Autophagy in neutrophils.

Autophagy is a highly conserved intracellular degradation and energy-recycling mechanism that contributes to the maintenance of cellular homeostasis. Extensive researches over the past decades have defined the role of autophagy innate immune cells. In this review, we describe the current state of knowledge regarding the role of autophagy in neutrophil biology and a picture of molecular mechanism underlying autophagy in neutrophils. Neutrophils are professional phagocytes that comprise the first line of defense against pathogen. Autophagy machineries are highly conserved in neutrophils. Autophagy is not only involved in generalized function of neutrophils such as differentiation in bone marrow but also plays crucial role effector functions of neutrophils such as granule formation, degranulation, neutrophil extracellular traps release, cytokine production, bactericidal activity and controlling inflammation. This review outlines the current understanding of autophagy in neutrophils and provides insight towards identification of novel therapeutics targeting autophagy in neutrophils.

Autophagy Primes Neutrophils for Neutrophil Extracellular Trap Formation during Sepsis.

RATIONALE: Neutrophils are key effectors in the host's immune response to sepsis. Excessive stimulation or dysregulated neutrophil functions are believed to be responsible for sepsis pathogenesis. However, the mechanisms regulating functional plasticity of neutrophils during sepsis have not been fully determined. OBJECTIVES: We investigated the role of autophagy in neutrophil functions during sepsis in patients with community-acquired pneumonia. METHODS: Neutrophils were isolated from patients with sepsis and stimulated with phorbol 12-myristate 13-acetate (PMA). The levels of reactive oxygen species generation, neutrophil extracellular trap (NET) formation, and granule release, and the autophagic status were evaluated. The effect of neutrophil autophagy augmentation was further evaluated in a mouse model of sepsis. MEASUREMENTS AND MAIN RESULTS: Neutrophils isolated from patients who survived sepsis showed an increase in autophagy induction, and were primed for NET formation in response to subsequent PMA stimulation. In contrast, neutrophils isolated from patients who did not survive sepsis showed dysregulated autophagy and a decreased response to PMA stimulation. The induction of autophagy primed healthy neutrophils for NET formation and vice versa. In a mouse model of sepsis, the augmentation of autophagy improved survival via a NET-dependent mechanism. CONCLUSIONS: These results indicate that neutrophil autophagy primes neutrophils for increased NET formation, which is important for proper neutrophil effector functions during sepsis. Our study provides important insights into the role of autophagy in neutrophils during sepsis.

Diabetes Primes Neutrophils for Neutrophil Extracellular Trap Formation through Trained Immunity.

Neutrophils are primed for neutrophil extracellular trap (NET) formation during diabetes, and excessive NET formation from primed neutrophils compromises wound healing in patients with diabetes. Here, we demonstrate that trained immunity mediates diabetes-induced NET priming in neutrophils. Under diabetic conditions, neutrophils exhibit robust metabolic reprogramming comprising enhanced glycolysis via the pentose phosphate pathway and fatty acid oxidation, which result in the accumulation of acetyl-coenzyme A. Adenosine 5'-triphosphate-citrate lyase-mediated accumulation of acetyl-coenzyme A and histone acetyltransferases further induce the acetylation of lysine residues on histone 3 (AcH3K9, AcH3K14, and AcH3K27) and histone 4 (AcH4K8). The pharmacological inhibition of adenosine 5'-triphosphate-citrate lyase and histone acetyltransferases completely inhibited high-glucose-induced NET priming. The trained immunity of neutrophils was further confirmed in neutrophils isolated from patients with diabetes. Our findings suggest that trained immunity mediates functional changes in neutrophils in diabetic environments, and targeting neutrophil-trained immunity may be a potential therapeutic target for controlling inflammatory complications of diabetes.

Extracellular Mechanisms of Neutrophils in Immune Cell Crosstalk.

