[Role of intercellular adhesion molecule-1 in adhesion and migration of mesenchymal stem cells in ankylosing spondylitis and its mechanisms].

Objective: To investigate the role and underlying mechanisms of intercellular adhesion molecule-1 (ICAM-1) in the adhesion and migration of mesenchymal stem cells (MSCs) in patients with ankylosing spondylitis (AS). Methods: Bone marrow and ligament tissues were collected during surgery from patients with AS and thoracolumbar fractures (as controls, HC) treated from October 2021 to October 2022 at Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital. MSCs were isolated and cultured from the bone marrow using the Ficoll separation method. Cell morphology was observed under high-resolution microscopy, and differences in the cytoskeletal features between AS-and HC-MSCs were analyzed through immunofluorescence staining. The expression of ICAM-1 was quantified in both groups using real-time quantitative polymerase chain reaction (RT-qPCR) and flow cytometry. Transwell migration assays and wound healing experiments were conducted to evaluate the differences in migration rates between the two groups of MSCs. Results: The interspinous ligament and bone marrow was acquired in AS (2 males and 1 female; 33, 37, 32 years old, respectively) and no-AS patients (2 males and 1 female; 35, 32, 38 years old, respectively). AS-MSCs exhibited broader cell morphology compared to HC-MSCs under bright field and fluorescence microscopy. Immunofluorescence staining of the interspinous ligament showed higher expression of ICAM-1 (68.38±3.42 vs 48.31±2.43) and CD105 (37.97±2.16 vs 23.36±2.06) in AS patients (both P<0.001). Western blot and RT-qPCR analysis revealed significantly stronger protein expression and transcription levels of ICAM-1 in AS-MSCs when compared to those in HC-MSCs (both P<0.001). Flow cytometry confirmed greater mean fluorescence intensity of ICAM-1 in AS-MSCs than in that in HC-MSCs (924.30±54.99 vs 636.47±40.03, P=0.002). Regarding cell adhesion efficiency, it showed no significant difference between AS-MSCs and HC-MSCs in the early stage of adhesion (0.5 h: 1 496±213 vs 1 205±163, P=0.133), but they were all significantly higher in AS-MSCs in the later stage (1 h: 2 894±172 vs 1 908±155, P=0.002; 2 h: 4 540±286 vs 3 334±188, P=0.004; 3 h: 5 212±281 vs 4 208±303, P=0.014). Finally, cell migration experiments demonstrated a stronger migration capability of AS-MSCs compared to HC-MSCs (5 449±172 vs 4 016±155, P<0.001), and the inhibition efficiency of A-205804 on the migration rate of AS-MSCs was stronger than that on HC-MSCs (2 145±239 vs 3 539±316, P=0.004). Conclusions: The aberrant expression of ICAM-1 markedly influences the adhesion and migration dynamics of MSCs. Elevated ICAM-1 levels in MSCs derives from patients with AS significantly enhance their migratory capabilities.

[Advances in the regulation of craniomandibular regeneration by topical application of parathyroid hormone].

Parathyroid hormone (PTH) is a polypeptide molecule synthesized and secreted by parathyroid principal cells. It is an important hormone to maintain the balance of calcium and phosphorus metabolism in the body. It has the dual function of promoting bone formation and bone resorption. In clinic, it promotes osteogenesis by intermittent low-dose subcutaneous injection. In order to avoid the problems of subcutaneous injection, such as poor patient compliance, low utilization of target organs and pain at the injection site, the local application of PTH has attracted much attention in recent years. However, how to realize the local application of PTH and the effect of the local application need to be confirmed by more experiments. This article reviews the local application of PTH and the promotion of jaw regeneration in recent years, in order to provide reference for the local application and research of PTH.

[Expression of osteoprotegerin and receptor activator of nuclear factor kappa-B ligand in mandibular ramus osteotomy healing with administration of different doses of parathyroid hormone].

