teleost fish are unknown. In this study, a CD6 homolog (designated OnCD6) was characterized from Nile tilapia (Oreochromis niloticus), and establishing its role as a PRRs that participates in immune recognition. OnCD6 contains an open reading frame of 1872 bp that encodes a peptide of 623 amino acids, and contains two conserved SR domain. Multiple sequence alignment revealed that OnCD6 shares a relatively high level of identity with those of other species. Transcriptional expression analysis revealed that OnCD6 was constitutively expressed in immunes tissues such as head kidney and thymus. The expression level of OnCD6 in mainly immune tissues were found significantly upregulated after the injection of Streptococcus agalactiae (S. agalactiae). Moreover, OnCD6 protein was located in the head kidney and brain, mainly over the plasma membrane of lymphocytes in these immune tissues. In vitro experiments showed that CD6 extracellular protein bound to and aggregated several Gram-positive and -negative bacterial strains through the recognition of bacterial surface conserved components LPS and LTA etc. In vivo experiments demonstrated that overexpression OnCD6 before S. agalactiae challenge significantly improved tilapia survival, and this was concomitant with reduced bacterial load and pro-inflammatory cytokines (IL-1β and TNF-α). Taken together, our results illustrated the function of CD6 molecular pattern recognition receptors (PRRs) is conserved and plays an important role in antibacterial infection.

Blebbing of the plasma membrane of lymphocytes as a marker of endothelial dysfunction in the development of post-resection insufficiency in patients with focal liver lesions

Purpose of the study. To evaluate the intensity of blebbing of the plasma membrane of lymphocytes and the formation of microparticles of lymphocytic origin in patients with postresection hepatic failure. Material and methods. The study involved 54 patients with focal liver diseases. The study involved 32 (59%) women, 22 (41%) men. The average age was 59.5 [49.75; 66,00]. Patients were divided into groups, 1 group - 10 patients with developed acute liver failure and 2 group - 44 patients with a favorable course of postoperative period. The following surgical interventions were performed: 36 (67%) patients underwent surgery - laparotomy, segmental liver resection, 10 (18%) - left-sided hemihepatectomy and 8 (15%) - right-sided hemihepatectomy. Results. Predictors of early postoperative hepatic failure are a decrease in initial blebbing and an increase in terminal blebbing and free microparticles of lymphocytic origin. Conclusions. Thus, changes in lymphocyte blebbing and the presence of free microparticles of lymphocytic origin are due to the development of endothelial insufficiency and apoptosis of lymphocytes in the context of liver failure and are determined by the reduced function of residual liver volume.

Membrane organization by tetraspanins and galectins shapes lymphocyte function.

Immune receptors are not randomly distributed at the plasma membrane of lymphocytes but are segregated into specialized domains that function as platforms to initiate signalling, as exemplified by the B cell or T cell receptor complex and the immunological synapse. 'Membrane-organizing proteins' and, in particular, tetraspanins and galectins, are crucial for controlling the spatiotemporal organization of immune receptors and other signalling proteins. Deficiencies in specific tetraspanins and galectins result in impaired immune synapse formation, lymphocyte proliferation, antibody production and migration, which can lead to impaired immunity, tumour development and autoimmunity. In contrast to conventional ligand-receptor interactions, membrane organizers interact in cis (on the same cell) and modulate receptor clustering, receptor dynamics and intracellular signalling. New findings have uncovered their complex and dynamic nature, revealing shared binding partners and collaborative activity in determining the composition of membrane domains. Therefore, immune receptors should not be envisaged as independent entities and instead should be studied in the context of their spatial organization in the lymphocyte membrane. We advocate for a novel approach to study lymphocyte function by globally analysing the role of membrane organizers in the assembly of different membrane complexes and discuss opportunities to develop therapeutic approaches that act via the modulation of membrane organization.

Major histocompatibility complex class IIα and IIβ of pufferfish (Takifugu obscurus): Identification and functional characterization in response to bacterial challenge.

