Activity Of Leukocytes Research Articles

Modulation of the 5-Lipoxygenase Pathway by Chalcogen-Containing Inhibitors of Leukotriene A4 Hydrolase.

The 5-lipoxygenase (5-LOX) pathway gives rise to bioactive inflammatory lipid mediators, such as leukotrienes (LTs). 5-LOX carries out the oxygenation of arachidonic acid to the 5-hydroperoxy derivative and then to the leukotriene A4 epoxide which is converted to a chemotactic leukotriene B4 (LTB4) by leukotriene A4 hydrolase (LTA4H). In addition, LTA4H possesses aminopeptidase activity to cleave the N-terminal proline of a pro-inflammatory tripeptide, prolyl-glycyl-proline (PGP). Based on the structural characteristics of LTA4H, it is possible to selectively inhibit the epoxide hydrolase activity while sparing the inactivating, peptidolytic, cleavage of PGP. In the current study, chalcogen-containing compounds, 4-(4-benzylphenyl) thiazol-2-amine (ARM1) and its selenazole (TTSe) and oxazole (TTO) derivatives were characterized regarding their inhibitory and binding properties. All three compounds selectively inhibit the epoxide hydrolase activity of LTA4H at low micromolar concentrations, while sparing the aminopeptidase activity. These inhibitors also block the 5-LOX activity in leukocytes and have distinct inhibition constants with recombinant 5-LOX. Furthermore, high-resolution structures of LTA4H with inhibitors were determined and potential binding sites to 5-LOX were proposed. In conclusion, we present chalcogen-containing inhibitors which differentially target essential steps in the biosynthetic route for LTB4 and can potentially be used as modulators of inflammatory response by the 5-LOX pathway.

Dynamics of changes in immunological parameters in adults with COVID-19

Aim. To evaluate the immune response in patients with COVID-19 and to track the dynamics of changes in immunological parameters 2-3 weeks after the onset of the disease. Materials and methods. The study included the results of a survey of 55 people aged 19 to 55 years with a new coronavirus infection (NCVI, COVID-19) in 2020. 27 of them were re-examined. The first sample (n=55) was taken on average 4.3 ± 0.49 days from the onset of the disease, the second (n=27) – 12.7 ± 1.12 days. The control group consisted of practically healthy individuals (n=41). The following indicators were determined in all patients: the presence of SARSCoV-2 RNA, the parameters of the general blood test, the main subpopulations of lymphocytes, the level of total immunoglobulins (IgM, IgG, IgA), the number of CEC, the absorption and bactericidal activity of leukocytes, the level of specific IgM and IgG to SARS-CoV-2. Results. At the onset of the disease, a statistically significant decrease in the level of lymphocytes (p<0.001) and their subpopulations (TNK cells (p<0.001), T-helper lymphocytes (<0.001), cytotoxic T-lymphocytes (p<0.001), NK cells (<0.001) and T-lymphocytes (<0.001)). At the same time, the level of activated T-lymphocytes was statistically significantly higher (p<0.001) than the same indicator in the control group. When analyzing immunological parameters after 12.7±1.12 days, an increase in the total number of lymphocytes (p<0.001), T-lymphocytes (p=0.003), T-helper cells (p=0.015) was found; an increased content of activated T-lymphocytes (p<0.001) and a low level of TNK cells (p<0.001). The indicators of the phagocytic link in patients with COVID-19 in the first days of the disease were within the reference range (similar indicators in the control group). Repeated examination of patients (sample 2) showed an increase in the phagocytic activity of leukocytes and monocytes (p<0.003 and p<0.09, respectively). At the beginning of the disease, already on the 4th-5th day of the disease, specific IgM and IgG antibodies to SARS-CoV-2 were detected in the blood serum of patients, by the second week of the disease their level was significantly higher than in the first study (p<0.009). Conclusion. At the onset of the disease, insufficiency of the cellular link of immunity was noted, which is probably as sociated with a violation of immunoregulatory mechanisms. At the same time, an increase in the level of activated T-lymphocytes indicates an overactivation of immune reactions, i.e. it can be said that there is an imbalance in the immune response in COVID-19.