Neutrophils are professional phagocytes that provide defense against invading pathogens through phagocytosis, degranulation, generation of ROS, and the formation of neutrophil extracellular traps (NETs). Although long been considered as short-lived effector cells with limited biosynthetic activity, recent studies have revealed that neutrophils actively communicate with other immune cells. Neutrophils employ various types of soluble mediators, including granules, cytokines, and chemokines, for crosstalk with immune cells. Additionally, ROS and NETs, major arsenals of neutrophils, are utilized for intercellular communication. Furthermore, extracellular vesicles play a crucial role as mediators of neutrophil crosstalk. In this review, we highlight the extracellular mechanisms of neutrophils and their roles in crosstalk with other cells.

Clinical profile and biochemical abnormalities in brucellosis: A cross-sectional study.

Introduction: Brucellosis is the commonest zoonotic disease worldwide and a common public health problem in Nepal. Because of the highly variable clinical presentation and non-specific manifestations, it remains a big challenge for clinicians from developing countries. Brucellosis has a tropism for the reticuloendothelial system, the liver is frequently involved. There is a paucity of data about the laboratory and clinical findings of human Brucellosis from Nepal. To address this knowledge gap, we conducted this study to find out the clinical profile and biochemical abnormalities of patients with brucellosis at a tertiary-care teaching hospital in western Nepal. Methods: A cross-sectional study was carried out at Gandaki Medical College Teaching Hospital, Pokhara, Nepal. All patients admitted to the in-patient department of our hospital with probable or definitive diagnoses of brucellosis were included. We excluded those who did not consent to their participation in our study, those who were under 18 years of age, and those who had deranged liver function due to other pre-existing illnesses. Descriptive statistics were used to analyze the data in terms of demography, clinical manifestations, and laboratory parameters. Results: There was a total of 40 confirmed cases of Brucellosis (age: 18-66 years) during the study period. More than half (55%, n = 22) of the study participants were males and most of them lived in a rural setting (77.5%, n = 31). Most of them (70%, n = 28) gave history of ingestion of high-risk food. The commonest clinical findings were fever with/out chills (90%, n = 36) followed by nausea/vomiting (72.5%, n = 29), headache (40%, n = 16) and malaise (37.5, n = 15). Liver function was deranged in a majority of the patients, the common parameters being Alkaline phosphatase in 96% (n = 38) cases, followed by SGOT (62.5%, n = 25), leukocytosis (57.5%, n = 23), total bilirubin (52.5%, n = 21) and SGPT (37.5%, n = 15). Characteristic increment (more than two folds of the upper limit of normal) was observed for alkaline phosphatase. Conclusion: The reticuloendothelial system is frequently involved in brucellosis. Notable changes were observed in liver function and hematological parameters in a majority of the participants in our study. These findings highlight the need for the implementation of effective control programs to address this problem in the Nepalese context.

Clinical profile and biochemical abnormalities in Scrub Typhus: A cross-sectional study.

Introduction: Scrub Typhus (ST) is an acute febrile illness caused by obligate intracellular bacteria of the family Rickettsia. It is often unrecognized and neglected but prevalent in tropical regions of endemic areas. The tragedy behind this diagnostic dilemma is non-specific clinical signs and symptoms, limited awareness, unavailability of diagnostic facilities, and low index of suspicion among the physicians. To address the knowledge gap, we tried to find out a proper panel of laboratory investigations to diagnose the disease and predict its progression because of the uncertainty of the course of the disease in a tertiary care hospital in western Nepal. Methods: This is a hospital laboratory-based prospective study conducted at Gandaki Medical College- Teaching Hospital (GMC-TH) for a period of two years. Among 988 cases of acute febrile illness, 40 seropositive cases of ST were enrolled in the study. We excluded those who did not give consent for the participation, those who were under 17 years of age, and those who had preexisting liver dysfunctions and other co-morbidities and dual seropositive with other infectious etiologies. We used descriptive statistics to analyze the data in terms of demography, clinical features, and laboratory parameters. Results: Out of 988 febrile patients, we included 40 confirmed cases of ST aged between 17 and 70 years during the study-period. Maximum seropositive cases were from Tanahun district 14 (35%), with predominance among the women (70%). The cases were prevalent in the age group 30-60 years, 19 (47.5%), and in the month of October 15 (37.5%). The commonest complaints were fever in 40 (100%), headache in 20 (50%), eschar in 11 (27.5%). Laboratory parameters showed anemia in 22 (55%), hypoalbuminemia in 11 (27.5%), leukopenia in 5 (12.5%), leukocytosis in 9 (22.5%), thrombocytopenia in 13 (32.5%), raised transaminase levels, SGPT in 21 (52.5%) and SGOT in 14 (26%) ST patients. Conclusion: We found clinical and laboratory profiles in patients with ST were varied and nonspecific. However, knowledge of these findings may evoke the recognition of ST and give a clue to the progression of the disease.