Classical major histocompatibility complex (MHC) class II molecules play an essential role in immune system. In this study, MHC IIα (Pf-MHC IIα) and MHC IIβ (Pf-MHC IIβ) homology genes from pufferfish (Takifugu obscurus) were cloned and their functional characterization in response to bacterial challenge was identified. The nucleotide sequences of the open reading frames (ORFs) of pufferfish Pf-MHC IIα and Pf-MHC IIβ were 708 bp and 750 bp, encoding 235 aa and 249 aa, respectively. The structure of Pf-MHC IIα or Pf-MHC IIβ contained a signal peptide, an α1/β1 domain, an α2/β2 domain, a transmembrane region and a cytoplasmic region. Multiple sequence alignment and phylogenetic analysis showed that Pf-MHC IIα and Pf-MHC IIβ molecules had the highest similarity with Fugu rubripes (Takifugu rubripes). Cellular localization analysis indicated that the distribution of Pf-MHC IIα and Pf-MHC IIβ was in the lymphocyte membrane and cytoplasm. qRT-PCR results showed that Pf-MHC IIα and Pf-MHC IIβ expressed relatively high in skin, gills and gut. In addition, after stimulation challenge in vitro (lipopolysaccharide, or polyinosinic: polycytidylic acid) and in vivo (A. hydrophila), the mRNA expressions of Pf-MHC IIα and Pf-MHC IIβ were significantly up-regulated in lymphocytes and in tissues of skin, gills, gut and head kidney. Moreover, Pf-MHC IIα or Pf-MHC IIβ neutralization reduced the ability of A. hydrophila to induce the expressions of lymphocyte cytokines (TNF-α, IL-1β and IL-10). Overall, it is speculated that Pf-MHC IIα and Pf-MHC IIβ may play an important role in the host response against A. hydrophila in pufferfish.

#6333 LARGE B CELL RENAL INFILTRATION IN A PATIENT WITH WALDESTRöM MACROGLOBULINEMIA: A CASE REPORT

Abstract Background and Aims Waldeström Macroglobulinemia (WM) is a clinicopathologic entity demonstrating lymphoplasmacytic lymphoma (LPL) in the bone marrow with an IgM monoclonal gammopathy in the blood. Only 15% of patients with WM may develop a renal insufficiency due to either glomerular or tubulointerstitial pathologies. Most common findings include deposits of IgM in the glomerular basement membrane and infiltration of lymphocytes or plasmacytoid cells; light chain cast nephropathy; nephrotic syndrome led by AL amyloid deposition; immune-mediated glomerulonephritis (typically due to IgM deposition or cryoglobulinemia) and non-amyloid nephrotic syndrome (minimal change-like). Moreover, WM may evolve in hematological malignancies with renal localization. Therefore, a renal biopsy may be needed in patients who have recent unexplained renal dysfunction. Method A 72-year-old male with a history of WM is referred to our nephrology center for declining renal function. He had been diagnosed with Waldeström Macroglobulinemia IgM Kappa 4 years prior to presentation, at onset serum protein electrophoresis showed an M spike of 4900. After diagnosis confirmation, he underwent treatment with 6 cycles of rituximab and bendamustine resulting in a complete response and M spike at serum protein electrophoresis lowering to 23. At WM diagnosis his renal function was normal, he had no proteinuria nor urinary Bence-Jones. His medical background also included hypertension and diabetes mellitus type 2, both in good pharmacologic control and Sars-Cov2 infection. Two years later, we witnessed to progressive presentation of anemia, fatigue and cough along with hepatomegaly, axillary lymphomegaly, pulmonary basal thickenings and IgM levels elevation. Serum creatinine was 1,47 mg/dl, proteinuria 1,75 g/die, no urinary Bence-Jones proteinuria. A renal ultrasound showed poor cortico-medullary differentiation in both kidneys and a left kidney slightly bigger than the other one. Respiratory symptoms and pulmonary thickenings persisted after antibiotics therapy, also cytology and culturing exams performed on broncho-alveolar lavage resulted negative. To exclude a possible relapse of WM, a BOM and a lymphnodal mass biopsy were performed. Both exams showed no disease progression. Due to lack of WM progression, the patient is referred to renal biopsy execution. Results Renal biopsy showed morpho-phenotypic findings for localization of mainly large B peripherical cells, partial plasmocytic differentiation and high proliferation levels. Further exams revealed a double monoclonal component IgM Kappa with normal Kappa/Lambda, also a total Body TC was performed, and it evidenced a possible renal and pulmonary lymphoproliferative disease involvement. In the figure an image of the patient's renal biopsy showing IgM immunofluorescence. Our patient is still fulfilling all the supplementary analysis needed to evaluate the best therapeutic strategies. We also reviewed all renal biopsies performed in our center in patients with hematological diseases in the last 10 years and a neoplastic renal infiltration was documented in just 6 cases out of 650 biopsies. Conclusion Although lymphomatous infiltration of the kidney in WM is a rare event, it should be considered in patients presenting with abnormal renal function. It is important during follow-up to monitor kidney function in patients with plasma cell dyscrasias, even if patients appear to have stable lymphoproliferative disease.

