Platelet Storage-Problems, Improvements, and New Perspectives.

Platelet transfusions are routine procedures in clinical treatment aimed at preventing bleeding in critically ill patients, including those with cancer, undergoing surgery, or experiencing trauma. However, platelets are susceptible blood cells that require specific storage conditions. The availability of platelet concentrates is limited to five days due to various factors, including the risk of bacterial contamination and the occurrence of physical and functional changes known as platelet storage lesions. In this article, the problems related to platelet storage lesions are categorized into four groups depending on research areas: storage conditions, additive solutions, new testing methods for platelets (proteomic and metabolomic analysis), and extensive data modeling of platelet production (mathematical modeling, statistical analysis, and artificial intelligence). This article provides extensive information on the challenges, potential improvements, and novel perspectives regarding platelet storage.

Evaluation of selected hematological, biochemical and oxidative stress parameters in stored canine CPDA-1 whole blood.

Blood transfusions are mainly given to intensive care patients; therefore, additional complications that could arise from storage lesions in preserved blood should be avoided. It has been shown that human stored red blood cells are subject to changes that are considered to be a number of interdependent processes involving metabolic disarrangement and oxidative stress. The aim of our study was to determine alterations in selected hematological and biochemical parameters and to assess whether and when oxidative stress is a significant phenomenon in stored dog CPDA-1 whole blood. Ten ½ unit bags of whole blood donated from dogs and preserved with CPDA-1 (anticoagulant containing citrate, phosphate, dextrose and adenine) were stored for 5 weeks. Each week, a 9 ml sample was drawn aseptically to measure hematological parameters, selected metabolites, free hemoglobin content, osmotic fragility, antioxidant enzyme activity, total antioxidant capacity, malondialdehyde concentration and protein carbonyl content.The results revealed an MCV decrease in the first week of storage and then a gradual increase; osmotic fragility decreased at that time and remained low throughout the study period. Leukodepletion became significant in the fourth week of storage. The free hemoglobin concentration continuously increased, with the greatest changes observed in the last two weeks of storage. The total antioxidant capacity changed in a reverse manner. Superoxide dismutase and glutathione peroxidase activities decreased from week 0 to week 3, and catalase activity tended to decrease over time. The highest malondialdehyde concentrations in blood supernatant were measured in the first week of storage, and the carbonyl concentration increased after 35 days.Hematological changes and oxidative stress are already present in the first week of storage, resulting in depletion of the antioxidant system and subsequent accumulation of oxidation products as well as erythrocyte hemolysis, which are most pronounced at the end of the storage period.

Near infrared light induces post-translational modifications of human red blood cell proteins.

There is a growing body of evidence that near infrared (NIR) light exerts beneficial effects on cells. Its usefulness in the treatment of cancer, acute brain injuries, strokes and neurodegenerative disorders has been proposed. The mechanism of the NIR action is probably of photochemical nature, however it is not fully understood. Here, using a relatively simple biological model, human red blood cells (RBCs), and a polychromatic non-polarized light source, we investigate the impact of NIR radiation on the oxygen carrier, hemoglobin (Hb), and anion exchanger (AE1, Band 3). The exposure of intact RBCs to NIR light causes quaternary transitions in Hb, dehydration of proteins and decreases the amount of physiologically inactive methemoglobin, as detected by Raman spectroscopy. These effects are accompanied by a lowering of the intracellular pH (pHi) and changes in the cell membrane topography, as documented by atomic force microscopy (AFM). All those changes are in line with our previous studies where alterations of the membrane fluidity and membrane potential were attributed to NIR action on RBCs. The rate of the above listed changes depends strictly on the dose of NIR light that the cells receive, nonetheless it should not be considered as a thermal effect.

Biphasic dose-response and effects of near-infrared photobiomodulation on erythrocytes susceptibility to oxidative stress in vitro.

