Investigating neutrophil cell death in TB pathogenesis

Background: Neutrophils are one of the major early role players in antimycobacterial immunity. Upon infection, neutrophils can undergo NETosis, a cell death characterized by release of neutrophil extracellular traps (NETs). The role of NETosis in TB progression remains poorly characterized. We aim to characterize mechanisms underlying NETosis during TB pathogenesis by identifying genes that drive the cell death, and to determine their potential as markers of disease progression in high-risk individuals. Finally, we intend to evaluate neutrophil associated genes as targets for host directed therapy to reduce pathological damage caused by NETosis. Methods: Quantitative PCR will be used to quantify expression of specific genes identified in the blood of individuals with active lung disease (n=30), compared to those from healthy (n=30) and latently infected individuals (LTBI) (n=30). In addition, temporal events associated with NETosis will be measured using live microscopy in a neutrophil in vitro model of Mycobacterium tuberculosis (Mtb) infection. Candidate genes found to be associated with NETosis will be targeted with pharmaceutical inhibitors. Conclusion: Genes associated with neutrophil mediated cell death may serve as potential biomarkers of pathological damage and disease progression, as well as targets for host-directed therapy.

Autoantibodies against unmodified and citrullinated human endogenous retrovirus K envelope protein in rheumatoid arthritis patients

Objective Autoantibodies against proteins encoded by human endogenous retrovirus K (HERV-K) have been reported in patients with rheumatoid arthritis (RA), but their relevance, if any, has remained unresolved. We revisited this question and tested if such autoantibodies may react with citrullinated epitopes on the envelope (Env) protein of HERV-K. Methods Immunoblotting and ELISAs were conducted with unmodified Env protein and with Env citrullinated by protein arginine deiminase (PAD) 4. Sera from 100 RA patients, plasma from 32 juvenile idiopathic arthritis (JIA) patients, and healthy adult and pediatric controls were included. Antibody reactivity was evaluated for correlations with clinical and laboratory parameters of the patients. Results We replicated and expanded upon published data that patients with RA or JIA have autoantibodies against HERV-K Env, some with high titers. Anti-HERV-K antibodies correlated with cigarette smoking and with circulating DNA-myeloperoxidase complexes indicative of nonapoptotic neutrophil cell death. Furthermore, most of the RA patients, but not JIA patients, had autoantibodies that reacted more strongly with Env that was citrullinated by PAD4. These anticitrullinated Env autoantibodies correlated with seropositivity and tended to be higher in patients with erosive disease. Conclusion Our data suggest that anti-HERV-K immunity is elevated in RA and JIA and may have a connection with pathogenic protein citrullination in RA.

Anti-inflammatory Activity of Lefamulin in a Lipopolysaccharide-Induced Lung Neutrophilia Model

ABSTRACTLefamulin is a novel pleuromutilin antibiotic approved for the treatment of community-acquired bacterial pneumonia. This study demonstrated anti-inflammatory activity of lefamulin in a murine lipopolysaccharide-induced lung neutrophilia model. Pretreatment of mice at clinically relevant lefamulin subcutaneous doses (35, 70, 140 mg/kg [free base]) followed by intranasal lipopolysaccharide challenge (5 μg/50 μL/mouse) demonstrated significant, dose-dependent reductions in total and neutrophil cell counts in bronchoalveolar lavage fluid samples, with reductions comparable to oral dexamethasone (0.5 mg/kg) pretreatment.