Lymphocyte Membrane‐ and 12p1‐Dual‐Functionalized Nanoparticles for Free HIV‐1 Trapping and Precise siRNA Delivery into HIV‐1‐Infected Cells (Adv. Sci. 10/2023)

Precise siRNA Delivery Precise delivery of siRNA into HIV-1-infected cells is realized by the lymphocyte membraneand 12p1-dual-functionalized nanoparticles. These nanoparticles also display the ability of trapping free HIV-1 and inhibiting the gp120-induced bystander T cell death. More details can be found in article number 2300282 by Zhiping Li, Lin Li, Chunsheng Gao, and co-workers.

Lymphocyte Membrane- and 12p1-Dual-Functionalized Nanoparticles for Free HIV-1 Trapping and Precise siRNA Delivery into HIV-1-Infected Cells.

Despite the success of small interfering RNA (siRNA) in clinical settings and its potential value in human immunodeficiency virus (HIV) therapy, the rapid clearance and absence of precise delivery to target cells still hinder the therapeutic effect of siRNA. Herein, a new system, which can escape immune recognition, has HIV-1 neutralizing capacity, and the ability to deliver siRNA specifically into HIV-1-infected cells, is constructed by functionalizing siRNA delivery lipid nanoparticles with the lymphocyte membrane and 12p1. The constructed system is shown to escape uptake by the mononuclear phagocyte system. The constructed system exhibits strong binding ability with gp120, thus displaying distinguished neutralizing breadth and potency. The constructed system neutralizes all tested HIV-1 pseudotyped viruses with a geometric mean 80% inhibitory concentration (IC80) of 29.75µg mL-1 and inhibits X4-tropic HIV-1 with an IC80 of 64.20µg mL-1 , and R5-tropic HIV-1 with an IC80 of 16.39µg mL-1 . The new system also specifically delivers siRNA into the cytoplasm of HIV-1-infected cells and exhibits evident gene silencing of tat and rev. Therefore, this new system can neutralize HIV-1 and deliver siRNA selectively into HIV-1-infected cells and may be a promising therapeutic candidate for the precise therapy of HIV.

BLEBBING OF THE PLASMA MEMBRANE OF LYMPHOCYTES IN THE POSTOPERATIVE PERIOD OF UROLITHIASIS

Urolithiasis is an urgent problem of surgical urology, about 200 thousand operations are performed annually in the Russian Federation. The complicated course of the postoperative period is accompanied by changes in homeostasis associated with immune activation and dysregulation of the endothelial system. The study evaluated the parameters of lymphocyte plasma membrane blebbing in patients with complicated and uncomplicated postoperative urolithiasis. 240 patients suffering from urolithiasis took part, who underwent surgical treatment. Patients were divided into two clinical groups: I clinical group (n = 130) – patients with a favorable course of the postoperative period of urolithiasis, II clinical group (n = 110) – patients with a complicated course of the postoperative period of urolithiasis. The control group was – 25 practically healthy persons. Lymphocyte membrane status was assessed by phase contrast microscopy. The maximum frequency of plasma membrane changes was recorded in patients with postoperative complications and amounted to 15.92 [13.20; 17.01] when evaluating initial blebbing and 21.93 [17.67; 30.45] for terminal blebbing. With a favorable course of postoperative period, patients with urolithiasis did not have statistically significant changes in the parameters of blebbing of the plasma membrane of lymphocytes.