The effect of simultaneous application of tert-butyl hydroperoxide (tBHP) and polychromatic near-infrared (NIR) radiation on bovine blood was examined to determine whether NIR light decreases the susceptibility of red blood cells (RBCs) to oxidative stress. The study assessed various exposure methods, wavelength ranges, and optical filtering types. Continuous NIR exposure revealed a biphasic response in cell-free hemoglobin changes, with antioxidative effects observed at low fluences and detrimental effects at higher fluences. Optimal exposure duration was identified between 60 s and 15 min. Protective effects were also tested across wavelengths in the range of 750-1100 nm, with all of them reducing hemolysis, notably at 750 nm, 875 nm, and 900 nm. Comparing broadband NIR and far-red light (750 nm) showed no significant difference in hemolysis reduction. Pulse-dosed NIR irradiation allowed safe increases in radiation dose, effectively limiting hemolysis at higher doses where continuous exposure was harmful. These findings highlight NIR photobiomodulation's potential in protecting RBCs from oxidative stress and will be helpful in the effective design of novel medical therapeutic devices.

Changes in the blood redox status of horses subjected to combat training.

Combat training of police horses, involving physical activity in the presence of environmental stressors, poses a risk of oxidative stress. This study compared the oxidative imbalance after combat training in horses in the regular police service and in horses that had just been schooled. Blood collection was performed immediately after training and after 16 h rest. The activity of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), and total antioxidant status (TAS) were determined as the markers of enzymatic antioxidant defence. At the same time, lipid peroxidation (TBARS) and protein carbonylation (Carb) were assessed as oxidation biomarkers. Additionally, oxidative imbalance indexes such as SOD/CAT, SOD/GPx, TBARS/TAS and TBARS/GPx were calculated. Animals during schooling had significantly lower SOD activity in erythrocytes than those experienced. CAT activity in erythrocytes was insignificantly higher immediately after training than during recovery. The SOD/GPx ratio was higher in experienced animals, which may reflect the intra-erythrocyte imbalance between enzymes producing and degrading hydrogen peroxide towards the first one. The concentration of carbonyl groups was significantly higher after the combat training compared to the recovery period in all horses. In inexperienced animals slight increase in TBARS/TAS and TBARS/GPx indexes were observed during the recovery time after exercises, contrary to experienced horses, in which these markers decreased slightly. These results suggest that the oxidative imbalance in inexperienced horses, although less pronounced just after combat training, was more prolonged as compared to horses in regular service.

Biostimulation by methanol enables the methylotrophic yeasts Hansenula polymorpha and Trichosporon sp. to reveal high formaldehyde biodegradation potential as well as to adapt to this toxic pollutant.

The methylotrophic yeasts Hansenula polymorpha and Trichosporon sp. revealed enhanced biodegradation capability of exogenously applied formaldehyde (Fd) upon biostimulation achieved by the presence of methanol, as compared to glucose. Upon growth on either of the above substrates, the strains proved to produce the activity of glutathione-dependent formaldehyde dehydrogenase-the enzyme known to control the biooxidative step of Fd detoxification. However, in the absence of methanol, the yeasts' tolerance to Fd was decreased, and the elevated sensitivity was especially pronounced for Trichosporon sp. Both strains responded to the methanol and/or Fd treatment by increasing their unsaturation index (UI) at xenobiotic levels below minimal inhibitory concentrations. This indicated that the UI changes effected from the de novo synthesis of (poly) unsaturated fatty acids carried out by viable cells. It is concluded that the yeast cell response to Fd intoxication involves stress reaction at the level of membranes. Fluidization of the lipid bilayer as promoted by methanol is suggested as a significant adaptive mechanism increasing the overall fitness enabling to cope with the formaldehyde xenobiotic via biodegradative pathway of C1-compound metabolism.

[Are disinfectant residues remained after cleaning hemodialysis machine procedure safe for patients?]. / Czy sladowe stezenia srodków dezynfekujqcych sztuczna nerke sa bezpieczne dla pacjentów hemodializowanych?