Teaching NeuroImages: Infantile-onset Krabbe disease with tigroid appearance of the white matter

An 8-month-old girl presented with a 1-month history of developmental regression, irritability, and opisthotonic posturing. A brain MRI revealed abnormal T2 hyperintensities in the periventricular and deep cerebral white matter and peridentate cerebellar white matter. Reduced galactocerebrosidase activity in leukocytes confirmed the diagnosis of Krabbe disease.

Regeneration of an infected skin wound under conditions of experimental steroid hyperglycemia

Purpose. To evaluate the regeneration of an infected skin wound in experimental steroid hyperglycemia in rats.Materials and methods. Regeneration of an infected rat skin wound (n = 30) was evaluated on the background of steroid hyperglycemia. Depending on the type of wound surface treatment, all observed animals were divided into 3 groups: the first group (n = 10) was treated with an ointment containing Cymbopogon proximus per os administration; the second group (n = 10) was treated with an ointment containing sodium fusidate 2 %; the third group (n = 10, control) the wound was treated with vaseline. After five days, local changes in the wound area and the activity of its regeneration were evaluated. We also studied the blood changes: the composition of the shaped elements and the content of total protein, albumins, transaminase activity (ALT, AST), total cholesterol, urea and creatinine.Results. The use of Cymbopogon proximus orally by rats of the first group, with induced diabetes mellitus, contributes to a decrease in serum glucose levels by almost two times compared with the second and third groups. Ointment containing Cymbopogon proximus has immunomodulatory properties: it prevents the development of leukopenia, promotes the activation of agranular immunocytes. The use of Cymbopogon proximus extract has a positive effect on the rate of wound regeneration due to several factors: reduction of hyperemia and edema, stimulation of leukocyte activity, and as a consequence activation of regeneration. We did not find a significant difference in the rate of wound regeneration when treated with an ointment based on Cymbopogon proximus extract and an ointment containing sodium fusidate (p > 0.05).Conclusion. The regeneration activity of an infected wound in rats with steroid hyperglycemia does not significantly differ when treated with antimicrobial ointment and Cymbopogon proximus. The extract of Cymbopogon proximus has a double effect in vivo: it reduces glucose levels and promotes wound healing in the condition of hyperglycemia, which is promising in the treatment of complications in diabetes mellitus.

Myeloid Progenitor Inhibitory Factor-1 (CCL23) Inhibits Lung Leukocyte Recruitment in a Primate Cardiopulmonary Bypass-Induced Pulmonary Ischaemia Model.

Bone marrow (BM)-derived polymorphonuclear leukocytes (PMNs) and monocytes (MO) induced by cardiopulmonary bypass (CPB) are highly proteolytic and cause postoperative lung injury. Although CCL23/Myeloid progenitor inhibitory factor-1 is a human CC chemokine with potent suppressor effects on myeloid progenitor cells, invivo inhibitory effects on BM-derived leukocyte kinetics associated with CPB are unknown. Two-hour CPB was surgically performed in cynomolgus monkeys and BM-derived leukocytes kinetics were monitored postoperatively by flow cytometry with 5'-bromo-2'-deoxyuridine (BrdU) and cytokine ELISA. Monkeys were given CCL23 (n=5) or saline (control, n=5) intravenously daily for 3 days before BrdU labelling and peripheral blood/bronchoalveolar lavage fluid (BALF) timepoint sampling to reveal BrdU-labelled cells. Levels of cytokines, CD11b, and L-selectin were considered leukocytic activation markers. The CCL23 treatment significantly prolonged BM transit of leukocytes (PMNs, 118.4±11.7-95.5±4.1 hours [control]; MO, 91.6±5.0-62.0±3.0 hours [control]) and reduced their alveolar appearance. The BM pool size of MO was decreased by CCL23 but PMNs were unaffected. CD11b, L-selectin expression of PMNs and MO during CPB, and post-surgical increases of interleukin (IL)-6, IL-8, TNF-α, MCP-1, and PMN elastase in the BALF were not suppressed. CCL23 treatment slows turnover of PMN and MO progenitors in BM and suppresses their circulatory release and lung recruitment. CCL23 has inhibitory effects specifically on the CPB-induced BM response and could hold value for preventing CPB-induced lung injury.