Two new immature and dysfunctional neutrophil cell subsets define a predictive signature of sepsis useable in clinical practice

Sepsis is the leading cause of death in adult intensive care units. At present, sepsis diagnosis relies on non-specific clinical features. It could transform clinical care to have objective immune cell biomarkers that could predict sepsis diagnosis and guide treatment. For decades, neutrophil phenotypes have been studied in sepsis, but a diagnostic cell subset has yet to be identified. Here, high dimensional mass cytometry was used to reveal for the first time a specific neutrophil signature of sepsis severity that does not overlap with other inflammatory biomarkers, and that distinguishes patients with sepsis from those with non-infectious inflammatory syndrome. Unsupervised analysis of 42-dimesional mass cytometry data characterized previously unappreciated heterogeneity within the CD64+ immature neutrophils and revealed two new subsets distinguished by CD123 and PD-L1 expression. These immature neutrophils exhibited diminished activation and phagocytosis functions. The proportion of CD123-expressing neutrophils also correlated with clinical severity. Critically, this study showed that these two new neutrophil subsets were specific to sepsis and detectable by routine flow cytometry using seven markers. The demonstration here that a simple blood test distinguishes sepsis from other inflammatory conditions represents a key biological milestone that can be immediately translated into improvements in patient care.One Sentence SummaryCD123+ and/or PD-L1+ immature and dysfunctional neutrophil subsets identified by mass cytometry, define an early human blood signature of sepsis

Importance of haemogram parameters for prediction of the time of birth in women diagnosed with threatened preterm labour

Objective This study aimed to estimate the importance of complete blood count parameters for predicting the timing of birth in threatened preterm labour cases. Methods We performed a retrospective study of 92 patients who were diagnosed with threatened preterm labour (24–34 gestational weeks). The patients were divided into two groups according to the time of birth (group 1: delivered within the first week after diagnosis; group 2: delivered later than 1 week). We compared characteristics and complete blood count parameters between these two groups. Results There were no significant differences in maternal age, body mass index, gravida, parity, haemoglobin levels, and gestational weeks between the two groups. The mean cervical length was 24.24 ± 3.60 mm in group 1 and 30.70 ± 5.32 mm in group 2. There were significant differences in the neutrophil to lymphocyte ratio, white blood cell count, red cell distribution width (RDW), absolute lymphocyte cell count, and absolute neutrophil cell count between the two groups. Conclusion Maternal serum RDW, the neutrophil to lymphocyte ratio, white blood cell count, absolute lymphocyte cell count, and the absolute neutrophil cell count profile could guide clinicians in predicting the time of birth in threatened preterm labour cases.

Comparison Of The Function Of Neutrophil Cells Oxidative Burst Among Various Group Ages

Background: In old ages there is a change in the immune system along with the aging process called the term immunosenescence. Neutrophil cells play an important role in natural immunity because they are the first immune cells to be deployed in the body's defenses.Objective: To prove that there is a difference in the function of neutrophil cell oxidative burst in older age group compared with the younger age group as well as the tendency of decreased oxidative burst function of neutrophil cells with increasing age.Method: This is a cross-sectional observational analytic study involving 48 healthy subjects. The subjects were divided into 3 age groups: young age (18-40 years old), middle age (41-59 years old), and old age ≥60 years old). Each of them were examined for the function of neutrophil cells oxidative burst. The data were then analyzed using one-way ANOVA test. The result was considered significant if p<0.05.Results: The obtained mean age 59.26±8.03 years old. The mean age for young age group was 28.75±6.66 years old. The mean of middle age group was 50.19±5.46 years old. The mean age of old group was 66.38±3.83 years old. The mean of netrofil oxidative burst cell function was 96.83±2.7% with mean of young age being 98.57±0.98%, middle age 97.71±1.64%, and old age 94.20±3.56%. One way ANOVA comparison analysis showed a significant difference with p = 0.000 (significant when p <0.05). The result of Rank Spearman test showed significant result with r=-0.590 (p=0.000).Conclusion: There are differences in the function of neutrophil cells oxidative burst between young and old age groups. There is a negative trend between the age group and the function of neutrophil cell oxidative burst. The increase of age causes decrease in the function of neutrophil cells oxidative burst.

Autologous plasma versus fetal calf serum as a supplement for the culture of neutrophils.