МИТОХОНДРИАЛЬНО-НАПРАВЛЕННЫЙ АНТИОКСИДАНТ SKQ1 РЕГУЛИРУЕТ СИГНАЛЬНУЮ СИСТЕМУ KEAP1/NRF2/ARE И АПОПТОЗ В ЛЕЙКОЦИТАХ КРОВИ ПРИ ОКИСЛИТЕЛЬНОМ СТРЕССЕ

This study demonstrated that hyperoxia induced (0.5 MPa, 90 min) oxidative stress decreased mRNA level of Nrf2 transcription factor and Nrf2 induced genes encoding antioxidant enzymes (SOD1, CAT, GPx4) in leukocytes of rat blood. The change in gene expression profiles in hyperoxia was accompanied by disbalance of antioxidant enzyme activities in the leukocytes – activation of superoxide dismutase, and inhibition of catalase, glutathione peroxidase. The administration of SkQ1 (50 nmol/kg during 5 days) significantly increased mRNA level of Nrf2 transcription factor and Nrf2 induced genes encoding antioxidant enzymes SOD2 and GPx4 and nor malized the transcriptional activity of the SOD1 and CAT genes in the leukocytes under the hyperoxia induced oxidative stress. Catalase and glutathione peroxidase activity in the leukocytes increased concurrently with reversion of superoxide dismutase activity to the control level. The protective effect of SkQ1 in hyperoxia induced oxidative stress may be realized via direct antioxidant activity and the stimulation of the Keap1/Nrf2/ARE defense system. Administration of SkQ1 in extremely low amounts (50 nmol/kg for 5 days) effectively inhibits the accumulation of lipid peroxidation molecular products, normalizes the structural state of lymphocyte membranes and the level of their apoptosis under the condition of physiological standard and oxidative stress.

Блеббинг плазматической мембраны лимфоцитов при инфекционных осложнениях уролитиаза

About 200 thousand operations for urolithiasis are performed annually in Russia, but the frequency of postoperative complications reaches 30%. Infectious complications occur with a frequency of up to 40% in the form of various forms of pyelonephritis, urosepsis is recorded in 3% of patients. The aim of the study determine the intensity of blebbing of the plasma membrane of lymphocytes in infectious complications of urolithiasis.A prospective study examined 1,240 patients with urolithiasis. Inflammatory complications were characterized by the development of bacteriuria, serous pyelonephritis, purulent pyelonephritis and urosepsis. Complications of infectious nature were characterized by the development of bacteriuria, serous pyelonephritis, purulent pyelonephritis and urosepsis. During the study, complications of an infectious nature were detected in 148 patients, which amounted to 11,93% of cases. The control group consisted of - 25 persons with urolithiasis and a favorable course of the postoperative period. Lymphocyte membrane condition was evaluated by phase contrast microscopy.Changes in plasma membrane structure were more frequently reported in patients with postoperative purulent pyelonephritis and urosepsis. When assessing the presence of circulating microparticles, the highest number was recorded in patients with urolithiasis complicated by purulent pyelonephritis or urosepsis - 1318 [982; 2007] and 1531 [1028; 1963], respectively. A relationship was established between the degree of severity of terminal blebbing of the plasma membrane of lymphocytes and the nature of inflammatory complications in urolithiasis.

BLOOD FLOW CONDITION IN LOWER EXTREMITY VESSELS AND ENDOTHELIAL DYSFUNCTION IN PATIENTS WITH TYPE 2 DIABETES MELLITUS DEPENDING ON DISEASE DURATION

The purpose of the study. Study arterial blood flow and blebbing of the plasma membrane of lymphocytes in the neuroischemic form of diabetic foot syndrome (SDS) depending on the age of the disease.
 Material and methods. In a prospective study, 150 patients of type 2 DM were examined, of which 50 people with a neuroischemic form of SDS. Blood flow condition on vessels of lower extremities and plasma membrane of lymphocytes was examined. There were 30 women (60%), 20 men (40%). Vascular channel condition was evaluated by ultrasound dopplerography of lower extremity arteries, degree of diabetes mellitus compensation was determined by glycated hemoglobin level, lymphocyte membrane condition was evaluated by phase contrast microscope imaging results.
 Results of the study. 72% of patients had complete occlusion of the tibia-foot segment of the arteries of the lower extremity, 28% had stenosis. The glycated hemoglobin level averaged 10.3 [8.5; 14.3]%, i.e. all observed patients had insufficient compensation for carbohydrate metabolism disorders. The total and terminal lymphocyte blebbing was 30.1 [25.4; 33.2]% and 15.8 [11.8; 18.4]%, which was 3 and 5 times higher than the control group, respectively. A high positive correlation between the duration of DM disease and the total (rS=0,72; p = 0.001) and terminal (rS=0,78; p = 0.001) blebbing of lymphocyte membrane.
 Conclusion. The high correlation between the timing of DM disease and the blebbing of the lymphocyte plasma membrane allows these indicators to be used as markers of endothelial dysfunction in neuroischemic and ischemic forms of SDS. The level of summary and terminal blebbing can be considered as a prognostic marker of the development of macroangiopathy in patients with type 2 DM.