An analysis of the dynamic role of lipids in the progression and development of cardiovascular disease

The main cause of mortality around the globe is cardiovascular disease (CVD). There are several indications followed by experimental validation that dysfunctional lipotoxicity is one of the primary causes of cardiovascular illnesses such as atherosclerosis, “coronary heart disease”, (CHD), and hypertension. Obesity and diabetes raise the number of circulating lipids, which may enhance the production of harmful intermediates and cause issues related to cardiovascular disorders. Effective and affordable medicines to lower cardiovascular risk are desperately required. Lipids or lipoprotein particles play a critical role in atherosclerosis, the pathophysiology behind CVD. They also have an effect on inflammatory processes, vascular or cardiac cell function, leukocyte activity, and ultimately the health of the heart and blood arteries. In this paper, an attempt was made to identify the cause of lipid buildup that led to the pathophysiology of cardiovascular disorders as well as the processes behind lipotoxicity. A summary of current and developing treatment approaches to decrease the incidence of lipid-induced cardiovascular disease is also compiled through previous studies.

Pompe disease - What do we currently know about the disease?


 Introduction: Pompe disease (PD) is a rare metabolic disorder caused by a partial or complete deficiency of acid α-glucosidase (GAA) which leads to lysosomal accumulation of glycogen. Excessive amounts of glycogen accumulate mainly in the cells of the heart and skeletal muscles and cause dysfunction of these tissues. It is inherited in an autosomal recessive manner. The most common diagnostic methods include genetic tests and the measurement of enzyme activity in leukocytes or fibroblasts. A screening test is also available that tests the enzyme activity in dried blood spot (DBS). The treatment of PD is mostly based on synthetic GAA enzyme supply to the patients. The therapy reduces glycogen storage and improves muscle function, decreases heart size and prolongs the lives of those with infantile form of PD. In the adult onset of the disease treatment increases physical efficiency and reduces the progression of respiratory failure.
 
 
 Aim of the study: Systematization of current knowledge about Pompe disease with particular emphasis on possible clinical presentations, diagnostics and therapeutic options.
 Material and methods: Literature review based on PubMed data using the following keywords: Pompe disease, Glycogenosis type ii, glucosidase alpha
 Summary : Pompe disease is a rare disease with many problems related to diagnosis and possible therapies. Its symptoms can be very misleading and cause a delay in diagnosis . It is critical to start the therapy as soon as possible to best manage the disease. The prognosis of PD is poor, especially in children under 12 months of age. Gene therapy research is currently underway and early results are very promising. There is still much to learn about dealing with this disease.

Porcine extraintestinal pathogenic Escherichia coli delivers two serine protease autotransporters coordinately optimizing the bloodstream infection.