The dialysis machine shall be cleaned and disinfected after each patient treatment or after every 72 hours break in working. An acceptable disinfectants such as Puristeril plus or Puristeril 340, Citrosteril, Diasteril and Sporotal are used for decontamination. Puristeril 340 is designed for cold disinfection and due to the low pH value, the necessary decalcification of hemodialysis machines is easily achieved. It can be used for all haemodialysis systems like hemodialysis machines, water treatment devices and circuit pipes. Diluted Puristeril decomposes in a non-toxic way. Degradation products of peracetic acid, which is main component of Puristeril are: hydrogen peroxide and acetic acid. Peracetic acid is widely used for disinfection due to its exceptionally broad spectrum of microbiocidal activity at low concentrations and short exposure times. After use Puristeril is easily removable by rinsing with water. This paper deals with the effect of the Puristeril toxicity on blood as a function of its concentration and incubation time. Concentration range of 3.5-70 ppm was used, with particular emphasis on concentrations close to 5 ppm, a value is the limit of sensitivity of strips of starch potassium iodide, the tests for detection of peracetic acid. There was a strong increase in autohaemolysis and malondialdehyde concentrations with increasing concentration of Puristeril. There were also changes in dependence on the parameters of the incubation time, with the greatest effects obtained after 2 hours incubation with Puristeril. The detection limit of peracetic acid used strips of starch potassium iodide does not guarantee the safety of a patient undergoing hemodialysis. Even the residual concentration of Puristeril plus cause increased lipid peroxidation of membrane, and therefore suggest the routine use of stripes on the lower limit of detection of peracetic acid or implement measurement of hydrogen peroxide residues performed with sensitivity 1 ppm.

Three in-one fenestrated approaches of yolk-shell, silver-silica nanoparticles: A comparative study of antibacterial, antifungal and anti-cancerous applications.

Yolk-shell-based silica-coated silver nanoparticles are prominently used in the biomedical field aas well as bare silver nanoparticles for various biological applications. The present work narrates the synthesis and silica coating of metallic silver nanoparticles and investigates their antibacterial, antifungal, and anticancerous activity. Both synthesized nanoparticles were characterized by TEM, and SEM-EDX. The average size of silver nanoparticles was 50 nm, while after coating with silica, the average size of silica-coated silver nanoparticles was 80 nm. The nanoparticles' antibacterial, antifungal, and anticancer properties were comparatively examined in vitro. Agar well diffusion method was employed to explore the antibacterial activity against gram-positive bacteria (Bacillus cereus) and gram-negative bacteria (Escherichia coli) at different concentrations and antifungal activity against Candida Albicans. To understand the minimum concentration of both nanoparticles, we employed the minimum inhibitory concentration (MIC) test, against bacterial and fungal strains, which was dose dependent. We learned that bare silver nanoparticles showed high antibacterial activity, whereas silica-coated silver nanoparticles surpassed their antifungal capability over bare silver nanoparticles against Candida albicans. The anticancer activity of the as-prepared nanoparticles was executed in opposition to the prostate cancer cell (PC-3) line by MTT assay, which showed meaningful activity. Following this, flow cytometry was also effectuated to learn about the number of apoptotic and necrotic cells. The results of this study demonstrate the dynamic anti-cancerous, antibacterial, and antifungal activities of bare silver nanoparticles and silica-coated silver nanoparticles for a long-lasting period.

Micromotion Derived Fluid Shear Stress Mediates Peri-Electrode Gliosis through Mechanosensitive Ion Channels.