Objective Currently the replacement of fetal calf serum (FCS) by a more suitable alternative is a sought aim in the field of tissue and cell culture research. Autologous plasma (AP) and especially autologous serum (AS) have been shown to be effective substitutes of FCS in culture media for some of cell types. Nevertheless, there is no comparative data on the most appropriate supplement for cell media in neutrophil studies, it is now unclear whether AP have relatively an equal, superior or inferior performance to FCS in neutrophil cell culture. In the present study, human blood neutrophils were isolated and cultured in FCS- or AP-supplemented medium. After 12, 36 and 60 hours of incubation, cell viability, oxidative burst and CD11b expression were determined by flow cytometry.Results Compared to the culture of neutrophils in FCS 10% medium, the culture of neutrophils in a medium with AP 10% could prolong their life span without affecting their function. The findings introduce AP as a better supplement for human neutrophil cell culture than FCS and propose a simple and economical procedure for neutrophil isolation and culture.

Autologous plasma versus fetal calf serum as a supplement for the culture of neutrophils

Objective Currently the replacement of fetal calf serum (FCS) by a more suitable alternative is a sought aim in the field of tissue and cell culture research. Autologous plasma (AP) and especially autologous serum (AS) have been shown to be effective substitutes of FCS in culture media for some of cell types. Nevertheless, there is no comparative data on the most appropriate supplement for cell media in neutrophil studies, it is now unclear whether AP have relatively an equal, superior or inferior performance to FCS in neutrophil cell culture. In the present study, human blood neutrophils were isolated and cultured in FCS- or AP-supplemented medium. After 12, 36 and 60 hours of incubation, cell viability, oxidative burst and CD11b expression were determined by flow cytometry.Results Compared to the culture of neutrophils in FCS 10% medium, the culture of neutrophils in a medium with AP 10% could prolong their life span without affecting their function. The findings introduce AP as a better supplement for human neutrophil cell culture than FCS and propose a simple and economical procedure for neutrophil isolation and culture.

Autologous plasma versus fetal calf serum as a supplement for the culture of neutrophils.

Objective Currently the replacement of fetal calf serum (FCS) by a more suitable alternative is a sought aim in the field of tissue and cell culture research. Autologous plasma (AP) and especially autologous serum (AS) have been shown to be effective substitutes of FCS in culture media for some of cell types. Nevertheless, there is no comparative data on the most appropriate supplement for cell media in neutrophil studies, it is now unclear whether AP have relatively an equal, superior or inferior performance to FCS in neutrophil cell culture. In the present study, human blood neutrophils were isolated and cultured in FCS- or AP-supplemented medium. After 12, 36 and 60 hours of incubation, cell viability, oxidative burst and CD11b expression were determined by flow cytometry.Results Compared to the culture of neutrophils in FCS 10% medium, the culture of neutrophils in a medium with AP 10% could prolong their life span without affecting their function. The findings introduce AP as a better supplement for human neutrophil cell culture than FCS and propose a simple and economical procedure for neutrophil isolation and culture.

Autologous plasma versus fetal calf serum as a supplement for the culture of neutrophils.

Objective Currently the replacement of fetal calf serum (FCS) by a more suitable alternative is a sought aim in the field of tissue and cell culture research. Autologous plasma (AP) and especially autologous serum (AS) have been shown to be effective substitutes of FCS in culture media for some of cell types. Nevertheless, there is no comparative data on the most appropriate supplement for cell media in neutrophil studies, it is now unclear whether AP have relatively an equal, superior or inferior performance to FCS in neutrophil cell culture. In the present study, human blood neutrophils were isolated and cultured in FCS- or AP-supplemented medium. After 12, 36 and 60 hours of incubation, cell viability, oxidative burst and CD11b expression were determined by flow cytometry.Results Compared to the culture of neutrophils in FCS 10% medium, the culture of neutrophils in a medium with AP 10% could prolong their life span without affecting their function. The findings introduce AP as a better supplement for human neutrophil cell culture than FCS and propose a simple and economical procedure for neutrophil isolation and culture.