Extraintestinal pathogenic Escherichia coli (ExPEC) is one of the leading causes of bloodstream infections in a broad spectrum of birds and mammals, thus poses a great threat to public health, while its underlying mechanism causing sepsis is not fully understood. Here we reported a high virulent ExPEC strain PU-1, which has a robust ability to colonize within host bloodstream, while induced a low level of leukocytic activation. Two serine protease autotransporters of Enterobacteriaceae (SPATEs), VatPU-1 and TshPU-1, were found to play critical roles for the urgent blood infection of strain PU-1. Although the Vat and Tsh homologues have been identified as virulence factors of ExPEC, their contributions to bloodstream infection are still unclear. In this study, VatPU-1 and TshPU-1 were verified to interact with the hemoglobin (a well-known mucin-like glycoprotein in red blood cell), degrade the mucins of host respiratory tract, and cleave the CD43 (a major cell surface component sharing similar O-glycosylated modifications with other glycoprotein expressed on leukocytes), suggesting that these two SPATEs have the common activity to cleave a broad array of mucin-like O-glycoproteins. These cleavages significantly impaired the chemotaxis and transmigration of leukocytes, and then inhibited the activation of diverse immune responses coordinately, especially downregulated the leukocytic and inflammatory activation during bloodstream infection, thus might mediate the evasion of ExPEC from immune clearance of blood leukocytes. Taken together, these two SPATEs play critical roles to cause a heavy bacterial load within bloodstream via immunomodulation of leukocytes, which provides a more comprehensive understanding how ExPEC colonize within host bloodstream and cause severe sepsis.

Effect of Pemafibrate on Hemorheology in Patients with Hypertriglyceridemia and Aggravated Blood Fluidity Associated with Type 2 Diabetes or Metabolic Syndrome

Persistent high serum triglyceride (TG) and free fatty acid (FFA) levels, which are common in metabolic syndrome and type 2 diabetes, are risk factors for cardiovascular events because of exacerbated hemorheology. To explore the effects of pemafibrate, a selective peroxisome proliferator-activated receptor alpha modulator, on hemorheology, we performed a single-center, nonrandomized, controlled study in patients with type 2 diabetes (HbA1c 6-10%) or metabolic syndrome, with fasting TG levels of ≥ 150 mg/dL and a whole blood transit time of > 45 s on a microarray channel flow analyzer (MCFAN). Patients were divided into a study group, receiving 0.2 mg/day of pemafibrate (n = 50) for 16 weeks, and a non-pemafibrate control group (n = 46). Blood samples were drawn 8 and 16 weeks after entry to the study to evaluate whole blood transit time as a hemorheological parameter, leukocyte activity by MCFAN, and serum FFA levels. No serious adverse events were observed in either of the groups. After 16 weeks, the pemafibrate group showed a 38.6% reduction in triglycerides and a 50.7% reduction in remnant lipoproteins. Pemafibrate treatment did not significantly improve whole blood rheology or leukocyte activity in patients with type 2 diabetes mellitus or metabolic syndrome complicated by hypertriglyceridemia and exacerbated hemorheology.

Peptidoglycan recognition protein MsPGRP in largemouth bass (Micropterus salmoides) mediates immune functions with broad nonself recognition ability

Peptidoglycan (PGN) recognition proteins (PGRPs) are important immune factors in innate immunity that function in recognising pathogens and activating the immune system. These ubiquitous proteins are conserved in invertebrates and vertebrates. In this study, a PGRP gene (MsPGRP) from largemouth bass (Micropterus salmoides) was identified and characterised, and its transcription distribution was explored. Recombinant protein (rMsPGRP) exhibited dose-dependent binding to PGN and glucan (GLU), but weak binding to lipopolysaccharide (LPS). MsPGRP exhibited agglutinating activity against several Gram-negative bacteria, Gram-positive bacteria and fungi, and it promoted phagocytosis activity of leukocytes against Micrococcus luteus and Aeromonas hydrophila. The protein also possessed amidase activity in the presence of Zn2+, degraded PGN, and disrupted the M. luteus cell wall. The results suggest that MsPGRP plays an important role in pathogen recognition, and acts as a opsonin during immune system responses and elimination of invading pathogens.

