Ascaris lumbricoides cystatin (Al-CPI) prevents the development of allergic airway inflammation and dextran-induced colitis in mice models. It has been suggested that helminth-derived cystatins inhibit cathepsins in dendritic cells (DC), but their immunomodulatory mechanisms are unclear. We aimed to analyze the transcriptional profile of human monocyte-derived DC (moDC) upon stimulation with Al-CPI to elucidate target genes and pathways of parasite immunomodulation.
moDC were generated from peripheral blood monocytes from six healthy human donors of Denmark, stimulated with 1 µM of Al-CPI, and cultured for 5 hours at 37°C. RNA was sequenced using TrueSeq RNA libraries and the NextSeq 550 v2.5 (75 cycles) sequencing kit (Illumina, Inc). After QC, reads were aligned to the human GRCh38 genome using Spliced Transcripts Alignment to a Reference (STAR) software. Differential expression was calculated by DESEq2 and expressed in fold changes (FC). Cell surface markers and cytokine production by moDC were evaluated by flow cytometry.
Compared to unstimulated cells, Al-CPI stimulated moDC showed differential expression of 444 transcripts (|FC| ≥1.3). The top significant differences were in Kruppel-like factor 10 (KLF10, FC 3.3, PBH = 3 x 10-136), palladin (FC 2, PBH = 3 x 10-41), and the low-density lipoprotein receptor (LDLR, FC 2.6, PBH = 5 x 10-41). Upregulated genes were enriched in regulation of cholesterol biosynthesis by sterol regulatory element-binding proteins (SREBP) signaling pathways and immune pathways. Several genes in the cholesterol biosynthetic pathway showed significantly increased expression upon Al-CPI stimulation, even in the presence of lipopolysaccharide (LPS). Regarding the pathway of negative regulation of immune response, we found a significant decrease in the cell surface expression of CD86, HLA-DR, and PD-L1 upon stimulation with 1 µM Al-CPI.
Al-CPI modifies the transcriptome of moDC, increasing several transcripts encoding enzymes involved in cholesterol biosynthesis and SREBP signaling. Moreover, Al-CPI target several transcripts in the TNF-alpha signaling pathway influencing cytokine release by moDC. In addition, mRNA levels of genes encoding KLF10 and other members of the TGF beta and the IL-10 families were also modified by Al-CPI stimulation. The regulation of the mevalonate pathway and cholesterol biosynthesis suggests new mechanisms involved in DC responses to helminth immunomodulatory molecules.

Stefin B (cystatin B) is an inhibitor of lysosomal and nuclear cysteine cathepsins. The gene for stefin B is located on human chromosome 21 and its expression is upregulated in the brains of individuals with Down syndrome. Biallelic loss-of-function mutations in the stefin B gene lead to Unverricht-Lundborg disease-progressive myoclonus epilepsy type 1 (EPM1) in humans. In our past study, we demonstrated that mice lacking stefin B were significantly more sensitive to sepsis induced by lipopolysaccharide (LPS) and secreted higher levels of interleukin 1-β (IL-1β) due to increased inflammasome activation in bone marrow-derived macrophages. Here, we report lower interleukin 1-β processing and caspase-11 expression in bone marrow-derived macrophages prepared from mice that have an additional copy of the stefin B gene. Increased expression of stefin B downregulated mitochondrial reactive oxygen species (ROS) generation and lowered the NLR family pyrin domain containing 3 (NLRP3) inflammasome activation in macrophages. We determined higher AMP-activated kinase phosphorylation and downregulation of mTOR activity in stefin B trisomic macrophages-macrophages with increased stefin B expression. Our study showed that increased stefin B expression downregulated mitochondrial ROS generation and increased autophagy. The present work contributes to a better understanding of the role of stefin B in regulation of autophagy and inflammasome activation in macrophages and could help to develop new treatments.

Cystatins are a family of intracellular and extracellular protease inhibitors that inhibit cysteine cathepsins-a group of lysosomal cysteine proteases that participate in multiple biological processes, including protein degradation and post-translational cleavage. Cysteine cathepsins are associated with the development of autoimmune diseases, tumor progression, and metastasis. Cystatins are categorized into three subfamilies: type 1, type 2, and type 3. The type 2 cystatin subfamily is the largest, containing 10 members, and consists entirely of small secreted proteins. Although type 2 cystatins have many shared biological roles, each member differs in structure, post-translational modifications (e.g., glycosylation), and expression in different cell types. These distinctions allow the type 2 cystatins to have unique biological functions and properties. This review provides an overview of type 2 cystatins, including their biological similarities and differences, their regulatory effect on human immune responses, and their roles in tumor progression, immune evasion, and metastasis.

