OBJECTIVE: The purpose of this study was to investigate the dynamic changes in serum (1-3)-β-D-glucan (BDG) caused by intravenous immunoglobulins (IVIG) infusion in adults.
METHODS: This study included patients who received IVIG infusion from October 2021 to October 2022 during hospitalization. We randomly examined two IVIG samples for every patient. Serum samples were collected at nine time points: before (Tpre), immediately (T1-0), 6h (T1-1) and 12h (T1-2) later on the first day; immediately (T2-0) and six hours later (T2-1) on the second day during IVIG infusion, and within three days after IVIG infusion (Ta1, Ta2, and Ta3, respectively). The Friedman test was used for statistical analysis.
RESULTS: A total of 159 serum BDG from 19 patients were included in the analysis. The BDG content of IVIG ranged from 249 pg/ml to 4812 pg/ml. Patients had significantly elevated serum BDG on T1-0 (176 (113, 291) pg/ml, p = 0.002) and Ta1 (310 (199, 470) pg/ml, p < 0.001), compared with Tpre (41 (38, 65) pg/ml). The increments of serum BDG (ΔBDG) were associated with BDG concentration of IVIG (Spearman r = 0.59, p = 0.02). Individuals with abnormal renal function indexes showed higher serum ΔBDG values at Ta1 (403 (207, 484) pg/ml) than patients with normal renal function (172 (85, 316) pg/ml, p = 0.036).
CONCLUSIONS: Patients who received IVIG had significantly higher serum BDG values. Elevated BDG levels correlate with BDG content of IVIG and abnormal renal function indexes.

The process of neovascularization during cell-based pulp regeneration is difficult to study. Here we developed a tube model that simulates root canal space and allows direct visualization of the vascularization process in vitro. Endothelial-like cells (ECs) derived from guiding human dental pulp stem cells (DPSCs) into expressing endothelial cell markers CD144, vWF, VEGFR1, and VEGFR2 were used. Human microvascular endothelial cells (hMVECs) were used as a positive control. DPSC-ECs formed tubules on Matrigel similar to hMVECs. Cells were mixed in fibrinogen/thrombin or mouse blood and seeded into wells of 96-well plates or injected into a tapered plastic tube (14 mm in length and 1 or 2 mm diameter of the apex opening) with the larger end sealed with MTA to simulate root canal space. Cells/gels in wells or tubes were incubated for various times in vitro and observed under the microscope for morphological changes. Samples were then fixed and processed for histological analysis to determine vessel formation. Vessel-like networks were observed in culture from 1 to 3 d after cell seeding. Cells/gels in 96-well plates were maintained up to 25 d. Histologically, both hMVECs and DPSC-ECs in 96-well plates or tubes showed intracellular vacuole formation. Some cells showed merged large vacuoles indicating the lumenization. Tubular structures were also observed resembling blood vessels. Cells appeared healthy throughout the tube except some samples (1 mm apical diameter) in the coronal third. Histological analysis also showed pulp-like soft tissue throughout the tube samples with vascular-like structures. hMVECs formed larger vascular lumen size than DPSC-ECs while the latter tended to have more lumen and tubular structure counts. We conclude that DPSC-ECs can form vascular structures and sustained in the 3-dimensional fibrin gel system in vitro. The tube model appears to be a proper and simple system simulating the root canal space for vascular formation and pulp regeneration studies.

Unlike the adult meniscus, the fetal meniscus possesses robust healing capacity. The dense and stiff matrix of the adult meniscus provides a biophysical barrier for cell migration, which is not present in the fetal meniscus. Inspired by developmental characteristics, modifying the matrix of the adult meniscus into a fetal-like, loose and soft microenvironment holds opportunity to facilitate repair, especially in the avascular zone.
Modifying the dense and stiff matrix of the adult meniscus into a fetal-like, loose and soft microenvironment could enhance cell migration to the tear interface and subsequent robust healing capacity.
Controlled laboratory study.
Fresh porcine menisci were treated with hyaluronidase or collagenase. The density and arrangement of collagen fibers were assessed. The degradation of proteoglycans and collagen was evaluated histologically. Cell migration within the meniscus or the infiltration of exogenous cells into the meniscus was examined. Dendritic silica nanoparticles with relatively large pores were used to encapsulate hyaluronidase for rapid release. Mesoporous silica nanoparticles with relatively small pores were used to encapsulate transforming growth factor-beta 3 (TGF-β3) for slow release. A total of 24 mature male rabbits were included. A longitudinal vertical tear (0.5 cm in length) was prepared in the avascular zone of the medial meniscus. The tear was repaired with suture, repaired with suture in addition to blank silica nanoparticles, or repaired with suture in addition to silica nanoparticles releasing hyaluronidase and TGF-β3. Animals were sacrificed at 12 months postoperatively. Meniscal repair was evaluated macroscopically and histologically.
The gaps between collagen bundles increased after hyaluronidase treatment, while collagenase treatment resulted in collagen disruption. Proteoglycans degraded after hyaluronidase treatment in a dose-dependent manner, but collagen integrity was maintained. Hyaluronidase treatment enhanced the migration and infiltration of cells within meniscal tissue. Last, the application of fibrin gel and the delivery system of silica nanoparticles encapsulating hyaluronidase and TGF-β3 enhanced meniscal repair responses in an orthotopic longitudinal vertical tear model.
The gradient release of hyaluronidase and TGF-β3 removed biophysical barriers for cell migration, creating a fetal-like, loose and soft microenvironment, and enhanced the fibrochondrogenic phenotype of reparative cells, facilitating the synthesis of matrix and tissue integration.
Modifying the adult matrix into a fetal-like, loose and soft microenvironment via the local gradient release of hyaluronidase and TGF-β3 enhanced the healing capacity of the meniscus.

Highly organized collagen fibrils interlacing with proteoglycans form the crucial architecture of the cornea and facilitate its transparency. Corneal scarring from accidental injury, surgery, or infection alters this highly organized tissue, causing severe consequences, including blindness. There are no pharmacological or surgical methods to effectively and safely treat excessive corneal scarring. Thus, we tested the anticorneal scarring utility of a rationally designed anticollagen antibody (ACA) whose antifibrotic effects have already been demonstrated in nonocular models. Utilizing a rabbit model with an incisional corneal wound, we analyzed ACA\'s effects on forming collagen and proteoglycan-rich extracellular matrices in scar neotissue. We used microscopic and spectroscopic techniques to quantify these components and measure crucial parameters characterizing the structure and organization of collagen fibrils. Moreover, we analyzed the spatial distribution of collagen and proteoglycans in normal and healing corneas. Our study demonstrated significant changes in the quality and quantity of the analyzed molecules synthesized in scar neotissue. It showed that these changes extend beyond incision margins. It also showed ACA\'s positive impact on some crucial parameters defining proper cornea structure. This pilot study provides a stepping stone for future tests of therapeutic approaches that target corneal extracellular scar matrix assembly.

BACKGROUND: Sepsis-associated destruction of the pulmonary microvascular endothelial glycocalyx (EGCX) creates a vulnerable endothelial surface, contributing to the development of acute respiratory distress syndrome (ARDS). Constituents of the EGCX shed into circulation, glycosaminoglycans and proteoglycans, may serve as biomarkers of endothelial dysfunction. We sought to define the patterns of plasma EGCX degradation products in children with sepsis-associated pediatric ARDS (PARDS), and test their association with clinical outcomes.
METHODS: We retrospectively analyzed a prospective cohort (2018-2020) of children (≥1 month to <18 years of age) receiving invasive mechanical ventilation for acute respiratory failure for ≥72 h. Children with and without sepsis-associated PARDS were selected from the parent cohort and compared. Blood was collected at time of enrollment. Plasma glycosaminoglycan disaccharide class (heparan sulfate, chondroitin sulfate, and hyaluronan) and sulfation subtypes (heparan sulfate and chondroitin sulfate) were quantified using liquid chromatography tandem mass spectrometry. Plasma proteoglycans (syndecan-1) were measured through an immunoassay.
RESULTS: Among the 39 mechanically ventilated children (29 with and 10 without sepsis-associated PARDS), sepsis-associated PARDS patients demonstrated higher levels of heparan sulfate (median 639 ng/mL [interquartile range, IQR 421-902] vs 311 [IQR 228-461]) and syndecan-1 (median 146 ng/mL [IQR 32-315] vs 8 [IQR 8-50]), both p = 0.01. Heparan sulfate subtype analysis demonstrated greater proportions of N-sulfated disaccharide levels among children with sepsis-associated PARDS (p = 0.01). Increasing N-sulfated disaccharide levels by quartile were associated with severe PARDS (n = 9/29) with the highest quartile including >60% of the severe PARDS patients (test for trend, p = 0.04). Higher total heparan sulfate and N-sulfated disaccharide levels were independently associated with fewer 28-day ventilator-free days in children with sepsis-associated PARDS (all p < 0.05).
CONCLUSIONS: Children with sepsis-associated PARDS exhibited higher plasma levels of heparan sulfate disaccharides and syndecan-1, suggesting that EGCX degradation biomarkers may provide insights into endothelial dysfunction and PARDS pathobiology.

BACKGROUND: Histology of human oral mucosa is closely related with its function and anatomical location, and a proper characterization of the human masticatory oral mucosa could be very useful in periodontal pathology.
OBJECTIVE: In the present work, we have carried out a comprehensive study in order to determine the main histological features of parakeratinized (POM) and orthokeratinized (OOM) masticatory human oral mucosa using light and electron microscopy.
METHODS: To perform this, we have used several histological, histochemical and immunohistochemical methods to detect key markets at the epithelial, basement membrane and connective tissue levels.
RESULTS: Our results demonstrated that POM and OOM share many histological similarities, as expected. However, important differences were observed at the epithelial layer of POM, that was significantly thicker than the epithelial layer found in OOM, especially due to a higher number of cells at the stratum spinosum. The expression pattern of CK10 and filaggrin revealed intense signal expression in OOM as compared to POM. Collagen and proteoglycans were more abundant in OOM stroma than in POM. No differences were found for blood vessels and basement membrane.
CONCLUSIONS: These results may contribute to a better understanding of the pathological conditions affecting the human masticatory oral mucosa. In addition, these findings could be useful for the generation of different types of oral mucosa by tissue engineering techniques.
UNASSIGNED: Microscopical features of parakeratinized and orthokeratinized masticatory human oral mucosa showed important differences at both, epithelial and stromal levels. Parakeratinized masticatory human oral mucosa exert thicker epithelial layer, especially, at the stratum spinosum in comparison to orthokeratinized human oral mucosa. Cytokeratin 10 and filaggrin human epithelial markers were intensively expressed in orthokeratinized masticatory human oral mucosa in comparison to parakeratinized masticatory human oral mucosa. At the stromal level, orthokeratinized masticatory human oral mucosa exhibit higher levels of collagen and proteoglycans than parakeratinized masticatory oral mucosa. The deep knowledge of histological features of masticatory oral mucosa could lead to a better understanding of oral mucosa pathology and advanced treatments.

OBJECTIVE: Mandibular condylar cartilage (MCC) of the rat was examined with the Fourier-transform infrared (FITR) spectroscopic imaging to study the effects of ageing, oestrogen level and altered dietary loading on the structure of MCC.
METHODS: The Sprague-Dawley rats (n = 96) aged 5 and 14 months were divided into 12 subgroups according to age, oestrogen status (ovariectomized [OVX], non-ovariectomized [non-OVX)]) and diet (hard, normal, soft). Specimens of the MCC were examined with FTIR spectroscopic imaging to quantify the distribution of collagens and proteoglycans. MCC was divided sagittally into three segments: anterior, most superior and posterior. From each segment, the collagen and proteoglycan contents at different depths of cartilage were statistically compared between the groups using an N-way analysis of variance (ANOVA).
RESULTS: The amount of collagen content was significantly associated with old age in the deep layer of the anterior segment and in the middle layer of the posterior segment of MCC. In the deep layer of the most superior segment, the collagen content also increased with ageing. The amount of proteoglycan content increased significantly when dietary loading increased, and the oestrogen level decreased in the deep layer of the most superior segment of MCC.
CONCLUSIONS: Ageing, oestrogen level and altered dietary loading have a significant effect on the location and content of collagens and proteoglycans of rat MCC. Ageing significantly increased the amount of collagen content in the superior and posterior segments, being highest in the older soft-diet rats. Decreased oestrogen levels and increased dietary loading increased the amount of proteoglycan content.

OBJECTIVE: Beta-D-Glucan (BDG) testing has been suggested to support the diagnosis of candidemia and invasive candidiasis. The actual benefit in critically ill high-risk patients in intensive care units (ICU) has not been verified so far.
METHODS: In ICU patients receiving empirical echinocandin treatment for suspected invasive candidiasis (IC), serial BDG testing using the Fujifilm Wako Beta-Glucan Test was performed, starting on the first day of echinocandin administration and every 24-48 h afterwards. Diagnostic accuracy was determined for single testing and serial testing strategies using a range of cut-off values. In addition, we compared the added value of these testing strategies when their results were introduced as additional predictors into a multivariable logistic regression model controlling for established risk factors of IC.
RESULTS: A total of 174 ICU patients, forty-six of which (25.7%) classified as cases of IC, were included in our study. Initial BDG testing showed moderate sensitivity (74%, 95%CI 59-86%) and poor specificity (45%, 95% CI 36-54%) for IC which could hardly be improved by follow-up testing. While raw BDG values or test results obtained with very high thresholds improved the predictive performance of our multivariable logistic regression model for IC, neither single nor serial testing with the manufacturer-proposed low-level cut-off showed substantial benefit.
CONCLUSIONS: In our study of critically ill intensive care patients at high risk for candidemia or invasive candidiasis, diagnostic accuracy of BDG testing was insufficient to inform treatment decisions. Improved classification was only achieved for cases with very high BDG values.

Current experimental approaches cannot elucidate the effect of maladaptive changes on the main cartilage constituents during the degeneration process in osteoarthritis (OA). In silico approaches, however, allow creating \'virtual knock-out\' cases to elucidate these effects in a constituent-specific manner. We used such an approach to study the main mechanisms of cartilage degeneration in different mechanical loadings associated with the following OA etiologies: (1) physiological loading of degenerated cartilage, (2) injurious loading of healthy intact cartilage and (3) physiological loading of cartilage with a focal defect.
We used the recently developed Cartilage Adaptive REorientation Degeneration (CARED) framework to simulate cartilage degeneration associated with primary and secondary OA (OA cases (1)-(3)). CARED incorporates numerical description of tissue-level cartilage degeneration mechanisms in OA, namely, collagen degradation, collagen reorientation, fixed charged density loss and tissue hydration increase following mechanical loading. We created \'virtual knock-out\' scenarios by deactivating these degenerative processes one at a time in each of the three OA cases.
In the injurious loading of intact and physiological loading of degenerated cartilage, collagen degradation drives degenerative changes through fixed charge density loss and tissue hydration rise. In contrast, the two later mechanisms were more prominent in the focal defect cartilage model.
The virtual knock-out models reveal that injurious loading to intact cartilage and physiological loading to degenerated cartilage induce initial degenerative changes in the collagen network, whereas, in the presence of a focal cartilage defect, mechanical loading initially causes proteoglycans (PG) depletion, before changes in the collagen fibril network occur.

Subcellular fractionation is an introductory step in a variety of experimental approaches designed to study intracellular components, like membranes and organelle systems. Subcellular fractions enriched in membranes of the Golgi apparatus of mammalian cells have been isolated to address localization and activity of proteins, including enzymes, to study intracellular membrane transport mechanisms, and to reconstitute in vitro cellular processes associated with the Golgi apparatus. Here, I describe methods to purify Golgi membranes by subcellular fractionation, to assay nucleotide sulfate (PAPS) uptake into Golgi vesicles, and to measure sulfate incorporation into in vitro synthesized glycosaminoglycans.