An immunodeficiency tale: same mutation, different phenotype

Actin-related protein 2/3 complex subunit 1B (ARPC1B) deficiency is an autosomal-recessive inborn error of immunity resulting in impaired actin cytoskeletal function. This condition presents with similar features to Wiskott-Aldrich Syndrome, including leukocytoclastic vasculitis, thrombocytopenia, eczema and colitis. An absence of ARPC1B protein leads to impaired actin filament formation, which is critical for leukocyte activity in mediating cytoskeletal processes, including migration, adhesion, endocytosis, and phagocytosis.

Modulation of the Effect of Doxorubicin and Medroxyprogesterone Acetate on Cytokine and Oxidant Activity of Human Leukocytes by Hanks' Cold Plasma-Treated Solution

Cold plasma and plasma-treated solutions (PTS) have recently become widely adopted for the treatment of cancer and inflammatory diseases, particularly in concert with other medications. These complexes affect cytokine and oxidative activity of leukocytes, which are important in the pathogenesis of a variety of diseases. In this work, we study the variations in the immune response of healthy leukocytes when doxorubicin, medroxyprogesterone acetate, and PTS affect them separately or in combination. Employing the "CAPKO-JET" plasma source, we achieve an increase in the anti-tumor immune response from the combination of PTS with doxorubicin and MPA at treatment times shorter than 2.5 min. In contrast, the anti-inflammatory immune response is amplified at treatment times of 7.5 min or more. Applying PTS without the above medications upon the completion of the first phase of the inflammation therapy at the injury site is suggested to accelerate healing.

Phagocytotic activity and gene expression of leukocytes isolated from Astyanax lacustris.

The constant intensification of aquaculture has considerable increased the stress levels of farmed fish and, consequently, the number and intensity of diseases outbreaks. Thus, studies on fish immune response, especially regarding the interaction of fish leukocytes with potential pathogens and xenobiotics are of great importance in order to develop new prophylactic and curative strategies. We isolated leukocytes from the head kidney of Astyanax lacustris-an important Neotropical fish species for aquaculture and a potential model for Neotropical aquaculture research-using a Percoll centrifugation protocol. The isolated leukocytes were incubated with lipopolysaccharide (LPS), and the expression of genes IL-1β, IL-8, LysC, and LysG were measured. We assessed the phagocytotic activity of leukocytes using Congo red-dyed yeast, a novel and cost-effective protocol that has been developed in this study. The isolated leukocytes responded to LPS induction, exhibiting strong IL-1β and IL-8 upregulation, two of the most important pro-inflammatory interleukins for vertebrates immune reponse. The optimal concentration of yeast for the phagocytic assay was 106 cells mL-1, resulting in acceptable phagocytic capacity (PC) but without excess of yeasts during the counting process, ensuring a high precision and accuracy of the method. To the best of our knowledge, the present study is the first to investigate the in vitro gene expression and phagocytic activity of leukocytes isolated from A. lacustris. Our findings will serve as a reference for future studies on the immunology and toxicology of Neotropical fish.

Иммунобиологическая активность инденопирролов и инденопиридазинов

The availability of highly effective drugs with minimal side effects is essential in medical practice. In this regard, the search for pharmacologically active substances of plant and chemical origin is being actively pursued. Promising in terms of the search for biologically active compounds are derivatives of pyrrole and pyridazine - indenopyrroles and indenopyridazines. Evaluation of the effect of these compounds on the functional activity of the immune system is relevant, since the immune system plays one of the key roles in maintaining homeostasis in the body. As objects of study, we took indenopyrroles (1a-d) and indenopyridazines (2a-d), which differ in the presence of various substituents in the molecular structure. o assess the effect of the test compounds on the phagocytic activity of rat peripheral blood leukocytes in the in vivo system, the test substances were administered to animals intraperitoneally in 2% starch mucus at a dose of 100 mg/kg. Blood samples were taken from the tail vein. Determination of the phagocytic activity of leukocytes was carried out by the standard method in modification. The results were taken into account microscopically. The cellularity of the spleen was counted, the number of antibody-forming cells in the spleen was assessed by local hemolysis in agarose gel according to Jerne. The effect of the studied compounds on cellular immunity was assessed using a delayed-type hypersensitivity reaction. We have revealed a predominantly stimulating effect, first of all, of indenopyrroles on the phagocytic activity of neutrophils and general leukocyte phagocytosis. Indenopyrroles decreased the cellularity of the spleen and reduced the number of antibody-forming cells. Indenopyridazines had a multidirectional effect on antibody genesis. The test compounds did not change the severity of the delayed-type hypersensitivity reaction.