Method for Identifying Galectin Ligands on Lymphocyte Membrane Glycoproteins.

Glycosylation is one of the most common protein posttranslational modifications. Most human lymphocyte membrane receptors are modified by diverse glycan structures, and functional studies have indicated that a family of glycan-binding proteins, galectins, can significantly modulate lymphocyte development and function by interacting with these glycans. Several galectins have a varying degree of affinity for the N-acetyllactosamine (LacNAc) disaccharide, and some critical lymphocyte receptors can be modified by glycan structures carrying this motif. However, the site-specific glycan composition on primary lymphocyte membrane receptors in healthy individuals is largely limited. The main reason for the limitation is low abundance of available material from a single donor and compositional heterogeneity in glycan structures that can potentially modify a protein. Donor-dependent variability in N-glycan structures on CD16a isolated from primary NK cells of healthy human donors was recently reported. NK cell CD16a is glycosylated at five N-glycosylation sites, and two of the five sites are modified, almost exclusively, by N-glycans with multiple LacNAc repeats which can serve as ligands for endogenous galectins. Thus, the protocol described in this section can be utilized to identify galectin ligands at specific glycosylation sites of endogenous membrane receptor from circulating primary human lymphocytes.

Дослідження глікобіому поверхні лімфоцитів хворих на В-клітинний хронічний лімфолейкоз

The purpose of the study was to investigate the intensity of exposure of peripheral blood lymphocyte surface glycans in patients with B-cell chronic lymphocytic leukemia by measuring the density of lectin- or antigen-positive epitopes under antitumor therapy in order to evaluate it for a more reasonable selection of qualitative and quantitative composition of therapy. Materials and methods. The objects of the study were blood lymphocytes of patients with chronic lymphocytic leukemia (n=15) aged 58-66 years before and after a course of standard chemotherapy according to the COP scheme. The control group consisted of healthy volunteers (n=15) aged 55 to 65 years. Isolation of lymphocytes was performed by a modified method of A. Boyum. Polyclonal antibodies to α1-acid glycoprotein and fibronectin were used. Exposure to Tn antigen and CD43 on blood lymphocytes was determined with secondary antibodies to mouse immunoglobulins conjugated to FITC (Millipore, USA). To study the exposure of glycans on the surface of lymphocytes, we used a set of seven lectins labeled with FITC. Data recording was performed on a Beckman Flower EPICS flow cytometer. Processing of the results was done using the program FCS3 Express. Results and discussion. Compared with the group of hematologically healthy donors on the surface of lymphocytes in patients with chronic lymphocytic leukemia, a 20-fold increase in the density of exposure to ConA epitopes, 10 times – UEAI- and SNA-positive epitopes were shown; MAA II epitope, Tn, and CD43 antigen densities were increased 100-fold (p <0.01). Exposure densities of MAA II-, Tn-, and CD43-positive epitopes on the plasma membrane of lymphocytes in patients with chronic lymphocytic leukemia receiving alkylation therapy decreased 10-fold relative to treatment data, but remained 10-fold higher than in the group of healthy hematologists. Conclusion. On the plasma membranes of lymphocytes in patients with chronic lymphocytic leukemia, the density of exposure of mannose and neuraminic acid residues was significantly increased. COP therapy reduced the density of these epitopes to control values. A significant increase in the density of carcinogenesis markers – Tn- and CD43-antigens on the plasma membranes of lymphocytes in patients with chronic lymphocytic leukemia has been shown. COP therapy provided only a partial decrease in their density, which indicates the insufficient effectiveness of COP therapy, its inability to completely stop the oncological process in patients with chronic lymphocytic leukemia

Colon cancer cells acquire immune regulatory molecules from tumor-infiltrating lymphocytes by trogocytosis

