Monomeric serum immunoglobulin A (IgA) can contribute to the development of various autoimmune diseases, but the regulation of serum IgA effector functions is not well defined. Here, we show that the ...two IgA subclasses (IgA1 and IgA2) differ in their effect on immune cells due to distinct binding and signaling properties. Whereas IgA2 acts pro-inflammatory on neutrophils and macrophages, IgA1 does not have pronounced effects. Moreover, IgA1 and IgA2 have different glycosylation profiles, with IgA1 possessing more sialic acid than IgA2. Removal of sialic acid increases the pro-inflammatory capacity of IgA1, making it comparable to IgA2. Of note, disease-specific autoantibodies in patients with rheumatoid arthritis display a shift toward the pro-inflammatory IgA2 subclass, which is associated with higher disease activity. Taken together, these data demonstrate that IgA effector functions depend on subclass and glycosylation, and that disturbances in subclass balance are associated with autoimmune disease.
Alterations of cell death pathways, including apoptosis and the neutrophil specific kind of death called NETosis, can represent a potential source of autoantigens. Defects in the clearance of ...apoptotic cells may be responsible for the initiation of systemic autoimmunity in several chronic inflammatory diseases, including systemic lupus erythematosus (SLE). Autoantigens are released mainly from secondary necrotic cells because of a defective clearance of apoptotic cells or an inefficient degradation of DNA-containing neutrophil extracellular traps (NETs). These modified autoantigens are presented by follicular dendritic cells to autoreactive B cells in germinal centers of secondary lymphoid organs. This results in the loss of self-tolerance and production of autoantibodies, a unifying feature of SLE. Immune complexes (IC) are formed from autoantibodies bound to uncleared cellular debris in blood or tissues. Clearance of IC by blood phagocytes, macrophages, and dendritic cells leads to proinflammatory cytokine secretion. In particular, plasmacytoid dendritic cells produce high amounts of interferon-α upon IC uptake, thereby contributing to the interferon signature of patients with SLE. The clearance of antinuclear IC via Fc-gamma receptors is considered a central event in amplifying inflammatory immune responses in SLE. Along with this, the accumulation of cell remnants represents an initiating event of the etiology, while the subsequent generation of autoantibodies against nuclear antigens (including NETs) results in the perpetuation of inflammation and tissue damage in patients with SLE. Here, we discuss the implications of defective clearance of apoptotic cells and NETs in the development of clinical manifestations in SLE.
Coronavirus induced disease 2019 (COVID-19) can be complicated by severe organ damage leading to dysfunction of the lungs and other organs. The processes that trigger organ damage in COVID-19 are ...incompletely understood.
Samples were donated from hospitalized patients. Sera, plasma, and autopsy-derived tissue sections were examined employing flow cytometry, enzyme-linked immunosorbent assays, and immunohistochemistry.
Here, we show that severe COVID-19 is characterized by a highly pronounced formation of neutrophil extracellular traps (NETs) inside the micro-vessels. Intravascular aggregation of NETs leads to rapid occlusion of the affected vessels, disturbed microcirculation, and organ damage. In severe COVID-19, neutrophil granulocytes are strongly activated and adopt a so-called low-density phenotype, prone to spontaneously form NETs. In accordance, markers indicating NET turnover are consistently increased in COVID-19 and linked to disease severity. Histopathology of the lungs and other organs from COVID-19 patients showed congestions of numerous micro-vessels by aggregated NETs associated with endothelial damage.
These data suggest that organ dysfunction in severe COVID-19 is associated with excessive NET formation and vascular damage.
Deutsche Forschungsgemeinschaft (DFG), EU, Volkswagen-Stiftung
Activating mutations in the estrogen receptor (ER) α-gene (
) result in constitutive transcriptional activity in the absence of estrogen and are associated with endocrine resistance in metastatic ...ER-positive (+) breast cancer. It is not known how activating
mutations may alter the predictive values of molecular imaging agents for endocrine therapy response. This study investigated the effect of an activating
mutation on pretreatment
F-fluoroestradiol (
F-FES) uptake and early assessment of endocrine therapy response using
F-FDG and
F-fluorofuranylnorprogesterone (
F-FFNP) PET/CT imaging of tumor glucose metabolism and progesterone receptor (PR) expression, respectively.