The development of bioelectronic neural implant technologies has advanced significantly over the past 5 years, particularly in brain-machine interfaces and electronic medicine. However, neuroelectrode-based therapies require invasive neurosurgery and can subject neural tissues to micromotion-induced mechanical shear, leading to chronic inflammation, the formation of a peri-electrode void and the deposition of reactive glial scar tissue. These structures act as physical barriers, hindering electrical signal propagation and reducing neural implant functionality. Although well documented, the mechanisms behind the initiation and progression of these processes are poorly understood. Herein, in silico analysis of micromotion-induced peri-electrode void progression and gliosis is described. Subsequently, ventral mesencephalic cells exposed to milliscale fluid shear stress in vitro exhibited increased expression of gliosis-associated proteins and overexpression of mechanosensitive ion channels PIEZO1 (piezo-type mechanosensitive ion channel component 1) and TRPA1 (transient receptor potential ankyrin 1), effects further confirmed in vivo in a rat model of peri-electrode gliosis. Furthermore, in vitro analysis indicates that chemical inhibition/activation of PIEZO1 affects fluid shear stress mediated astrocyte reactivity in a mitochondrial-dependent manner. Together, the results suggest that mechanosensitive ion channels play a major role in the development of a peri-electrode void and micromotion-induced glial scarring at the peri-electrode region.

Near-infrared photobiomodulation of blood reversibly inhibits platelet reactivity and reduces hemolysis.

Photobiomodulation (PBM) in the red/near-infrared (R/NIR) spectral range has become widely recognized due to its anti-inflammatory and cytoprotective potential. We aimed to assess the effects of blood PBM on platelets function and hemolysis in an in vitro setting. Porcine blood samples were separated into four aliquots for this study, one of which served as a control, while the other three were subjected to three different NIR PBM dosages. The platelet count and functions and the plasma free haemoglobin and osmotic fragility of red blood cells were measured during the experiment. The control group had a considerable drop in platelet number, but the NIR exposed samples had more minimal and strictly dose-dependent alterations. These modifications were consistent with ADP and collagen-induced platelet aggregation. Furthermore, red blood cells that had received PBM were more resistant to osmotic stress and less prone to hemolysis, as seen by a slightly lower quantity of plasma free hemoglobin. Here we showed under well-controlled in vitro conditions that PBM reversibly inhibits platelet activation in a dose-dependent manner and reduces hemolysis.

Therapeutic potential of intranasal photobiomodulation therapy for neurological and neuropsychiatric disorders: a narrative review.

The application of photobiomodulation therapy (PBMT) for neuronal stimulation is studied in different animal models and in humans, and has shown to improve cerebral metabolic activity and blood flow, and provide neuroprotection via anti-inflammatory and antioxidant pathways. Recently, intranasal PBMT (i-PBMT) has become an attractive and potential method for the treatment of brain conditions. Herein, we provide a summary of different intranasal light delivery approaches including a nostril-based portable method and implanted deep-nasal methods for the effective systemic or direct irradiation of the brain. Nostril-based i-PBMT devices are available, using either lasers or light emitting diodes (LEDs), and can be applied either alone or in combination to transcranial devices (the latter applied directly to the scalp) to treat a wide range of brain conditions such as mild cognitive impairment, Alzheimer's disease, Parkinson's disease, cerebrovascular diseases, depression and anxiety as well as insomnia. Evidence shows that nostril-based i-PBMT improves blood rheology and cerebral blood flow, so that, without needing to puncture blood vessels, i-PBMT may have equivalent results to a peripheral intravenous laser irradiation procedure. Up to now, no studies were conducted to implant PBMT light sources deep within the nose in a clinical setting, but simulation studies suggest that deep-nasal PBMT via cribriform plate and sphenoid sinus might be an effective method to deliver light to the ventromedial part of the prefrontal and orbitofrontal cortex. Home-based i-PBMT, using inexpensive LED applicators, has potential as a novel approach for neurorehabilitation; comparative studies also testing sham, and transcranial PBMT are warranted.

The effect of red-to-near-infrared (R/NIR) irradiation on inflammatory processes.