Perception of Problem Based Learning by Undergraduate Dental Students in Basic Medical Science.

BACKGROUND: Problem based learning is self-directed form of learning. Problem of Diabetes Mellitus was chosen. The objective was to evaluate perception of students towards Problem based learning and test their understanding. METHODS: A descriptive study was conducted from November 2019 till October 2020. An online Problem based learning session of a week was conducted to second-year Bachelors of Dental Surgery students using online applications. Tutors facilitated students in a group of five to six each. Pre-post testing of evaluation questions was done. At end of session, feedback of students on Problem based learning and tutor of Problem based learning were received with 'Dolmans and Schmidt' and 'Dolmans and Ginns' questionnaire. RESULTS: There was increase in correct response in nine out of 12 evaluation questions. Most students agreed to influence of discussion, content tested, course objectives, lectures, tutor and reference literature. The students agreed that tutors facilitated active, self-directed, contextual and collaborative learning. CONCLUSIONS: The influence of discussion among participants, content tested, course objectives, reference literature during the Problem based learning session were agreed upon by majority of the students. The tutors' role was accepted by participants in terms of stimulation to self-directed, active, collaborative and contextual learning.

Extracellular vesicles from dHL-60 cells as delivery vehicles for diverse therapeutics.

Extracellular vesicles (EVs) are membrane-derived heterogeneous vesicles that mediate intercellular communications. They have recently been considered as ideal vehicles for drug-delivery systems, and immune cells are suggested as a potential source for drug-loaded EVs. In this study, we investigated the possibility of neutrophils as a source for drug-loaded EVs. Neutrophil-like differentiated human promyelocytic leukemia cells (dHL-60) produced massive amounts of EVs within 1 h. The dHL-60 cells are also easily loaded with various cargoes such as antibiotics (penicillin), anticancer drug (paclitaxel), chemoattractant (MCP-1), miRNA, and Cas9. The EVs derived from the dHL-60 cells showed efficient incorporation of these cargoes and significant effector functions, such as bactericidal activity, monocyte chemotaxis, and macrophage polarization. Our results suggest that neutrophils or neutrophil-like promyelocytic cells could be an attractive source for drug-delivery EVs.

Neutrophil-derived trail is a proinflammatory subtype of neutrophil-derived extracellular vesicles.