СОСТОЯНИЕ ИММУНИТЕТА У КОРОВ ПРИ ПОСЛЕРОДОВОМ ЭНДОМЕТРИТЕ В ХОЗЯЙСТВАХ ПРИМОРСКОГО КРАЯ

The objective of the research was to study the state of immunity in healthy and endometritic cows on farms in the Primorsky Krai of the Russian Federation. It was established that when studying the absolute and relative number of lymphocytes in sick and healthy cows, the total number of lymphocytes in the postpartum period did not significantly differ and did not change during all study periods, it was within 5.16-6.1 109/l. The number of T and B lymphocytes in healthy cows was significantly higher in all study periods than in cows with endometritis. In patients with postpartum endometritis (the initial stage of body disease, the concentration of class G immunoglobulins on the first day after delivery was by 13.7% lower than in healthy animals, by day 7 it decreased by 25.9%, by day 14 – by 32.7%, by day 28 – increased by 26.7%. The concentration of immunoglobulins M in sick animals on the first day was by 18.5% lower than in healthy animals, on the 7th day – by 30.1%, on the 14th day – by 25.8%, on the day 28 – by 20.83%. When studying non-specific cellular (phagocytic activity of neutrophils) and humoral (bactericidal and lysozyme activity of blood serum), a significantly increased phagocytic activity of leukocytes in healthy animals was established, as compared to sick animals, but the phagocytic number was higher in sick animals. The amount of lysozyme in the serum of healthy animals was at a high level) and the bactericidal activity of serum during all study periods.

Особенности нетотической активности лейкоцитов у больных с хроническим тонзиллитом

The proportion of patients with chronic tonsillitis (CT) who visit otorhinolaryngologists in outpatient practice ranges from 4 to 35%. Increasingly, opportunistic microflora acts as a source of inflammation. An aggravating factor in the development of a chronic focus of inflammation is a change in the activity of the immune response. A few years ago, a new mechanism of behavior of human phagocytic cells in the implementation of nonspecific immunoreactivity was discovered – that is, formation of neutrophil extracellular traps (NETosis), associated with the adoption of a suicidal decision by the cells, in which chromatin is deconcentrated, and neutrophil extracellular traps (NEТs) are formed, which are released into the surrounding tissues for capturing pathogens and exposing them to high doses of antimicrobial proteins and enzymes. The significance of this immune mechanism and its role in the pathogenesis of СT remains unexplored. To date, laboratory methods for assessing the health status of patients with CT demonstrate insufficient specificity and accuracy. It remains relevant to develop a method for examining CT patients, which would allow the doctor to clearly establish the form of the disease, select the treatment, and adequately assess the health of the patient with CT during the follow-up care. Such a method, in our opinion, can be an assessment of the level of NETs in the blood and on the surface of the lacunae of the palatine tonsils (PT). 71 patients (median 38±9.86 years) were examined. The control group consisted of 54 practically healthy volunteers (median 38±10,9 years). The NETosis activity of leukocytes was evaluated in parallel in whole blood and smears from the surface of the lacunae of the PT. The data obtained indicate the undeniable diagnostic significance of the NETosis activity, considering the presence of a direct relationship with the form of CT. Therefore, the assessment of the level of NETosis can be proposed as a promising laboratory criterion for the severity of the patient’s condition, predicting the course of the disease and the effectiveness of the treatment.