Introduction: Near-infrared (NIR) and red-to-near-infrared (R/NIR) radiation are increasingly applied for therapeutic use. R/NIR-employing therapies aim to stimulate healing, prevent tissue necrosis, increase mitochondrial function, and improve blood flow and tissue oxygenation. The wide range of applications of this radiation raises questions concerning the effects of R/NIR on the immune system. Methods: In this review, we discuss the potential effects of exposure to R/NIR light on immune cells in the context of physical parameters of light. Discussion: The effects that R/NIR may induce in immune cells typically involve the production of reactive oxygen species (ROS), nitrogen oxide (NO), or interleukins. Production of ROS after exposure to R/NIR can either be inhibited or to some extent increased, which suggests that detailed conditions of experiments, such as the spectrum of radiation, irradiance, exposure time, determine the outcome of the treatment. However, a wide range of immune cell studies have demonstrated that exposure to R/NIR most often has an anti-inflammatory effect. Finally, photobiomodulation molecular mechanism with particular attention to the role of interfacial water structure changes for cell physiology and regulation of the inflammatory process was described. Conclusions: Optimization of light parameters allows R/NIR to act as an anti-inflammatory agent in a wide range of medical applications.

Applicability of FTIR-ATR Method to Measure Carbonyls in Blood Plasma after Physical and Mental Stress.

INTRODUCTION: Oxidative stress is a state of imbalance between the production of reactive oxygen species and antioxidant defenses. It results in the oxidation of all cellular elements and, to a large extent, proteins, causing inter alia the formation of carbonyl groups in their structures. The study focused on assessment of changes in the plasma protein-bound carbonyls in police horses after combat training and after rest and the applicability of infrared spectroscopy with a Fourier transform, utilizing the attenuated total reflectance (FTIR-ATR) in detecting plasma protein oxidation. METHODS: We evaluated the influence of both the different concentrations of hydrogen peroxide and combat training on protein carbonylation in horse blood plasma. The oxidation of plasma proteins was assessed using a spectrophotometric method based on the carbonyl groups derivatization with 2,4-dinitrophenylhydrazine (DNPH). The measured values were correlated with the carbonyl groups concentrations determined by means of the FTIR-ATR method. RESULTS: The linear correlation between the DNPH and FTIR-ATR methods was shown. The concentration of plasma protein-bound carbonyls significantly deceased in police horses after one-day rest when compared to the values measured directly after the combat training (a drop by 23%, p<0.05 and 29%, p<0.01 measured by DNPH and FTIR-ATR methods, respectively). These results were consistent with the proteins phosphorylation analysis. CONCLUSION: The FTIR-ATR method may be applied to measure the level of plasma proteins peroxidation.

Publisher Correction: Low-level light therapy reduces platelet destruction during extracorporeal circulation.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Sheep model of haemodialysis treatment.

More than two million patients received haemodialysis (HD) in 2013. Many methods for improving dialysis therapy outcomes have been tested. Nevertheless, patients continue to experience high morbidity and mortality rates. We aimed to develop an animal model of HD treatment to study methods that would prevent the adverse effects of renal replacement therapy. The study was conducted using six male Merino sheep. The animals underwent a two-step bilateral nephrectomy, and a permanent dual-lumen catheter was inserted into the jugular vein. In each animal, 10 short, daily HD treatments were conducted. The dialysis prescription was adjusted individually to each animal. Measures of dialysis adequacy (spKt/V and urea reduction ratio [URR]) were calculated for each HD treatment. All animals remained in a good clinical state during the experiment. However, a sustained decrease in red blood cell count was detected. The average URR was 0.65 ± 0.01, whereas the calculated spKt/V was approximately 1.16 ± 0.03. Neither hyperphosphataemia nor a significant decline in serum albumin concentrations were detected during the study. A sustained increase in serum potassium concentrations was detected on consecutive days of the experiment. All sheep survived the treatment and were euthanized at the end of the experiment. In conclusion, we developed a reproducible sheep model of HD treatment. The gentle nature and specific anatomical features of sheep provided easy blood access and allowed us to perform HD without pharmacological intervention. However, some differences in sheep physiology relative to human physiology must be considered when interpreting the results of the study.

Low-Level Light Therapy Protects Red Blood Cells Against Oxidative Stress and Hemolysis During Extracorporeal Circulation.