Aims: Extracellular vesicles (EVs) are membrane-derived vesicles that mediate intercellular communications. Neutrophils produce different subtypes of EVs during inflammatory responses. Neutrophil-derived trails (NDTRs) are generated by neutrophils migrating toward inflammatory foci, whereas neutrophil-derived microvesicles (NDMVs) are thought to be generated by neutrophils that have arrived at the inflammatory foci. However, the physical and functional characteristics of neutrophil-derived EVs are incompletely understood. In this study, we aimed to investigate the differences between NDTRs and NDMVs. Methods: The generation of neutrophil-derived EVs were visualized by live-cell fluorescence images and the physical characteristics were further analyzed using nanotracking analysis assay, scanning electron microscopic analysis, and marker expressions. Functional characteristics of neutrophil-derived EVs were analyzed using assays for bactericidal activity, monocyte chemotaxis, phenotype polarization of macrophages, and miRNA sequencing. Finally, the effects of neutrophil-derived EVs on the acute and chronic inflammation were examined in vivo. Results: Both EVs share similar characteristics including stimulators, surface marker expression, bactericidal activity, and chemoattractive effect on monocytes via MCP-1. However, the integrin-mediated physical interaction was required for generation of NDTRs whereas NDMV generation was dependent on PI3K pathway. Interestingly, NDTRs contained proinflammatory miRNAs such as miR-1260, miR-1285, miR-4454, and miR-7975, while NDMVs contained anti-inflammatory miRNAs such as miR-126, miR-150, and miR-451a. Although both EVs were easily uptaken by monocytes, NDTRs enhanced proinflammatory macrophage polarization whereas NDMVs induced anti-inflammatory macrophage polarization. Moreover, NDTRs showed protective effects against lethality in a murine sepsis model and pathological changes in a murine chronic colitis model. Conclusion: These results suggest that NDTR is a proinflammatory subtype of neutrophil-derived EVs distinguished from NDMV.

Correlation of urinary cotinine with cardiovascular risk factors in pan masala tobacco users.

BACKGROUND: Tobacco consumption is considered as one of the major risk factors for cardiovascular (CV) morbidity. However, the effect of paan masala tobacco (PMT) (a type of smokeless tobacco) consumption has not been well studied in our context. Our study is aimed to find an association of CV risk factors between PMT users and nonusers and to correlate those parameters with urinary cotinine level, a degradation product of nicotine occurring in tobacco. METHODS: This comparative cross-sectional study was carried out among 200 participants. The effect of PMT use on CV risk factors such as blood pressure (BP), lipid profile, and body mass index was measured against urine cotinine level. Statistical tests used were χ2 test for categorical variable, independent t-test, Mann-Whitney U test, and Spearman's correlation applied for numerical variable, and multivariate regression analysis was performed as required. The level of significance was set at p < 0.05. RESULT: Mean BP, total cholesterol, triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and median cotinine level were found to be significantly higher in PMT users than in controls (p < 0.001). Urinary cotinine level was positively correlated with mean BP, TC, TG, and LDL-C in PMT users (p < 0.001). Similarly, the odds of having hypercholesterolemia and increased diastolic BP was also significantly higher in PMT users (p < 0.001). CONCLUSION: PMT use has an adverse effect on CV risk parameters and there is a rational of cotinine measurement for screening CV risk among PMT users.

Oxidant-antioxidant status and assessment of cardiovascular morbidity in Pan Masala containing Tobacco users: a cross-sectional study.

OBJECTIVE: Pan Masala containing Tobacco (PMT) use contributes significantly to the overall world tobacco burden especially in south Asian country like Nepal. Oxidative stress caused by it may leads to cardiovascular disease, peripheral vascular disease, hypertension, etc. Therefore, this work proposes to study the antioxidant and oxidative stress along with cardiovascular morbidity in PMT users. RESULTS: Hundred PMT users and 80 non-user controls with age and sex matched were enrolled. There was a significant difference in blood pressure, albumin, uric acid, vitamin C, vitamin E, malondialdehyde (MDA), total cholesterol, triglycerides, low density lipoprotein cholesterol between the two groups (p < 0.001). We observed statistically significant (p < 0.001) decrease in antioxidant and increase oxidative stress in PMT users. Duration and quantity of PMT user were significantly associated with the MDA level.

Retinoic acid induces hypersegmentation and enhances cytotoxicity of neutrophils against cancer cells.

