Introduction
Patients with ACLF are highly prone to bacterial infections .Bacterial infections frequently precipitates and complicate the course of ACLF . Increased bacterial infection is mainly due to defects in their defense mechanism . Deficiencies in the immune system are both in the innate and the adaptive responses, causing flaws in bactericidal capability, phagocytosis, opsonization, also causing flaws in chemotaxis of both mononuclear and polymorphonuclear cells (PMNs). PMNs are cells that constitute the first component of innate immune defense. They react rapidly in response to infection or damage, migrating to sites of inflammation sites by a guiding chemokine gradient, where they eliminate pathogens through diverse mechanisms, such as phagocytosis, degranulation, and NETosis . NETosis is a microbicidal mechanism recently described. The structures formed and released by neutrophils are known as neutrophil extracellular traps (NETs), which entrap and eliminate the microorganisms. Their structures are mainly formed by nuclear chromatin, associated histones and diverse granular proteins (elastase and myeloperoxides)[4]. It has been demonstrated that neutrophils in patients with hepatic diseases show defects in the production of oxygen radicals, mainly when producing superoxide anion and hydrogen peroxide, both essential in the elimination of bacteria and indispensable to the correct release of NETs. This can be reflected in the high incidence of bacterial infections that is observed in these patients.
In an attempt to elucidate the mechanism by which patients with acute decompensation of cirrhosis and ACLF patients have a higher susceptibility to infectious disease, we proposed this work, the main objective of which is to determine whether there is an association between patients with ACLF and their capability for the release of NETs.
Title: Alterations in neutrophil extracellular traps in patient with acute on chronic liver failure (ACLF).
Aims and Objectives
1. To find out NETs releasing capabilities of patients with ACLF.
2. Compare NETs releasing capabilities in ACLF patients with sepsis and ACLF patients without sepsis.
3. To compare the NETs releasing capabilities between patients with ACLF and patients with acute decompensation of cirrhosis
Study duration-1 and 1/2.
Methodology
Design and population of the research
A prospective observational study will be carried out in 80 individuals, who will be incorporated into four different groups as follows: healthy subjects (HS; n = 20), cirrhotic patients with acute decompensation (AD; n = 20), patients with ACLF who have sepsis (ACLF Sep; n = 20), and patients with ACLF but no sepsis (ACLF No Sep; n = 20).
All of the patients will be recruited from the Hepatology Services of the School of Digestive & Liver Diseases (SDLD) of IPGME&R Kolkata. NETs release capability and concentration of inflammatory cytokines will be determined and compared with the group of HS. Diagnosis of patients with acute decompensation and ACLF with or without sepsis will be performed by clinical, laboratory, and radiological analysis. ACLF will be defined as per EASL working definition of ACLF i.e. acute deterioration of pre-existing chronic liver disease usually related to a precipitating event and associated with increased mortality at 3 months due to multi-system organ failure, admitted to SDLD, IPGME&R, and Kolkata. Also, Patients presented with acute decompensation of cirrhosis will be included.
The patients included in this study will be informed of the purpose of the investigations and the importance of their participation. Written informed consent will be taken from patients or relatives incase patient is in encephalopathy or age <18 years.
Exclusion criteria
1. Patients with liver failure and without underlying cirrhosis
2. Pregnancy
3. Severe chronic extra hepatic diseases
4.HIV patients or patients receiving other immunosuppressive drugs
5.Unwillingness to participate in study .
Definitions -
ACLF (according to APASL)-acute hepatic insult manifesting as jaundice (serum bilirubin > 5mg/dl) & coagulopathy (INR >1.5 ) complicated within 4 weeks by clinical ascites and HE in a patient with CLD with or without cirrhosis who may have not had prior decompensation.
Acute decompensation (according to APASL)- Patients with decompensated cirrhosis who develop acute deterioration of their clinical status are considered as acute decompensation and not ACLF.
SIRS – Two or more of the following :
Fever- temperature > 38 c/100.4 F or hypothermia <36 c/ 96.8 F
Tachypnea > 24 breaths/min
Tachycardia – HR>90/min
Leukocytosis – 12000/dl or leukopenia < 4000 /dl or >10% band cells.
Sepsis-SIRS due to proven infection or when strongly suspected plus some degree of organ hypofunction i.e-Cardiovascular – Systolic BP <90 mmHg or mean arterial pressure <70 mmHg that responds to IV fluid .
Renal- U.O. < 0.5 ml/kg/hr for 1 hr despite fluid resuscitition.
Respiratory – paO/Fio2= <250 or if lung alone is involved <200
Hematologic- platelets < 80000/dl or 50 % decrease of platelet count from highest recorded count in past 3 days
Unexplained metabolic acidosis pH<7.30 or lactate >1.5 times of upper limit of normal, or base deficit of >5 meq /L .