Aim: An activation of non-specific inflammatory response, coagulation disorder, and blood morphotic elements damage are the main side effects of the extracorporeal circulation (ECC). Red-to-near-infrared radiation (R/NIR) is thought to be capable of stabilizing red blood cell (RBC) membrane through increasing its resistance to destructive factors. We focused on the development of a method using low-level light therapy (LLLT) in the spectral range of R/NIR which could reduce blood trauma caused by the heart-lung machine during surgery. Methods: R/NIR emitter was adjusted in terms of geometry and optics to ECC circuit. The method of extracorporeal blood photobiomodulation was tested during in vivo experiments in an animal, porcine model (1 h of ECC plus 23 h of animal observation). A total of 24 sows weighing 90-100 kg were divided into two equal groups: control one and LLLT. Blood samples were taken during the experiment to determine changes in blood morphology [RBC and white blood cell (WBC) counts, hemoglobin (Hgb)], indicators of hemolysis [plasma-free hemoglobin (PFHgb), serum bilirubin concentration, serum lactate dehydrogenase (LDH) activity], and oxidative stress markers [thiobarbituric acid reactive substances (TBARS) concentration, total antioxidant capacity (TAC)]. Results: In the control group, a rapid systemic decrease in WBC count during ECC was accompanied by a significant increase in RBC membrane lipids peroxidation, while in the LLLT group the number of WBC and TBARS concentration both remained relatively constant, indicating limitation of the inflammatory process. These results were consistent with the change in the hemolysis markers like PFHgb, LDH, and serum bilirubin concentration, which were significantly reduced in LLLT group. No differences in TAC, RBC count, and Hgb concentration were detected. Conclusion: We presented the applicability of the LLLT with R/NIR radiation to blood trauma reduction during ECC.

Low-level light therapy reduces platelet destruction during extracorporeal circulation.

Extracorporeal circulation causes many deleterious effects on blood cells. Low-level light therapy (LLLT) in the red/near-infrared spectral range is known for its cytoprotective properties but its use during cardiopulmonary bypass (CPB) has not yet been studied. We aimed to assess whether LLLT protects platelets during CPB. 24 pigs were connected to 1-hour-CPB and observed for the next 23 hours. In 12 animals, blood circulating through the oxygenator was treated with LLLT. Platelet count and function were monitored throughout the experiment. The decrease in platelet count was greater in the control group, especially during CPB and after 24 hours. In LLLT group CD62P expression remained quite stable up to the 12th hour of the experiment, whereas in the control group it continuously decreased till the end of observation. Platelets in the control group were more prone to aggregation in the postoperative period than at the beginning of the experiment, whereas platelets in the LLLT group aggregated similarly or less intense. Limitation of platelet loss, pattern of aggregation and CD62P expression suggest that LLLT may stabilize platelet function during CPB and diminish the negative effects associated with the interaction of cells with an artificial surface.

Diversity and functions of protein glycosylation in insects.

The majority of proteins is modified with carbohydrate structures. This modification, called glycosylation, was shown to be crucial for protein folding, stability and subcellular location, as well as protein-protein interactions, recognition and signaling. Protein glycosylation is involved in multiple physiological processes, including embryonic development, growth, circadian rhythms, cell attachment as well as maintenance of organ structure, immunity and fertility. Although the general principles of glycosylation are similar among eukaryotic organisms, insects synthesize a distinct repertoire of glycan structures compared to plants and vertebrates. Consequently, a number of unique insect glycans mediate functions specific to this class of invertebrates. For instance, the core α1,3-fucosylation of N-glycans is absent in vertebrates, while in insects this modification is crucial for the development of wings and the nervous system. At present, most of the data on insect glycobiology comes from research in Drosophila. Yet, progressively more information on the glycan structures and the importance of glycosylation in other insects like beetles, caterpillars, aphids and bees is becoming available. This review gives a summary of the current knowledge and recent progress related to glycan diversity and function(s) of protein glycosylation in insects. We focus on N- and O-glycosylation, their synthesis, physiological role(s), as well as the molecular and biochemical basis of these processes.