Hypersegmentation of nuclei is considered a distinct characteristic of the antitumoral phenotype of neutrophils. Retinoic acid, a metabolite of retinol, reorganizes and induces segmentation of the nucleus during the differentiation of neutrophils. However, the role of retinoic acid in the phenotype polarization of neutrophils has not been fully established. Here, we investigated the effect of retinoic acid on phenotype polarization of neutrophils. Retinoic acid-induced the hypersegmentation of human neutrophils via retinoic acid receptors and mTOR pathways. Retinoic acid-induced hypersegmented neutrophils enhanced neutrophil extracellular traps (NETs) formation in response to phorbol-12-myristate 13-acetate (PMA) and fMLP (N-Formylmethionine-leucyl-phenylalanine) stimulation, and increased cytotoxicity against various tumor cells. Moreover, retinoic acid treatment attenuated tumor growth in a murine model of tumor. Taken together, these results suggests that retinoic acid induces the phenotype polarization of neutrophils to exert antitumor effects.

Dataset on the changes of neutrophils treated with retinoic acid.

The data presented in this article are related to the research article entitled "Retinoic acid induces hypersegmentation and enhances cytotoxicity of neutrophils against cancer cells" (S. Shrestha, S.Y. Kim, Y.J. Young, J.K. Kim, J.M. Lee, M. Shin, D.K. Song, C.W. Hong, 2017) [1]. This article complements the potential of retinoic acid to induce changes in effector function of human neutrophils. Here the datasets describe the rate of apoptosis, changes in numbers of nuclear lobes, and the expressions of surface markers in human neutrophils in presence or absence of retinoic acid. The tumor growth in recipient mice with adoptive transfer of retinoic acid-treated neutrophils was evaluated. The included data is made publicly available to criticism and extended analysis.

Angiotensin converting enzyme inhibitors and angiotensin II receptor antagonist attenuate tumor growth via polarization of neutrophils toward an antitumor phenotype.

Tumor microenvironments polarize neutrophils to protumoral phenotypes. Here, we demonstrate that the angiotensin converting enzyme inhibitors (ACEis) and angiotensin II type 1 receptor (AGTR1) antagonist attenuate tumor growth via polarization of neutrophils toward an antitumoral phenotype. The ACEis or AGTR1 antagonist enhanced hypersegmentation of human neutrophils and increased neutrophil cytotoxicity against tumor cells. This neutrophil hypersegmentation was dependent on the mTOR pathway. In a murine tumor model, ACEis and AGTR1 antagonist attenuated tumor growth and enhanced neutrophil hypersegmentation. ACEis inhibited tumor-induced polarization of neutrophils to a protumoral phenotype. Neutrophil depletion reduced the antitumor effect of ACEi. Together, these data suggest that the modulation of Ang II pathway attenuates tumor growth via polarization of neutrophils to an antitumoral phenotype.

Chronic Visceral Right Upper Quadrant Pain Without Gallstones.

Patients with chronic visceral right upper quadrant pain without gallstones can be broadly categorized into two groups: patients with gallbladder dyskinesia, and patients with sphincter of Oddi dysfunction (SOD). Treating patients with these disorders is often challenging to clinicians due to the difficulty at arriving at a definite diagnosis, and the lack of efficacy of various treatment modalities. The only real treatment option for patients with gallbladder dyskinesia is cholecystectomy; however, the results are difficult to predict in an individual patient. Patients with SOD can be approached according to a classification that at least partially reflects the underlying pathophysiology. Patients with type I SOD have underlying papillary stenosis, and benefit from empiric sphincterotomy. Patients with type II SOD may have muscle spasm as predominant pathophysiology; this group of patients benefit from sphincterotomy only if increased sphincter pressure is demonstrated by sphincter of Oddi manometry. Patients with type III SOD may have visceral hyperalgesia; a trial of antidepressants or a therapeutic trial with botulinum toxin injection into the ampulla should be considered prior to more invasive endoscopic therapy.