ER+, PR+ T47D breast cancer cells expressing wild-type (WT) ER or an activating
mutation, Y537S-ER, were used to generate tumor xenografts in ovariectomized female immunodeficient mice supplemented with 17β-estradiol. Tumor growth curves were determined in the presence or absence of estrogen and for ethanol vehicle control or fulvestrant treatment, a selective ER degrader. Pretreatment
F-FES uptake was compared between Y537S-ER and WT-ER tumors. Longitudinal PET/CT imaging with
F-FFNP and
F-FDG was performed before and 7-9 d after the start of endocrine therapy with fulvestrant. Radiopharmaceutical uptake in Y537S-ER and WT-ER tumors was compared between baseline and follow-up scans. Statistical significance was determined using paired
testing for longitudinal imaging and 2-way ANOVA for the
F-FFNP tissue biodistribution assay.
Y537S-ER xenografts showed estrogen-independent growth, whereas WT-ER tumors grew only with estrogen. Fulvestrant treatment for 28 d significantly reduced tumor volumes for WT-ER but only stabilized volumes for Y537S-ER. Baseline
F-FES uptake did not significantly differ between WT-ER and Y537S-ER tumors. Fulvestrant treatment induced a similar early metabolic response for both WT-ER and Y537S-ER tumors.
F-FFNP uptake in WT-ER tumors was significantly reduced after 7 d of fulvestrant treatment; however, this reduction did not occur in Y537S-ER tumors, which showed no significant change between baseline and follow-up PET/CT.
Molecular imaging of PR expression dynamics could be a noninvasive approach for early identification of reduced effectiveness of endocrine therapy resulting from activating
mutations.
In this study, we identified and characterized the potential of a high ratio of bicarbonate to CO2 and a moderately alkaline pH to render neutrophils prone to undergo neutrophil extracellular trap ...(NET) formation. Both experimental settings increased the rate of spontaneous NET release and potentiated the NET-inducing capacity of phorbol esters (phorbol-2-myristate-13-acetate), ionomycin, monosodium urate, and LPS. In contrast, an acidic environment impaired NET formation both spontaneous and induced. Our findings indicate that intracellular alkalinization of neutrophils in response to an alkaline environment leads to an increase of intracellular calcium and neutrophil activation. We further found that the anion channel blocker DIDS strongly reduced NET formation induced by bicarbonate. This finding suggests that the effects observed are due to a molecular program that renders neutrophils susceptible to NET formation. Inflammatory foci may be characterized by an acidic environment. Our data indicate that NET formation is favored by the higher pH at the border regions of inflamed areas. Moreover, our findings highlight the necessity for strict pH control during assays of NET formation.
Summary The p16 tumor suppressor gene is a member of the INK4 class of cell cycle inhibitors and is located on chromosome 9p21. The p16 protein binds to cyclin-dependent kinases 4 and 6 and maintains ...the retinoblastoma gene product in its hypophosphorylated state, which in turn binds to E2F transcription factor and prevents cell cycle progression. Expression of p16 protein is increased in aging cells. Immunohistochemistry for p16ink4a is most widely used as a surrogate maker for high risk human papilloma virus (hrHPV) infection in formalin fixed paraffin embedded tissues. The most widely researched, accepted and practiced use of p16 immunostain is in the lower anogenital tract. In addition, p16 is also widely used in the oropharynx for oropharyngeal squamous cell carcinoma. Its applications have also been extended to gynecologic tumors which are unrelated to HPV. This article aims to review the literature on the diagnostic utility of p16 immunohistochemistry (IHC) and highlight the practical issues in the application and interpretation of this stain.
The small signal model of MOSFET is a must for implementation of analog/digital circuits. The non-quasi-static (NQS) model is well known and provides accurate parameters for MOSFET. In the present ...work, extrinsic and intrinsic NQS model parameters were extracted for 20 nm Gate Stacked Junction-less Accumulation Mode (GSJAM) MOSFET using two-port admittance parameters. Further, for analog/RF applications, the first order active high pass filter (HPF), low pass filter (LPF) and second order active HPF, LPF are implemented using 20 nm GSJAM MOSFET. The first order and second order active filters are implemented for 10 GHz circuit applications. For amplifying the processed filtered input signal, a CMOS single stage common source voltage amplifier is used in the output circuit. Filter circuits use GSJAM N-type MOSFET as an active load in conjunction with a passive capacitor. With cut-off frequency, further bode-plot analysis of the low pass filter and high pass filter circuits is conducted. For numerical simulation, the well-known silvaco CAD tool is used.