Body resistance of calves during the imprinting period of growth and development

The results of the conducted research allow us to assert the different level of resistance of the body of calves at the end of the imprinting period, depending on the functional state of the body at birth. In calves born with signs of hypoxia, during the imprinting period, the number of leukocytes in the blood was 1.54 times higher than in functionally active animals. The number of neutrophils in the blood of the calves of the second group was 2.39 times more (p>0.001), and lymphocytes were 1.49 times less (p<0.001). The phagocytic activity of leukocytes in the blood of functionally active calves in the imprinting period of life was significantly higher than in calves born with signs of hypoxia. The phagocytic number of leukocytes in the blood of the calves of the first group was 1.40 times higher, and the phagocytic index was 15.70% more than that of the animals of the control group. At the end of the imprinting period, the resistance index of calves of the control group was 3.47 times higher than this index of calves of the experimental group. The percentage of active leukocytes (AL) reached 38.35 ± 2.17% in the calves of the first group and 13.63 ± 1.01% in the calves of the experimental group. The number of active lymphocytes in the blood of calves of the control group was found to be 1.83 times higher, and microbes were neutralized 2.18 times more than the white blood cells of the calves of the experimental group. The research results indicate the negative impact of hypoxia on the growth and development of the fetus. In newborn calves, adaptation to new living conditions was significantly less than in functionally active calves. The decrease in the resistance of the body of calves born with signs of hypoxia is most pronounced in the imprinting period of growth and development of animals.

Wound-healing activity of polyhexamethyleneguanidine hydrochloride hydrogel and extract of Bergenia crassifolia on thermal burn simulation

The results of a study of the wound healing activity of a composition based on the hydrogel of polyhexamethylene guanidine hydrochloride and Bergenia crassifolia extract, under conditions of modeling thermal burns in laboratory animals (rats), are presented. It was found that the composition affects the change in the summary antioxidant and leukocyte activity towards the normalization of these indicators. Morphological analysis of the slices showed that, under the influence of a polyhexamethylene guanidine hydrogel composition and B.crassifolia extract, healing proceeds more intensively than in the control group and is manifested by a smaller thickness of the leukocyte-necrotic scab, accelerated epithelization, and complete closure of the skin defect.

Rapid metabolomic screening of cancer cells via high-throughput static droplet microfluidics

Effective isolation and in-depth analysis of Circulating Tumour Cells (CTCs) are greatly needed in diagnosis, prognosis and monitoring of the therapeutic response of cancer patients but have not been completely fulfilled by conventional approaches. The rarity of CTCs and the lack of reliable biomarkers to distinguish them from peripheral blood cells have remained outstanding challenges for their clinical implementation. Herein, we developed a high throughput Static Droplet Microfluidic (SDM) device with 38,400 chambers, capable of isolating and classifying the number of metabolically active CTCs in peripheral blood at single-cell resolution. Owing to the miniaturisation and compartmentalisation capability of our device, we first demonstrated the ability to precisely measure the lactate production of different types of cancer cells inside 125 pL droplets at single-cell resolution. Furthermore, we compared the metabolomic activity of leukocytes from healthy donors to cancer cells and showed the ability to differentiate them. To further prove the clinical relevance, we spiked cancer cell lines in human healthy blood and showed the possibility to detect the cancer cells from leukocytes. Lastly, we tested the workflow on 8 preclinical mammary mouse models including syngeneic 67NR (non-metastatic) and 4T1.2 (metastatic) models with Triple-Negative Breast Cancer (TNBC) as well as transgenic mouses (12-week-old MMTV-PyMT). The results have shown the ability to precisely distinguish metabolically active CTCs from the blood using the proposed SDM device. The workflow is simple and robust which can eliminate the need for specialised equipment and expertise required for single-cell analysis of CTCs and facilitate on-site metabolic screening of cancer cells.