Clinical record
1. Mode of presentation/presenting features.
2. Appearance of coagulopathy, encephalopathy or ascites/ACLF features.
3. Time duration from presentation to appearance of complications.
4. Clinical course following onset of complications.
5. Serial record of the biochemical parameters
6. Event of sepsis
7. Cause and duration of hospitalization
8. Whether critical care required or not
9. Immediate cause of death (e.g. bleeding diathesis/DIC, HE, sepsis etc)
Biochemical-
Complete blood count
Liver function test
PT / INR
Renal function test
Serum Na & K
RBS
Urine R / E
Blood and Urine culture
CRP
Pro- calcitonin
Ascitic Fluid
TLC
DLC (N/L)
Protein
Albumin
SAAG
C/S
NET LEVELS
Other investigations-
USG whole Abdomen
Cect abdomen if needed
UGIE
Patient management and follow up
All the patients will be managed as per standard clinical protocol for the management of Acute Liver Disease and Cirrhosis with portal hypertension. Clinical and laboratory details will be recorded during at the time of admission.
Blood samples
From all study groups, peripheral blood (PB) samples will be collected to obtain neutrophils
1. Isolation of Human Polymorphonuclear Leukocyte10–20 ml of peripheral blood will be collected from patients suffering from patients (ACLF and acute decompensation) and control. Neutrophils will be obtained by FicollHistopaque 1119/1077 density gradient (Sigma-Aldrich, Saint Louis, Missouri, USA). The cellular ring will be re-suspended in RPMI-1640 medium (Sigma-Aldrich, San Luis, Missouri, USA). The process of hemolysis will be skipped to avoid non-specific activation of neutrophils. Because neutrophils are the most abundant cells in PMNs, PMNs will be used as a source of neutrophils in the following studies.
2. NET Induction in VitroThe PMNs will be re suspended in RPMI 1640 medium containing 5% fetal bovine serum (FBS) (1 x 106/ml). After pre-incubation for 30 min at 378C, the cells will be exposed to 100 nM PMA (Sigma-Aldrich, St. Louis, MO) for 0–4 h at 378C.
3. Flow Cytometric Detection of SYTOX Green-Positive CellsThe PMA-treated PMNs will be subjected to react with a plasma membrane-impermeable DNA-binding dye, SYTOX Green (Life Technologies, Carlsbad, CA) according to the manufacturer’s instruction. After filtering out the debris with a mesh, the percolated cells were analyzed using Attune flow cytometer (Applied Biosystems, Foster City, CA). Because SYTOX Green expresses fluorescence only after binding to DNA, the step to remove unbound dye can be omitted. Granulocytes mainly composed of neutrophils will be selected by the properties of forward and side scatters in flow cytometry. This study will focus on SYTOX Green-positive neutrophils .
4. Fluorescent Image Analysis of NETsThe PMNs will be re suspended in RPMI 1640 medium containing 5% FBS and then seeded in wells of 4-well chamber slides (1x105/ml). After pre incubation for 30 min at 378C, the cells will be exposed to 100 nM PMA for 4 h at 378C. Thereafter, the medium will be removed, and the remaining cells will be washed with PBS. The cells were then fixed with 4% paraformaldehyde for 15 min at room temperature. After washing with PBS, the cells will be incubated in PBS containing 3% bovine serum albumin (BSA) for 30 min at room temperature to block non-specific binding of antibodies. Then, the cells will be allowed to react with 5 mg/ml of anti-human MPO antibody (Bio-Rad, Hercules, CA) or the isotype control mouse IgG2b (BioLegend, San Diego, CA) for 60 min at room temperature. After washing with PBS, the cells will be allowed to react with 5 mg/ml of Alexa Fluor 488-conjugated goat anti-mouse IgG (H1L) antibody (Thermo Fisher Scientific, Waltham, MA) for 60 min at room temperature. After removal of unbound antibodies as needed, the slides were mounted with40, 6-diamidino-2-phenylindole (DAPI)-containing solution (Sigma-Aldrich)[8]. NET formation will be observed under a fluorescent microscope.
5. Double-Staining of MPO and SYTOX Green in Flow CytometryThe PMA-treated PMNs will be washed with PBS and then incubated in PBS containing 3% BSA for 30 min at room temperature to block non-specific binding of antibodies (1 3 106/100 ml). Five mg/ml of anti-human MPO antibody (Bio-Rad) or the isotype control mouse IgG2b (Bio Legend) will be added into the solution, and the samples will be incubated for 30 min at room temperature. The cells will be washed with PBS and re-suspended in PBS containing 3% BSA (1 3 106/100 ml).Then, 4 mg/ml of PE-labeled anti-mouse IgG antibody (BioLegend) will be added into the solution, and the samples will be incubated for 30 min at room temperature. After washing with PBS, flow cytometric detection of SYTOX Green-positive cells will be carried out as aforementioned .
Statistical analysis
Data will be presented as means ± standard deviations. The statistical analysis will be performed utilizing SPSS version 20 statistical software package (IBM, Armonk, USA). Comparisons will be performed by non-parametric tests using the Friedman and/or the Mann-Whitney U test. These will be considered significant when p < 0.05.