Meibomian gland dysfunction (MGD) is a highly prevalent condition and the most common cause of evaporative dry eye disease. Studying the proteome of MGD can result in important advances in the management of the condition. Here, we collected tear film samples from treatment naïve patients with MGD (n = 10) and age-matched controls (n = 11) with Schirmer filtration paper. The samples were analyzed with label-free quantification nano liquid chromatography-tandem mass spectrometry. The proteins were considered differentially expressed if p < 0.05. A total of 88 proteins were significantly regulated. The largest change was observed in cystatin-SN, which was downregulated in MGD and correlated negatively with tear meniscus height. The downregulation of cystatin-SN was confirmed with targeted mass spectrometry by single reaction monitoring (SRM). Eighteen immunoglobulin components involved in B cell activation, phagocytosis, and complement activation were downregulated in MGD including Ig alpha-1 chain C region, immunoglobulin J chain, immunoglobulin heavy variable 3-15, and Ig mu chain C region. The changes in cystatin-SN and immunoglobulin chains are likely to result from the inflammatory changes related to tear film evaporation, and future studies may assess their association with the meibum quality.

UNASSIGNED: Use of cystatin C in addition to creatinine to estimate glomerular filtration rate (estimated glomerular filtration rate based on cystatin C [eGFRcys] and estimated glomerular filtration rate based on creatinine [eGFRcr], respectively) is increasing. When eGFRcr and eGFRcys are discordant, it is not known which is more accurate, leading to uncertainty in clinical decision making.
UNASSIGNED: Cross-sectional analysis.
UNASSIGNED: Four thousand fifty participants with measured glomerular filtration rate (mGFR) from 12 studies in North America and Europe.
UNASSIGNED: Serum creatinine and serum cystatin C.
UNASSIGNED: Performance of creatinine-based and cystatin C-based glomerular filtration rate estimating equations compared to mGFR.
UNASSIGNED: We evaluated the accuracy of eGFRcr, eGFRcys, and the combination (eGFRcr-cys) compared to mGFR according to the magnitude of the difference between eGFRcr and eGFRcys (eGFRdiff). We used CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) equations to estimate glomerular filtration rate. eGFRdiff was defined as eGFRcys minus eGFRcr and categorized as less than -15, -15 to <15, and ≥15 mL/min/1.73 m2 (negative, concordant, and positive groups, respectively). We compared bias (median of mGFR minus eGFR) and the percentage of eGFR within 30% of mGFR.
UNASSIGNED: Thirty percent of participants had discordant eGFRdiff (21.0% and 9.6% negative and positive eGFRdiffs, respectively). In the concordant eGFRdiff group, all equations displayed similar accuracy. In the negative eGFRdiff groups, eGFRcr had a large overestimation of mGFR (-13.4 [-14.5 to -12.2] mL/min/1.73 m2) and eGFRcys had a large underestimation (9.9 [9.1-11.2] mL/min/1.73m2), with opposite results in the positive eGFRdiff group. In both negative and positive eGFRdiff groups, eGFRcr-cys was more accurate than either eGFRcr or eGFRcys. These results were largely consistent across age, sex, race, and body mass index.
UNASSIGNED: Few participants with major comorbid conditions.
UNASSIGNED: Discordant eGFRcr and eGFRcys are common. eGFR using the combination of creatinine and cystatin C provides the most accurate estimates among persons with discordant eGFRcr or eGFRcys.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection results in coagulation activation although it is usually not associated with consumption coagulopathy. D-dimers are also commonly elevated despite systemic hypofibrinolysis. To understand these unusual features of coronavirus disease 2019 (COVID-19) coagulopathy, 64 adult patients with SARS-CoV-2 infection (36 moderate and 28 severe) and 16 controls were studied. We evaluated the repertoire of plasma protease inhibitors (Serpins, Kunitz, Kazal, Cystatin-like) targeting the fibrinolytic system: Plasminogen Activator Inhibitor-1 (PAI-1), Tissue Plasminogen Activator/Plasminogen Activator Inhibitor-1 complex (t-PA/PAI-1), α-2-Antiplasmin, Plasmin-α2-Antiplasmin Complex, Thrombin-activatable Fibrinolysis Inhibitor (TAFI)/TAFIa, Protease Nexin-1 (PN-1), and Neuroserpin (the main t-PA inhibitor of the central nervous system). Inhibitors of the common (Antithrombin, Thrombin-antithrombin complex, Protein Z [PZ]/PZ inhibitor, Heparin Cofactor II, and α2-Macroglobulin), Protein C ([PC], Protein C inhibitor, and Protein S), contact (Kallistatin, Protease Nexin-2/Amyloid Beta Precursor Protein, and α-1-Antitrypsin), and complement (C1-Inhibitor) pathways, in addition to Factor XIII, Histidine-rich glycoprotein (HRG) and Vaspin were also investigated by enzyme-linked immunosorbent assay. The association of these markers with disease severity was evaluated by logistic regression. Pulmonary expression of PAI-1 and Neuroserpin in the lungs from eight post-mortem cases was assessed by immunohistochemistry. Results show that six patients (10%) developed thrombotic events, and mortality was 11%. There was no significant reduction in plasma anticoagulants, in keeping with a compensated state. However, an increase in fibrinolysis inhibitors (PAI-1, Neuroserpin, PN-1, PAP, and t-PA/PAI-1) was consistently observed, while HRG was reduced. Furthermore, these markers were associated with moderate and/or severe disease. Notably, immunostains demonstrated overexpression of PAI-1 in epithelial cells, macrophages, and endothelial cells of fatal COVID-19, while Neuroserpin was found in intraalveolar macrophages only. These results imply that the lungs in SARS-CoV-2 infection provide anti-fibrinolytic activity resulting in a shift toward a local and systemic hypofibrinolytic state predisposing to (immuno)thrombosis, often in a background of compensated disseminated intravascular coagulation.

The poultry red mite (PRM; Dermanyssus gallinae) is a hematophagous ectoparasite that mainly infests chickens, and its infestation causes significant economic losses to the poultry industry. In this study, we examined the use of RNAscope-based in situ hybridization (ISH) to characterize gene expression in PRM. We analyzed the mRNA expression of Dermanyssus gallinae cathepsin D-1 (Dg-CatD-1) and Dermanyssus gallinae cystatin (Dg-Cys). RNAscope ISH analysis revealed that mRNA expression of Dg-CatD-1 was observed in the digestive tract, and Dg-Cystatin mRNA was expressed in the ovaries in addition to the digestive tract. RNAscope ISH could be applicable for the analysis of gene expression in each tissue of PRM and is an effective method to investigate the characteristics of target genes.

UNASSIGNED: The industrial yeast Pichia pastoris is widely used as a cell factory to produce proteins, chemicals and advanced biofuels. We have previously constructed P. pastoris strains that overexpress protein disulfide isomerase (PDI), which is a kind of molecular chaperone that can improve the expression of an exogenous protein when they are co-expressed. Chicken cystatin (cC) is a highly thermostable cysteine protease inhibitor and a homologous protein of human cystatin C (HCC). Wild-type cC and the two mutants, I66Q and ΔW (a truncated cC lacking the á-helix 2) represent proteins with different degrees of stability.
UNASSIGNED: Wild-type cC, I66Q and ΔW were each overexpressed in P. pastoris without and with the coexpression of PDI and their extracellular levels were determined and compared. Transcriptomic profiling was performed to compare the changes in the main signaling pathways and cell components (other than endoplasmic reticulum quality control system represented by molecular chaperones) in P. pastoris in response to intracellular folding stress caused by the expression of exogenous proteins with different stabilities. Finally, hub genes hunting was also performed.
UNASSIGNED: The coexpression of PDI was able to increase the extracellular levels of both wild-type cC and the two mutants, indicating that overexpression of PDI could prevent the misfolding of unstable proteins or promote the degradation of the misfolded proteins to some extent. For P. pastoris cells that expressed the I66Q or ΔW mutant, GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses of the common DEGs in these cells revealed a significant upregulation of the genes involved in protein processing, but a significant downregulation of the genes enriched in the Ribosome, TCA and Glycolysis/Gluconeogenesis pathways. Hub genes hunting indicated that the most downregulated ribosome protein, C4QXU7 in this case, might be an important target protein that could be manipulated to increase the expression of foreign proteins, especially proteins with a certain degree of instability.
UNASSIGNED: These findings should shed new light on our understanding of the regulatory mechanism in yeast cells that responds to intracellular folding stress, providing valuable information for the development of a convenient platform that could improve the efficiency of heterologous protein expression in P. pastoris.

Non-invasive biomarker analysis has made repetitive and painless sampling over time possible. Exosomes are being released from a parent cell and their cargo mirrors the cell micromilieu of the parent cell. Therefore, exosomes are promising surrogates for their parent cells. That is also why exosomes provide an improved signal-to-noise ratio. Current studies have identified valid non-invasive biomarkers that may be able to monitor disease severity. Exosomes are suggested to play an important role in interepithelial communication and are suggested to play a role in the initiation and maintenance of inflammation in CRS. They are, however, also involved simultaneously in several immunological processes including immune protection and immunosuppression. As the isolation of exosomes is time-consuming their value in everyday routine diagnostics has yet to be determined.

Poultry red mite (PRM; Dermanyssus gallinae) is a hazardous, blood-sucking ectoparasite of birds that constitutes a threat to poultry farming worldwide. Acaricides, commonly used in poultry farms to prevent PRMs, are not effective because of the rapid emergence of acaricide-resistant PRMs. However, vaccination may be a promising strategy to control PRM. We identified a novel cystatin-like molecule in PRMs: Dg-Cys. Dg-Cys mRNA expression was detected in the midgut and ovaries, in all stages of life. The PRM nymphs that were artificially fed with the plasma from chickens that were immunized with Dg-Cys in vitro had a significantly reduced reproductive capacity and survival rate. Moreover, combination of Dg-Cys with other antigen candidates, like copper transporter 1 or adipocyte plasma membrane-associated protein, enhanced vaccine efficacies. vaccination and its application as an antigen for cocktail vaccines could be an effective strategy to reduce the damage caused by PRMs in poultry farming.