Cancer cells can develop an immunosuppressive tumor microenvironment to control tumor-infiltrating lymphocytes. The underlying mechanisms still remain unclear. Here, we report that mouse and human colon cancer cells acquire lymphocyte membrane proteins including cellular markers such as CD4 and CD45. We observed cell populations harboring both a tumor-specific marker and CD4 in the tumor microenvironment. Sorted cells from these populations were capable of forming organoids, identifying them as cancer cells. Live imaging analysis revealed that lymphocyte membrane proteins were transferred to cancer cells via trogocytosis. As a result of the transfer invivo, cancer cells also acquired immune regulatory surface proteins such as CTLA4 and Tim3, which suppress activation of immune cells [T. L. Walunas etal, Immunity 1, 405-413 (1994) and L. Monney etal., Nature 415, 536-541 (2002)]. RNA sequencing analysis of ex vivo-cocultured splenocytes with trogocytic cancer cells showed reductions in Th1 activation and natural killer cell signaling pathways compared with the nontrogocytic control. Cancer cell trogocytosis was confirmed in the patient-derived xenograft models of colorectal cancer and head and neck cancer. These findings suggest that cancer cells utilize membrane proteins expressed in lymphocytes, which in turn contribute to the development of the immunosuppressive tumor microenvironment.

Analysis of CTLA-4 -318C/T Polymorphism in Thalassemia Patients Transfused at the Casablanca Children's Hospital (Morocco)

Red blood cells (RBC) alloimmunization is a delayed adverse transfusion reaction in thalassemia patients. The mechanisms behind the inhibition or tolerance of red blood cells are still poorly understood. Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) molecule is expressed on Treg -lymphocyte membrane and is an inhibitory molecule which plays the mediator role of peripheral tolerance and maintains tolerance to self-antigens. Recent studies have reported that defect in the CTLA-4 gene expression could affect its function and be involved in the development of various pathologies as autoimmune diseases and the outcome after Allogenic hematopoietic stem cell transplantation; indeed, the objective of our study is the search for the association of the CTLA-4 polymorphism with the susceptibility to red blood cells alloimmunizations. In this study we looked for the polymorphism of the CTLA-4 gene at the -318 C/T position in 35 β-thalassemic patients (15alloimmunized and 20 non alloimunized) followed at the children's hospital in Casablanca, and 20 healthy controls, by PCR-RFLP and Sanger sequencing. In our cohort, none of the group cases revealed the mutation carried out by PCR RFLP. Indeed, this result was confirmed by Sanger sequencing. This study does not find an association of -318C/T SNPs in CTLA-4 gene and RBC alloimmunization among our cohort. Following these preliminary results, an investigation of the other exons of the CTLA-4 gene on a large cohort is necessary to complete this study.

АНАЛИЗ ФИЗИКО-ХИМИЧЕСКИХ ХАРАКТЕРИСТИК МЕМБРАН КЛЕТОК КРОВИ ПАЦИЕНТОВ С РЕВМАТОИДНЫМ АРТРИТОМ

In the work reviewed and analyzed the results reflecting the physical and chemical state of the lymphocyte membranes of patients with rheumatoid arthritis. It was found that patients with rheumatoid arthritis did not show significant changes in the polarity of different areas of the membrane. At the same time discovered that annular lipid microviscosity of the plasma membrane of lymphocytes in patients with rheumatoid arthritis is reduced 2.5 times compared to control values in healthy patients, and the increased microviscosity in the general area of lipid bilayer was increased this by 25 % in relation to control values. The value of the degree of quenching of tryptophan fluorescence by pyrene in patients with rheumatoid arthritis is reduced by 35 % in relation to the control. In the membranes of red blood cells of patients with rheumatoid arthritis, there were no significant differences compared with the control values.

Влияние пуринергической сигнальной системы на свойства клеток крови человека при старении организма