For low-power circuit applications, the performance of the circuit is significantly influenced by the MOSFET's analog/RF and non-linearity properties. Gate-all-around junction-less accumulation mode ...MOSFETs (JAM) are now the perfect choice for low-power circuit applications. In the present work, the gate-stack engineering method is used for gate-all-around junction-less accumulation mode MOSFET (GSJAM) to improve the analog/RF characteristics. The analog/RF and linearity parameter analyses are numerically performed for a 20 nm channel length gate-all-around gate-stacked junction-less MOSFET using an ATLAS 3D device simulator. The comparative analysis of these analog/RF and linearity parameters is also done. The gate stack engineering shows significant improvements in analog/RF parameters, i.e., trans-conductance (g
m
), output resistance (R
out
), early voltage (V
ea
), intrinsic gain (A
V
), trans-conductance frequency product (TFP), gain frequency product (GFP), trans-conductance generation factor (TGF), gain bandwidth product (GBWP), gain trans-conductance frequency product (GTFP), and cut-off frequency (f
T
). The results of the simulation investigation show that when GSJM is compared to JAM MOSFET, the values of gm, R
out
, V
ea
, and TGF are all 26.37%, 2.24 times, 22.38%, and 20.78% higher, respectively. Also, the gate-stack junction-less MOSFET improves the linearity parameters, i.e., 2nd -order trans-conductance (g
m2
)
,
3rd -order trans-conductance (g
m3
), VIP
2
, VIP
3
, IIP
3
, IMD
3
, and 1-dBCP. When comparing GSJAM to JAM MOSFET, the values of VIP2 are 81.63% higher, VIP3 is 1.77 times higher, IIP3 is 6.72 times higher, and 1-dBCP is twice as high. Further, for analog/RF applications, a 20 nm CMOS common-source voltage amplifier circuit using gate-stake junction-less MOSFET has also been designed for a 20 mV and 1 GHz sinusoidal input signal.
Obstructive Meibomian gland dysfunction (MGD) is one of the leading causes of evaporative dry eye disease. Meibomian glands at the eyelid secrete lipids that prevent evaporation of the aqueous tear ...film. The pathogenesis of obstructive MGD is incompletely understood to date. Herein, we aim to investigate the pathogenesis of obstructive MGD using murine and human samples with various forms of ocular surface inflammation.
The presence of Neutrophil extracellular Traps (NETs) was detected with immunofluorescence analysis of ocular surface discharge and biopsy samples from patients with blepharitis. Tear fluid from patients with MGD and blepharitis were evaluated for the presence of inflammatory mediators using bead based immunoassay. Murine model of allergic eye disease (AED) was performed to investigate the role of NETs in MG occlusion.
we show that the ocular discharge from patients with blepharitis contains aggregated neutrophil extracellular traps (aggNETs). Furthermore, the ducts of human Meibomian glands affected by blepharitis were largely congested by aggNETs. Tear fluid from patients with MGD showed elevated neutrophil chemoattractants (C5a, IL6, IL8 and IL18). C5a and IL8 correlated with the degree of deficiency of tear fluid. In the murine model of allergic eye disease (AED), aggNETs accumulated in the MG leading to occlusion of their ducts and the retrograde pent-up of the fluid followed by acinar atrophy. Constraining aggNET formation by genetic or pharmacological inhibition of peptidyl arginine deiminase type 4 (PADI4) effectively reduced MG damage.
We conclude that aggNETs occlude MG causing MGD after ocular surface inflammation.
Neutrophils respond to various stimuli by decondensing and releasing nuclear chromatin characterized by citrullinated histones as neutrophil extracellular traps (NETs). This achieves pathogen ...immobilization or initiation of thrombosis, yet the molecular mechanisms of NET formation remain elusive. Peptidyl arginine deiminase-4 (PAD4) achieves protein citrullination and has been intricately linked to NET formation. Here we show that citrullination represents a major regulator of proteolysis in the course of NET formation. Elevated cytosolic calcium levels trigger both peptidylarginine deiminase-4 (PAD4) and calpain activity in neutrophils resulting in nuclear decondensation typical of NETs. Interestingly, PAD4 relies on proteolysis by calpain to achieve efficient nuclear lamina breakdown and chromatin decondensation. Pharmacological or genetic inhibition of PAD4 and calpain strongly inhibit chromatin decondensation of human and murine neutrophils in response to calcium ionophores as well as the proteolysis of nuclear proteins like lamin B1 and high mobility group box protein 1 (HMGB1). Taken together, the concerted action of PAD4 and calpain induces nuclear decondensation in the course of calcium-mediated NET formation.
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