Biochemical Engraftment and Clinical Outcomes Following Ex-Vivo Autologous Stem Cell Gene Therapy for Mucopolysaccharidosis Type IIIA

Background: Mucopolysaccharidosis type IIIA (MPSIIIA, Sanfilippo syndrome) is a lysosomal storage disease caused by a gene defect in the SGSH gene, producing the defective enzyme N-sulfoglucosamine sulfohydrolase. Children with MPSIIIA, accumulate the substrate of SGSH, heparan sulphate, in all cells in the body, causing developmental delay, regression of previously acquired skills, hyperactivity, seizures and progressive cognitive decline. The disease is life-limiting, with death occurring typically within the second decade of life. MPSIIIA is unresponsive to allogeneic stem cell transplant, presumed because insufficient enzyme is delivered via cross correction from donor-derived engrafted cells, particularly in the brain. Autologous ex vivo hematopoietic stem cell gene therapy (HSC-GT) is an experimental treatment for MPSIIIA, with the insertion of a functional copy of the SGSH gene into the patient's own CD34+ stem cells prior to transplant, to allow overexpression of the missing SGSH enzyme. In our pre-clinical work, we tested a lentiviral vector containing the SGSH gene driven by the human CD11b (myeloid-specific) promoter in a mouse model of MPSIIIA. This lentiviral vector was used to transduce CD34+ stem cells before transplant into fully-myeloablated busulfan-treated animals. Six-months post-transplant, SGSH enzyme was detected in the brain, with normalised behavior and heparan sulfate levels. Transduction and transplant of human CD34+ stem cells into humanized NSG mice demonstrated stable engraftment with no evidence of viral shedding or transformational potential, supporting the translation of this work to the clinic. Study Design: This is a phase I/II open label study (NCT04201405/EudraCT#2019-002051-42) with primary outcomes of safety and tolerability and peripheral expression of SGSH activity in total leukocytes at 12 months. Secondary endpoints include neurocognition with measures including the Bayley Scales of Infant and Toddler Development. Patients will be followed for a minimum of 3 years. Five patients with severe MPSIIIA, aged 6 to 24 months, have been treated with HSC-GT as part of this trial, completing enrolment. An additional patient was treated off-trial, at 30 months old, on a compassionate use basis. All children underwent stem cell mobilization and peripheral collection of CD34+ cells, followed by transduction with the lentiviral vector and cryopreservation. Patients received myeloablative busulfan condition before infusion of the drug product. Trial Status and Results: Three trial patients have >18 months follow-up post-transplant, one patient has >12 months and one patient has >9 months, with the off-trial 'special' patient >36 months. Vector copy number of the IMP ranged from 1.19 to 8.91 copies/cell, with a cell dose between 4.3 to 22.7x106 CD34+/kg. Engraftment was rapid with median time to neutrophil, platelet and red cell engraftment of 19, 28 and 25 days, respectively. In all patients, engraftment is sustained to date, with supra-physiological levels of SGSH enzyme measurable in leukocytes (38-91-fold above median normal range at one-month post-transplant; see Figure A). Supra-physiological SGSH levels were also rapidly detected in CD15+ lineages, plasma and bone marrow. CSF SGSH levels were within or above normal range by 6-months post-transplant. Abnormal heparan sulfate levels at baseline were rapidly reduced in both urine (>90%) and plasma (82%, see Figure B). Data from the specials patient, continues to show supra-physiological biochemical data and neurocognitive outcomes at 36 months post-transplant indicating an improved phenotype compared to MPSIIIA natural history. Neurocognitive assessments of trial patients continue and early outcomes will be presented suggesting an alteration in the neurologic phenotype of the disease in one patient, whilst three patients are currently within the normal DQ range but require longer follow-up. All trial patients show an improvement in SGSH expression in leukocytes and other lineages at early time points compared to that of the specials patient. Conclusions: Treatment with ex-vivo autologous HSC-GT is generally well tolerated and delivers supra-physiological levels of enzyme throughout the body. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal