Análisis inmunohistoquímico de MMP-1, MMP-2 y MMP-9 en ameloblastoma convencional / Immunohistochemical analysis of MMP-1, MMP-2, MMP-9 in conventional ameloblastoma

Introducción: las metaloproteinasas son enzimas que participan en la remodelación tisular y su función se relaciona con procesos fisiológicos y patológicos, como la invasión y la metástasis. El ameloblastoma convencional (AMC) es una neoplasia epitelial benigna odontogénica intraósea caracterizada por una progresión lenta y localmente invasiva, cuyo crecimiento se ha vinculado con el recambio ósea y la remodelación de la matriz extracelular. El objetivo del presente trabajo fue determinar la presencia inmunohistoquímica de MMP-1, MMP-2 y MMP-9 en el AMC. Material y métodos: se realizó un estudio piloto observacional analítico utilizando cinco muestras de AMC. Los especímenes fueron recolectados aleatoriamente del archivo del Departamento de Patología Oral y Maxilofacial, de la Escuela Nacional de Estudios Superiores (ENES) Unidad León, UNAM. Como grupo control se emplearon dos especímenes de folículo dental, obtenido de pacientes con indicación de su extracción por motivos ortodóncicos. Se realizó la técnica de inmunohistoquímica por peroxidasa, recolectando el nivel y proporción de inmunoexpresión de manera semicuantitativa. Resultados: cuatro pacientes fueron de género masculino y uno femenino, la edad promedio fue de 40.6 ± 14.9 años. Todas las muestras fueron obtenidas de la región mandibular posterior. Se observaron dos especímenes con patrón folicular y tres con plexiforme. Las MMP-2 y MMP-9 se detectaron sólo en uno de los cinco especímenes y únicamente en el parénquima de la lesión, con una proporción de 100%. Conclusión: según nuestro análisis inmunohistoquímico, las MMP-2 y MMP-9 son las metaloproteinasas que presentaron expresión positiva dentro de la patogénesis del AMC comparado a la MMP-1; no obstante, es necesario realizar este tipo de estudios en una población mayor (AU)

Introduction: metalloproteinases are enzymes involved in tissue remodeling and their function is related to physiological and pathological processes, such as invasion and metastasis. These enzymes are capable of degrading components of the extracellular matrix, which may promote tumor progression. Conventional ameloblastoma (CA) is described as a benign intraosseous epithelial odontogenic neoplasm characterized by a slow and locally invasive progression, whose growth has been linked to bone turnover and extracellular matrix remodeling. The aim of the present work was to determine the immunohistochemical presence of MMP-1, MMP-2 and MMP-9 in CA. Material and methods: an analytical observational pilot study was performed using 5 CA, randomly collected from the archive of the Department of Oral and Maxillofacial Pathology, Escuela Nacional de Estudios Superiores (ENES) Unidad León, UNAM. The control group used were two dental follicle samples, obtained from patients with extraction indication for orthodontic treatment. The peroxidase immunohistochemistry assay was performed, collecting semiquantitatively level and proportion of immunoexpression. Results: four patients were male and one female, the average age was 40.6 ± 14.9 years. All specimens were obtained from the posterior mandibular region. Two specimens were observed with follicular pattern and three with plexiform pattern. MMP-2 and MMP-9 were detected only in one of the five specimens, with presence in the parenchyma of the lesion, with a proportion of 100% of the cell analyzed. Conclusion: according to our immunohistochemical analysis, MMP-2 and MMP-9 are the metalloproteinases that presented positive expression within the pathogenesis of CA compared to MMP-1; however, it is necessary to perform this type of studies in a larger population (AU)

MMP-9/BDNF ratio predicts more severe COVID-19 outcomes.

COVID-19 clinically manifests from asymptomatic to the critical range. Immune response provokes the pro-inflammatory interactions, which lead to the cytokines, reactive oxygen/nitrogen species, peptidases, and arachidonic acid metabolites enlargement and activation of coagulation components. Matrix metalloproteinases (MMPs) contribute to tissue destruction in the development of COVID-19. Due to the endothelial, systemic course of the disease, VEGF A participates actively in COVID-19 development, while neurotrophic and metabolic effects of BDNF recommends for the prediction of complications in COVID-19 patients. Searching for a marker that would improve and simplify the ranking in COVID-19, the study intended to evaluate the relationship of MMP-9 with VEGF A, BDNF, and MMP-8 with the COVID-19 severity. Upon admission to the hospital and before the therapy administration, 77 patients were classified into a mild, moderate, severe, or critical group. Due to the inflammatory stage in COVID-19, a comparison between groups showed related differences in leukocytes, neutrophils, lymphocytes, and platelets counts as anticipated. Only in seriously ill patients, there is a significant increase in the serum concentration of MMP-9, MMP-8, and VEGF A, while BDNF values did not show significant variations between groups. However, all those parameters positively correlated with each other. The ratio of MMP-9/BDNF markedly decreased in the severe and critically patients compared to the mild group. Testing the capability of this ratio to predict the COVID-19 stage by ROC curves, we found the MMP-9/BDNF could be a suitable marker for differentiating stages I/II (AUC 0.7597), stage I/III (AUC 0.9011), and stage I/IV (AUC 0.7727). Presented data describe for the first time the high-level systemic MMP-9/BDNF ratio in patients with COVID-19. This parameter could contribute to a more precise determination of the phase of the disease.

Optimization strategies for expression of a novel bifunctional anti-PD-L1/TGFBR2-ECD fusion protein.

The novel anti-PD-L1/TGFBR2-ECD fusion protein (BR102) comprises an anti-PD-L1 antibody (HS636) which is fused at the C terminus of the heavy chain to a TGF-ß1 receptor Ⅱ ectodomain (TGFBR2-ECD), and which can sequester the PD-1/PD-L1 pathway and TGF-ß bioactivity in the immunosuppressive tumor microenvironment. In the expression of TGFBR2-ECD wild-type fused protein (BR102-WT), a 50 kDa clipped species was confirmed to be induced by proteolytic cleavage at a "QKS" site located in the N-terminus of the ectodomain, which resulted in the formation of IgG-like clipping. The matrix metalloproteinase-9 was determined to be associated with BR102-WT digestion. In addition, it was observed that the N-glycosylation modifications of the fusion protein were tightly involved in regulating proteolytic activity and the levels of cleavage could be significantly suppressed by MMP-inhibitors. To avoid proteolytic degradation, eliminating protease-sensitive amino acid motifs and introducing potential glycosylation were performed. Three sensitive motifs were mutated, and the levels of clipping were strongly restrained. The mutant candidates exhibited similar binding affinities to hPD-L1 and hTGF-ß1 as well as highly purified BR102-WT2. Furthermore, the mutants displayed more significant proteolytic resistance than that of BR102-WT2 in the lysate incubation reaction and the plasma stability test. Moreover, the bifunctional candidate Mu3 showed an additive antitumor effect in MC38/hPD-L1 bearing models as compared to that of with anti-PD-L1 antibody alone. In conclusion, in this study, the protease-sensitive features of BR102-WT were well characterized and efficient optimization was performed. The candidate BR102-Mutants exhibited advanced druggability in drug stability and displayed desirable antitumor activity.

Myeloid-Derived Suppressor Cells Mediate T Cell Dysfunction in Nonhuman Primate TB Granulomas.

Myeloid-derived suppressor cells (MDSCs) represent an innate immune cell population comprised of immature myeloid cells and myeloid progenitors with very potent immunosuppressive potential. MDSCs are reported to be abundant in the lungs of active tuberculosis (TB) patients. We sought to perform an in-depth study of MDSCs during latent TB infection (LTBI) and active TB (ATB) using the nonhuman primate (NHP) model of pulmonary TB. We found a higher proportion of granulocytic, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in the lungs of ATB animals compared to those with LTBI or naive control animals. Active disease in the lung, but not LTBI, was furthermore associated with higher proliferation, expansion, and immunosuppressive capabilities of PMN-MDSCs, as shown by enhanced expression of Ki67, indoleamine 2,3-dioxygenase (IDO1), interleukin-10 (IL-10), matrix metallopeptidase 9 (MMP-9), inducible nitric oxide synthase (iNOS), and programmed death-ligand 1 (PD-L1). These immunosuppressive PMN-MDSCs specifically localized to the lymphocytic cuff at the periphery of the granulomas in animals with ATB. Conversely, these cells were scarcely distributed in interstitial lung tissue and the inner core of granulomas. This spatial regulation suggests an important immunomodulatory role of PMN-MDSCs by restricting T cell access to the TB granuloma core and can potentially explain dysfunctional anti-TB responses in active granuloma. Our results raise the possibility that the presence of MDSCs can serve as a biomarker for ATB, while their disappearance can indicate successful therapy. Furthermore, MDSCs may serve as a potential target cell for adjunctive TB therapy. IMPORTANCE Myeloid cells are immunocytes of innate origin that orchestrate the first response toward pathogens via immune surveillance (uptake and killing), antigen presentation, and initiation of adaptive immunity by T cell stimulation. However, MDSCs are a subset of innate immunocytes that deviate to an immunoregulatory phenotype. MDSCs possess strong immunosuppressive capabilities that are induced in autoimmune, malignant neoplastic, and chronic inflammatory diseases. Induction of MDSCs has been found in peripheral blood, bronchoalveolar lavage (BAL) fluid, and pleural effusions of active TB patients, but their precise localization in lung tissue and in TB granulomas remains unclear due to challenges associated with sampling lungs and granulomas from active TB patients. Nonhuman primates (NHPs) are an important animal model with TB granulomas that closely mimic those found in humans and can therefore be used for studies that are otherwise challenging with patient material. Herein, we study MDSC localization in the lungs of NHPs exhibiting latent and active TB. Our findings reveal that MDSCs localize and exert their immunosuppressive roles at the periphery rather than in the core of TB granulomas.

Disrupted homeostasis of synovial hyaluronic acid and its associations with synovial mast cell proteases of rheumatoid arthritis patients and collagen-induced arthritis rats.

Hyaluronic acid (HA) is the main component of the extracellular matrix (ECM) of joints, and it is important for a lubricating joint during body movement. Degradation is the main metabolic process of HA in vivo. Hyaluronidases (HAase) were known for HA degradation. The inflammation-induced HA rapid-metabolism can reduce HA viscosity and concentration in joints. Mast cells (MC) containing their specific proteases were found in synovium tissue. It is unclear if MC-proteases could be involved in HA degradation pathways. This study aims to explore the correlations between HA concentration vs mast cell proteases, or matrix metalloproteinase-2/9 (MMP-2/9) and to investigate the association of MC-specific proteases with disrupted synovial HA homeostasis in rheumatoid arthritis (RA) or collagen-induced arthritis rats. The synovial fluid samples from no-RA and RA patients were collected; the collagen-induced arthritis (CIA) rat model was established; HA concentration and the activities of MC-protease and MMP-2/9 in the samples were detected, and the correlations were analyzed. In vitro interaction experiment was carried out by mixing MC-proteases with HA to observe the degradation speed. The HA concentrations in synovial fluids were decreased in RA patients and CIA rats compared with those in no-RA subjects or normal rats respectively. The activities of mast cell proteases in synovial fluids were increased and positively correlated with MMP-9, but negatively correlated with HA concentrations. In vitro study, the addition of MC-chymase and tryptase promoted the speed in HA degradation. MC-proteases may influence HA degradation pathway.

Expression of soluble recombinant human matrix metalloproteinase 9 and generation of its monoclonal antibody.

We have successfully produced a recombinant human matrix metalloproteinase 9 (hMMP9) antigen with high yield and purity and used it to generate a hybridoma cell-culture-based monoclonal anti-hMMP9 antibody. We selected the most effective antibody for binding antigens and successfully identified its nucleotide sequence. The entire antigen and antibody developmental procedures described herein can be a practical approach for producing large amounts of monoclonal antibodies against hMMP9 and other antigens of interest. Additionally, the nucleotide sequence information of the anti-hMMP9 monoclonal antibody revealed herein will be useful for the generation of recombinant antibodies or antibody fragments against hMMP9.

Expression of Surface-associated 82 kDa proMMP-9 in Lymphatic Leukemia Blast Cells Differentially Correlates With Prognosis.

BACKGROUND/AIM: Matrix metalloproteinases (MMPs) degrade extracellular matrix and process regulatory proteins. Recently, a membrane-bound 82kDa variant of proMMP-9 identified on myeloid blasts was shown to be associated with prognosis. PATIENTS AND METHODS: To investigate the role of 82kDa proMMP-9 with acute lymphoblastic leukemia (ALL) and chronic lymphoid leukemia (CLL), we performed flow-cytometry analysis of expression on ALL blasts (n=18) and CLL lymphocytes (n=21) from blood and correlated data with clinical parameters. RESULTS: In ALL, mature B-linear blasts expressed higher levels of 82kDa proMMP-9 compared to T-linear blasts. Elevated levels of 82kDa proMMP-9 were found in elderly patients and at patients with relapse. No correlation was observed on blood cells and extramedullary disease. In CLL, the 82kDa proMMP-9 expression did not correlate with any of the clinical parameters. CONCLUSION: Our findings suggest that higher levels of 82kDa proMMP-9 expression on blast cells may correlate with a more unfavorable ALL-subtype. Further studies are required to clarify the prognostic role of the 82kDa pro-MMP-9 expression.

Dual interleukin-17A/F deficiency protects against acute and chronic response to cigarette smoke exposure in mice.

IL-17A and IL-17F are both involved in the pathogenesis of neutrophilic inflammation observed in COPD and severe asthma. To explore this, mice deficient in both Il17a and Il17f and wild type (WT) mice were exposed to cigarette smoke or environmental air for 5 to 28 days and changes in inflammatory cells in bronchoalveolar lavage (BAL) fluid were determined. We also measured the mRNA expression of keratinocyte derived chemokine (Kc), macrophage inflammatory protein-2 (Mip2), granulocyte-macrophage colony stimulating factor (Gmcsf) and matrix metalloproteinase-9 (Mmp9 ) in lung tissue after 8 days, and lung morphometric changes after 24 weeks of exposure to cigarette smoke compared to air-exposed control animals. Macrophage counts in BAL fluid initially peaked at day 8 and again on day 28, while neutrophil counts peaked between day 8 and 12 in WT mice. Mice dual deficient with Il17a and 1l17f showed similar kinetics with macrophages and neutrophils, but cell numbers at day 8 and mRNA expression of Kc, Gmcsf and Mmp9 were significantly reduced. Furthermore, airspaces in WT mice became larger after cigarette smoke exposure for 24 weeks, whereas this was not seen dual Il17a and 1l17f deficient mice. Combined Il17a and Il17f deficiency resulted in significant attenuation of neutrophilic inflammatory response and protection against structural lung changes after long term cigarette smoke exposure compared with WT mice. Dual IL-17A/F signalling plays an important role in pro-inflammatory responses associated with histological changes induced by cigarette smoke exposure.

Human osteoclastogenesis in Epstein-Barr virus-induced erosive arthritis in humanized NOD/Shi-scid/IL-2Rγnull mice.

Many human viruses, including Epstein-Barr virus (EBV), do not infect mice, which is challenging for biomedical research. We have previously reported that EBV infection induces erosive arthritis, which histologically resembles rheumatoid arthritis, in humanized NOD/Shi-scid/IL-2Rγnull (hu-NOG) mice; however, the underlying mechanisms are not known. Osteoclast-like multinucleated cells were observed during bone erosion in this mouse model, and therefore, we aimed to determine whether the human or mouse immune system activated bone erosion and analyzed the characteristics and origin of the multinucleated cells in hu-NOG mice. Sections of the mice knee joint tissues were immunostained with anti-human antibodies against certain osteoclast markers, including cathepsin K and matrix metalloproteinase-9 (MMP-9). Multinucleated cells observed during bone erosion stained positively for human cathepsin K and MMP-9. These results indicate that human osteoclasts primarily induce erosive arthritis during EBV infections. Human osteoclast development from hematopoietic stem cells transplanted in hu-NOG mice remains unclear. To confirm their differentiation potential into human osteoclasts, we cultured bone marrow cells of EBV-infected hu-NOG mice and analyzed their characteristics. Multinucleated cells cultured from the bone marrow cells stained positive for human cathepsin K and human MMP-9, indicating that bone marrow cells of hu-NOG mice could differentiate from human osteoclast progenitor cells into human osteoclasts. These results indicate that the human immune response to EBV infection may induce human osteoclast activation and cause erosive arthritis in this mouse model. Moreover, this study is the first, to our knowledge, to demonstrate human osteoclastogenesis in humanized mice. We consider that this model is useful for studying associations of EBV infections with rheumatoid arthritis and human bone metabolism.

Inflammation and Antiviral Immune Response Associated With Severe Progression of COVID-19.

Coronavirus disease-2019 (COVID-19) is a novel respiratory disease induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It remains poorly understood how the host immune system responds to the infection during disease progression. We applied microarray analysis of the whole genome transcriptome to peripheral blood mononuclear cells (PBMCs) taken from severe and mild COVID-19 patients as well as healthy controls. Functional enrichment analysis of genes associated with COVID-19 severity indicated that disease progression is featured by overactivation of myeloid cells and deficient T cell function. The upregulation of TLR6 and MMP9, which promote the neutrophils-mediated inflammatory response, and the downregulation of SKAP1 and LAG3, which regulate T cells function, were associated with disease severity. Importantly, the regulation of these four genes was absent in patients with influenza A (H1N1). And compared with stimulation with hemagglutinin (HA) of H1N1 virus, the regulation pattern of these genes was unique in PBMCs response to Spike protein of SARS-CoV-2 ex vivo. Our data also suggested that severe SARS-CoV-2 infection largely silenced the response of type I interferons (IFNs) and altered the proportion of immune cells, providing a potential mechanism for the hypercytokinemia. This study indicates that SARS-CoV-2 infection impairs inflammatory and immune signatures in patients, especially those at severe stage. The potential mechanisms underpinning severe COVID-19 progression include overactive myeloid cells, impaired function of T cells, and inadequate induction of type I IFNs signaling.

Matrix metalloproteinases in the pathogenesis of dengue viral disease: Involvement of immune system and newer therapeutic strategies.

Globally, the burden due to dengue infection is increasing with a recent estimate of 96 million progressing to the disease every year. Dengue pathogenesis and the factors influencing it are not completely known. It is now widely speculated that there is an important role of matrix metalloproteinases (MMPs) in the initiation and progression of dengue pathogenesis; however, their exact roles are not fully understood. Overactivation of matrix metalloproteinases may contribute to the severity of dengue pathogenesis. Cytokines and various other mediators of inflammation interact with the vascular endothelium and matrix metalloproteinases may be one of the components among them. Extensive plasma leakage into tissue spaces may result in a shock. It is evident in the literature that MMP2 and MMP9 increase in dengue patients is correlated with the severity of the disease; however, the underlying mechanism is still unknown. Activation of innate cells and adaptive immune cells which include, B and T cells, macrophages or monocytes and dendritic cells also contribute to the dengue pathology. Newer therapeutic strategies include microRNAs, such as miR-134 (targets MMP3 and MMP1) and MicroRNA-320d, (targets MMP/TIMP proteolytic system). The use of antibodies-based therapeutics like (Andecaliximab; anti-matrix metalloproteinase-9 antibody) is also suggested against MMPs in dengue. In this review, we summarize some recent developments associated with the involvement of immune cells and their mediators associated with the matrix metalloproteinases mediated dengue pathogenesis. We highlight that, there is still very little knowledge about the MMPs in dengue pathogenesis which needs attention and extensive investigations.

Local Triamcinolone Treatment Affects Inflammatory Response in Seroma Exudate of Abdominoplasty Patients: A Randomized Controlled Trial.

BACKGROUND: As the leading complication of abdominoplasty, seroma formation might represent an inflammatory process in response to surgical trauma. This prospective randomized trial investigated whether local administration of the antiinflammatory agent triamcinolone could prevent seroma accumulation. METHODS: Weekly and cumulative seroma volumes were compared between the study groups A, B, and C over a 4-week follow-up (group A, with drain, without triamcinolone; group B, without drain, without triamcinolone; group C, without drain, with triamcinolone). Aspirated seroma samples were analyzed by enzyme-linked immunosorbent assay for selective inflammatory mediators. RESULTS: Triamcinolone significantly reduced cumulative seroma volume (n = 60; mA 845 ± SDA 578 ml, mC 236 ± SDC 381 ml, p = 0.001). The most accentuated suppressive effect of triamcinolone was observed shortly after the treatment (week 1) (mA1 616 ± SDA1 457 ml, mB1 153 ± SDB1 161 ml, mC1 22 ± SDC1 44 ml, pA1/C1 < 0.001, pB1/C1 = 0.014). Local triamcinolone administration resulted in a differential concentration of interleukin-6 (IL-6) and matrix metalloproteinase-9 (MMP-9 (week 1) in seroma exudate as measured by enzyme-linked immunosorbent assay (mIL-6A1 1239 ± SDA1 59 pg/ml, mIL-6C1 848 ± SDC1 80 pg/ml, p < 0.001; mMMP-9A1 2343 ± SDA1 484 pg/ml, mMMP-9C1 376 ± SDC1 120 pg/ml, p = 0.001). CONCLUSIONS: Local administration of 80 mg of triamcinolone reduced postabdominoplasty seroma accumulation significantly. Under triamcinolone treatment, suppressed levels of IL-6 and MMP-9 in seroma fluid were observed. Notably, inflammatory marker suppression correlated clinically with a decrease in seroma accumulation. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, II.

Effects of a gestational level of estradiol on cellular transition, migration, and inflammation in cervical epithelial and stromal cells.

PROBLEM: Estrogen (E2) is one of the main steroid hormones associated with pregnancy and parturition. High levels of E2 increase uterine contractions, promote fetal membrane weakening, and induce degradation of the cervical extracellular matrix (ECM). Current evidence supports the role of E2 in epithelial-to-mesenchymal transition (EMT) and inflammation in different cell types; however, its effects on the cellular components of the cervix are still unknown. METHOD OF STUDY: In this study, we assessed the effects of gestational levels of E2 in: (a) the cellular transition of endocervical epithelial cells (EEC) and cervical stromal cells (CSC) in vitro using immunocytochemical staining and Western blot analyses for EMT markers (cytokeratin-18, E-cadherin, N-cadherin, SNAIL, and vimentin); (b) cell migration using in vitro scratch assays; (c) inflammatory cytokine (interleukin 1ß and TNF-α) and MMP9 production under untreated and lipopolysaccharide (LPS)-treated conditions using immunoassays. RESULTS: E2 treatment and co-treatment with LPS as a proxy for infection maintained the metastate of EEC (expression of both cytokeratin and vimentin) and the mesenchymal state of CSC. E2 delayed wound healing, which mimics the tissue remodeling process, in EEC and CSC. E2 led to persistently elevated levels of vimentin throughout the EEC wound healing process. E2 did not affect inflammatory cytokine production by EEC and CSC but increased MMP9 production by EEC. CONCLUSION: Collectively, these results show that third trimester levels of E2 may permit localized inflammation, increase MMP-9 production, and cause an EMT-mediated impairment of the remodeling process in the cervix in vitro. These data suggest a potential contribution of E2 in cervical ripening.

The role of IL-10 in immune responses against Pseudomonas aeruginosa during acute lung infection.

Pseudomonas aeruginosa is considered an opportunistic pathogen of great clinical importance. The clearance of this bacterium occurs through recognition of the pathogen by innate immune system receptors, leading to a lung inflammatory response. However, this response must be controlled via immunoregulatory pathways. In this study, we evaluate the role of endogenous murine IL-10 after acute infection with the virulent strain P. aeruginosa PA14. To assess the role of IL-10, intratracheal infection with the PA14 strain was performed in C57BL/6 or IL-10 KO mice. The PA14 strain was recovered in both types of animals, although IL-10 KO mice presented a higher number of viable bacteria in the lung when compared to the C57BL/6 group. Histopathological and stereological analyses showed that IL-10 KO mice had higher tissue damage and inflammatory infiltrate when compared to control animals. The activity of MMP-9 but not MMP-2, as well as IL-6 and TNF-α expression, were augmented in the lungs of infected animals and was much more evident in IL-10 KO animals when compared to the other analyzed groups. This work indicates that endogenous IL-10 control P. aeruginosa infection, the expression of pro-inflammatory genes, MMP-9 activity and histopathological processes of the infectious process in question.

Phenotypic and functional characterization of first-trimester human placental macrophages, Hofbauer cells.