Adenosine triphosphate (ATP) molecules and nucleotide derivatives contribute to a wide range of cellular reactions through the activation of A2 and P2 purinergic receptors located on the cell surface. Almost any type of cells expresses such receptors, including red blood cells and white blood cells. The study of purinergic transmission of signals mediating calcium mobilization, actin polymerization, chemotaxis, release of mediators, tissue oxygenation, cell maturation, cytotoxicity, cell death and cell–cell interactions in dynamics at different stages of ontogenesis is an important issue in biology and medicine today. This paper aimed to explore the biophysical properties of blood cells during the activation of the purinergic signalling system in middle-aged people and older adults. To activate the elements of the purinergic signalling system, an in vitro model of mechanical stress was used. Blood samples were analysed using the methods of atomic force microscopy, namely, force spectroscopy and the Kelvin probe. When the purinergic signalling system is activated, an increase in ATP blood concentration is identified in the blood of both middle-aged people and older adults. In the blood of older adults, a smaller amount of ATP was recorded compared with the blood of middle-aged people, which can be due to changes in the expression of purinoreceptors on the surface of red blood cells at ageing. Erythrocytes and granulocytes in older adults are characterized by increased surface stiffness and potential upon activation of the purinergic signalling system, compared with cells of middle-aged people. In response to mechanical stress, lymphocyte membrane stiffness in older adults increases more significantly than in middle-aged people. Thus, the results indicate an important role of the ATP molecule and its derivatives in the regulation of functioning of blood cells through binding to receptors of the P2 family. Research on the changes in the biophysical properties of red blood cells and white blood cells at activation of the purinergic signalling system will allow us to identify new effects of purine compounds on intercellular interaction during ageing.

CD20 as a gatekeeper of the resting state of human B cells.

CD20 is a B cell-specific membrane protein and represents an attractive target for therapeutic antibodies. Despite widespread usage of anti-CD20 antibodies for B cell depletion therapies, the biological function of their target remains unclear. Here, we demonstrate that CD20 controls the nanoscale organization of receptors on the surface of resting B lymphocytes. CRISPR/Cas9-mediated ablation of CD20 in resting B cells resulted in relocalization and interaction of the IgM-class B cell antigen receptor with the coreceptor CD19. This receptor rearrangement led to a transient activation of B cells, accompanied by the internalization of many B cell surface marker proteins. Reexpression of CD20 restored the expression of the B cell surface proteins and the resting state of Ramos B cells. Similarly, treatment of Ramos or naive human B cells with the anti-CD20 antibody rituximab induced nanoscale receptor rearrangements and transient B cell activation in vitro and in vivo. A departure from the resting B cell state followed by the loss of B cell identity of CD20-deficient Ramos B cells was accompanied by a PAX5 to BLIMP-1 transcriptional switch, metabolic reprogramming toward oxidative phosphorylation, and a shift toward plasma cell development. Thus, anti-CD20 engagement or the loss of CD20 disrupts membrane organization, profoundly altering the fate of human B cells.

Lipid specificity of the immune effector perforin.

Perforin is a pore forming protein used by cytotoxic T lymphocytes to remove cancerous or virus-infected cells during the immune response. During the response, the lymphocyte membrane becomes refractory to perforin function by accumulating densely ordered lipid rafts and externalizing negatively charged lipid species. The dense membrane packing lowers the capacity of perforin to bind, and the negatively charged lipids scavenge any residual protein before pore formation. Using atomic force microscopy on model membrane systems, we here provide insight into the molecular basis of perforin lipid specificity.

Molecular cloning, characterization and expression analysis of major histocompatibility complex class I alpha gene of pufferfish (Takifugu obscurus).

The major histocompatibility complex (MHC) genes play a key role in immune response in vertebrates. In this study, an MHC I alpha homolog gene (PfMHC Ⅰα) from pufferfish (Takifugu obscurus) was identified and its subcellular localization and expression patterns of PfMHC Ⅰα after challenge in vivo and in vitro were analysed. The open reading frame of PfMHC Ⅰα was 1,089bp in length, encoding 362 aa. The immunofluorescence result revealed that PfMHC Ⅰα was presented on the membrane of lymphocytes. qRT-PCR analysis indicated that PfMHC Ⅰα was expressed in all examined tissues, with the highest expression in skin, followed by the expression in gills and whole blood. After challenge of Aeromonas hydrophila or polyinosinic: polycytidylic acid (Poly I:C) in vitro, the expression levels of PfMHC Ⅰα on pufferfish kidney lymphocytes were significantly up-regulated, with the highest expression level at 48hr post-challenge. After infection with A. hydrophila or Poly I:C in vivo, the expression levels of PfMHC Ⅰα in the skin, whole blood and kidneys were significantly up-regulated. Taken together, it is speculated that PfMHC Ⅰα associates with resistance to both intracellular and extracellular antigens and plays an important role in the host response against pathogen infection in pufferfish.