Toxicity, membrane binding and uptake of the Sclerotinia sclerotiorum agglutinin (SSA) in different insect cell lines.

The fungal lectin purified from Sclerotinia sclerotiorum, further referred to as Sclerotinia sclerotiorum agglutinin or SSA, possesses insecticidal activity against important pest insects such as pea aphids (Acyrthosiphon pisum). This paper aims at a better understanding of its activity at cellular level. Therefore, different insect cell lines were treated with SSA. These cell lines were derived from different tissues and represent the three major orders of insects important in agriculture: CF-203 (midgut Choristoneura fumiferana, Lepidoptera), GUTAW1 (midgut, Helicoverpa zea, Lepidoptera), High5 cells (ovary, Trichoplusia ni, Lepidoptera), Sf9 (ovary cells from Spodoptera frugiperda, Lepidoptera), S2 (hemocyte, Drosophila melanogaster, Diptera), and TcA (whole body, Tribolium castaneum, Coleoptera). Although the sensitivity to SSA differs between the cell lines, SSA clearly showed toxicity in all six cell lines with median effect concentrations (EC50) ranging between 9 and 42 µg/ml. An in-depth analysis of the mechanism of uptake in the cells revealed superior amounts of FITC-SSA at the membrane of CF-203 cells compared to Sf9 cells, while a similar small amount of SSA was internalized in both cell lines. Pre-incubation with the clathrin-mediated endocytosis inhibitor phenylarsine oxide inhibited the internalization of SSA into the CF-203 and Sf9 cells with a respective reduction of 6- and 1.7-fold. The data are discussed in relation to the importance of cellular uptake mechanism for SSA binding and cytotoxicity.

Distribution of Glycan Motifs at the Surface of Midgut Cells in the Cotton Leafworm (Spodoptera littoralis) Demonstrated by Lectin Binding.

Glycans are involved in many biological phenomena, including signal transduction, cell adhesion, immune response or differentiation. Although a few papers have reported on the role of glycans in the development and proper functioning of the insect midgut, no data are available regarding the localization of the glycan structures on the surface of the cells in the gut of insects. In this paper, we analyzed the spatial distribution of glycans present on the surface of the midgut cells in larvae of the cotton leafworm Spodoptera littoralis, an important agricultural pest insect worldwide. For this purpose, we established primary midgut cell cultures, probed these individual cells that are freely suspended in liquid medium with a selection of seven fluorescently labeled lectins covering a range of different carbohydrate binding specificities [mannose oligomers (GNA and HHA), GalNAc/Gal (RSA and SSA), GlcNAc (WGA and Nictaba) and Neu5Ac(α-2,6)Gal/GalNAc (SNA-I)], and visualized the interaction of these lectins with the different zones of the midgut cells using confocal microscopy. Our analysis focused on the typical differentiated columnar cells with a microvillar brush border at their apical side, which are dominantly present in the Lepidopteran midgut and function in food digestion and absorption, and as well as on the undifferentiated stem cells that are important for midgut development and repair. Confocal microscopy analyses showed that the GalNAc/Gal-binding lectins SSA and RSA and the terminal GlcNAc-recognizing WGA bound preferentially to the apical microvillar zone of the differentiated columnar cells as compared to the basolateral pole. The reverse result was observed for the mannose-binding lectins GNA and HHA, as well as Nictaba that binds preferentially to GlcNAc oligomers. Furthermore, differences in lectin binding to the basal and lateral zones of the cell membranes of the columnar cells were apparent. In the midgut stem cells, GNA and Nictaba bound more strongly to the membrane of these undifferentiated cells compared to the microvillar pole of the columnar cells, while SSA, HHA, WGA, and SNA-I showed stronger binding to the microvilli. Our results indicated that polarization of the midgut cells is also reflected by a specific distribution of glycans, especially between the basal and microvillar pole. The data are discussed in relation to the functioning and development of the insect midgut.