Hofbauer cells (HBCs) are a population of macrophages found in high abundance within the stroma of the first-trimester human placenta. HBCs are the only fetal immune cell population within the stroma of healthy placenta. However, the functional properties of these cells are poorly described. Aligning with their predicted origin via primitive hematopoiesis, we find that HBCs are transcriptionally similar to yolk sac macrophages. Phenotypically, HBCs can be identified as HLA-DR-FOLR2+ macrophages. We identify a number of factors that HBCs secrete (including OPN and MMP-9) that could affect placental angiogenesis and remodeling. We determine that HBCs have the capacity to play a defensive role, where they are responsive to Toll-like receptor stimulation and are microbicidal. Finally, we also identify a population of placenta-associated maternal macrophages (PAMM1a) that adhere to the placental surface and express factors, such as fibronectin, that may aid in repair.

An immune-based biomarker signature is associated with mortality in COVID-19 patients.

Immune and inflammatory responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contribute to disease severity of coronavirus disease 2019 (COVID-19). However, the utility of specific immune-based biomarkers to predict clinical outcome remains elusive. Here, we analyzed levels of 66 soluble biomarkers in 175 Italian patients with COVID-19 ranging from mild/moderate to critical severity and assessed type I IFN-, type II IFN-, and NF-κB-dependent whole-blood transcriptional signatures. A broad inflammatory signature was observed, implicating activation of various immune and nonhematopoietic cell subsets. Discordance between IFN-α2a protein and IFNA2 transcript levels in blood suggests that type I IFNs during COVID-19 may be primarily produced by tissue-resident cells. Multivariable analysis of patients' first samples revealed 12 biomarkers (CCL2, IL-15, soluble ST2 [sST2], NGAL, sTNFRSF1A, ferritin, IL-6, S100A9, MMP-9, IL-2, sVEGFR1, IL-10) that when increased were independently associated with mortality. Multivariate analyses of longitudinal biomarker trajectories identified 8 of the aforementioned biomarkers (IL-15, IL-2, NGAL, CCL2, MMP-9, sTNFRSF1A, sST2, IL-10) and 2 additional biomarkers (lactoferrin, CXCL9) that were substantially associated with mortality when increased, while IL-1α was associated with mortality when decreased. Among these, sST2, sTNFRSF1A, IL-10, and IL-15 were consistently higher throughout the hospitalization in patients who died versus those who recovered, suggesting that these biomarkers may provide an early warning of eventual disease outcome.

CD28 Autonomous Signaling Orchestrates IL-22 Expression and IL-22-Regulated Epithelial Barrier Functions in Human T Lymphocytes.

IL-22 is a member of the IL-10 cytokine family involved in host protection against extracellular pathogens, by promoting epithelial cell regeneration and barrier functions. Dysregulation of IL-22 production has also frequently been observed in acute respiratory distress syndrome (ARDS) and several chronic inflammatory and autoimmune diseases. We have previously described that human CD28, a crucial co-stimulatory receptor necessary for full T cell activation, is also able to act as a TCR independent signaling receptor and to induce the expression of IL-17A and inflammatory cytokines related to Th17 cells, which together with Th22 cells represent the main cellular source of IL-22. Here we characterized the role of CD28 autonomous signaling in regulating IL-22 expression in human CD4+ T cells. We show that CD28 stimulation in the absence of TCR strongly up-regulates IL-22 gene expression and secretion. As recently observed for IL-17A, we also found that CD28-mediated regulation of IL-22 transcription requires the cooperative activities of both IL-6-activated STAT3 and RelA/NF-κB transcription factors. CD28-mediated IL-22 production also promotes the barrier functions of epithelial cells by inducing mucin and metalloproteases expression. Finally, by using specific inhibitory drugs, we also identified CD28-associated class 1A phosphatidylinositol 3-kinase (PI3K) as a pivotal mediator of CD28-mediated IL-22 expression and IL-22-dependent epithelial cell barrier functions.

Prevention of Progression of Aortic Aneurysm by Peptide Vaccine Against Ang II (Angiotensin II) in a Rat Model.

There is no proven medical therapy to inhibit the progression of abdominal aortic aneurysm (AAA) in the clinical setting. To develop a novel therapeutic approach for the treatment of AAA, we focused on vaccination targeting Ang II (angiotensin II) and assessed the effect of an Ang II peptide vaccine on the progression of AAA using a rat model. Ang II peptide was conjugated with KLH (keyhole limpet hemocyanin) carrier protein to induce a sufficient immune response. Male rats were subcutaneously immunized with Ang II-KLH with an adjuvant on days 0, 14, and 28. Aortic dilatation was induced by intraluminal incubation with elastase on day 35. Treatment with Ang II vaccine successfully induced the production of a high titer of anti-Ang II antibodies. Immunization with Ang II vaccine resulted in a significant reduction in expansion of the aortic diameter compared with control rats, without a blood pressure-lowering effect. Four weeks after operation, the increase in Ang II in the aneurysm wall was significantly inhibited by treatment with Ang II vaccine. Inhibition of Ang II action led to suppression of the inflammatory response in the AAA wall through attenuation of the NFκB (nuclear factor kappa B) and c-jun N-terminal kinase signaling cascades. Treatment with Ang II vaccine inhibited accumulation of macrophages in the AAA wall. In addition, expression of TNF-α (tumor necrosis factor alpha) and activation of MMP (matrix metalloproteinase)-2 and MMP-9 were also inhibited by treatment with Ang II vaccine, resulting in protection against the destruction of elastic fibers. This vaccine therapy could become a potent therapeutic option to treat patients with AAA.

A novel serine protease inhibitor as potential treatment for dry eye syndrome and ocular inflammation.

Dry eye syndrome (DES), a multifactorial disorder which leads to ocular discomfort, visual disturbance and tear film instability, has a rising prevalence and limited treatment options. In this study, a newly developed trypsin-like serine protease inhibitor (UAMC-00050) in a tear drop formulation was evaluated to treat ocular inflammation. A surgical animal model of dry eye was employed to investigate the potential of UAMC-00050 on dry eye pathology. Animals treated with UAMC-00050 displayed a significant reduction in ocular surface damage after evaluation with sodium fluorescein, compared to untreated, vehicle treated and cyclosporine-treated animals. The concentrations of IL-1α and TNF-α were also significantly reduced in tear fluid from UAMC-00050-treated rats. Additionally, inflammatory cell infiltration in the palpebral conjunctiva (CD3 and CD45), was substantially reduced. An accumulation of pro-MMP-9 and a decrease in active MMP-9 were found in tear fluid from animals treated with UAMC-00050, suggesting that trypsin-like serine proteases play a role in activating MMP-9 in ocular inflammation in this animal model. Comparative qRT-PCR analyses on ocular tissue indicated the upregulation of tryptase, urokinase plasminogen activator receptor (uPAR) and protease-activated receptor 2 (PAR2). The developed UAMC-00050 formulation was stable up to 6 months at room temperature in the absence of light, non-irritating and sterile with compatible pH and osmolarity. These results provide a proof-of-concept for the in vivo modifying potential of UAMC-00050 on dry eye pathology and suggest a central role of trypsin-like serine proteases and PAR2 in dry eye derived ocular inflammation.

In vitro validation of the tear matrix metalloproteinase 9 in-situ immunoassay.

We aimed to validate a tear MMP-9 in-situ immunoassay (InflammaDry) and to identify factors that could affect results or interpretation. Three factors were examined: sample concentration, volume, and time. Recombinant human (rh) MMP-9 (10 or 20 µl; 0, 12.5, 25, 50, 100, 200, 500, and 1,000 ng/ml) was applied to the kit and the detection limit and assay reproducibility were examined. At a rhMMP-9 volume of 10 µl (≥ 50 ng/ml), all positive results were identified by densitometry at 10 and 20 min; however, after 20 min, more than half of the nine ophthalmologists interpreted a positive result. At a rhMMP-9 volume of 20 µl (≥ 25 ng/ml), ophthalmologists and densitometry identified almost all test lines at 10 and 20 min. At 10 µl, densitometry showed a linear dose-response pattern. At 20 µl, densitometry showed a linear dose-response pattern at concentrations up to 500 ng/ml; however, full saturation was achieved at concentrations ≥ 500 ng/ml. When the same amount of rhMMP-9 was applied, the density result increased significantly upon doubling of the solvent volume (i.e., by adding the same volume of PBS to a sample). InflammaDry showed a high inter- and intra-assay coefficient of variation at 10 min (28.4% and 24.7%, respectively). The results of the MMP-9 in-situ immunoassay varied significantly depending on sample volume. Therefore, when interpreting the results, careful attention must be paid to tear volume.