Objective: To investigate the effect of parathyroid hormone (PTH) on the bone healing of mandibular ramus osteotomy. Methods: The mandibular ramus osteotomy model was established in sixty rabbits and these rabbits were randomly divided into experimental group A, experimental group B and control group. In the experimental group A and experimental group B, the rabbits were given PTH (20 and 40 µg/kg respectively) every other day after operation. In the control group, 1 ml saline was given. The animals were sacrificed at 1 week, 2 weeks, 3 weeks and 4 weeks postoperatively. The new bone formation was observed by histology and cone bone CT. The expression of osteoprotegerin and receptor activator of nuclear factor kappa-B (RANKL) in the new bone was detected by real-time quantitative PCR. Results: The experimental groups has better osteogenesis and the bone mineral density than the control group in osteotomy area. The experimental group B showed the best osteogenesis.Osteoprotegerin mRNA expression in experimental group A (1.127±0.035, 1.742±0.049, 1.049±0.062, 1.063±0.036) was significantly higher than that in the control group in each period (0.965±0.082, 1.254±0.071, 0.793±0.061, 0.684±0.055) (P=0.010, P=0.000, P=0.001, P=0.020), while group B (1.416±0.205, 2.648±0.168, 1.652±0.091, 1.712±0.070) was significantly higher than group A (P=0.000, P=0.010, P=0.023, P=0.003). RANKL mRNA expression in control group (1.666±0.086, 1.058±0.105, 0.885±0.124, 0.972±0.136) was significantly higher than that of the group A (0.788±0.036, 0.585±0.017, 0.692±0.017, 0.527±0.051) (P=0.001, P=0.006, P=0.003, P=0.028) in each period, while group A was significantly higher than group B(0.247±0.022, 0.240±0.034, 0.134±0.011, 0.103±0.050) (P=0.000, P=0.001, P=0.002, P=0.012). Conclusions: PTH can upregulate the expression of osteoprotegerin and reduce expression of RANKL, thus promoting new bone formation. Intermittent administration of high dose of parathyroid hormone can further promote the healing process after mandibular ramus osteotomy.

Recurrent splice-site mutation in MBTPS2 underlying IFAP syndrome with Olmsted syndrome-like features in a Chinese patient.

Mutations in MBTPS2 have been reported to cause a broad phenotypic spectrum of X-linked genodermatoses, including IFAP (ichthyosis follicularis; atrichia and photophobia) syndrome (OMIM 308205) with or without BRESHECK (brain anomalies, retardation of mentality and growth, ectodermal dysplasia, skeletal malformations, Hirschsprung disease, ear deformity and deafness, eye hypoplasia, cleft palate, cryptorchidism, and kidney dysplasia/hypoplasia) syndrome, keratosis follicularis spinulosa decalvans (KFSD; OMIM 308800) and an X-linked form of Olmsted syndrome. We report a recurrent intronic mutation in MBTPS2 (c.671-9T>G) in a Chinese patient with the typical triad of IFAP syndrome (i.e. ichthyosis, atrichia and photophobia), along with pachyonychia, palmoplantar and periorificial keratoderma, which were reminiscent of Olmsted syndrome. Interestingly, this mutation was previously reported in two cases of IFAP without keratoderma, which suggests clinical heterogeneicity of the same mutation in MBTPS2. The concomitance of Olmsted syndrome-like features in this patient with IFAP may challenge the existence of the X-linked form of Olmsted syndrome as an independent condition.

Effects of the preoperative administration of Yunnan Baiyao capsules on intraoperative blood loss in bimaxillary orthognathic surgery: a prospective, randomized, double-blind, placebo-controlled study.

Yunnan Baiyao is a well-known Chinese herbal medicine that has been used as a haemostatic drug for nearly 100 years. The aim of this clinical trial was to evaluate the efficacy and safety of Yunan Baiyao capsules on the reduction of blood loss in bimaxillary orthognathic surgery. 87 consecutive patients scheduled for simultaneous maxillary Le Fort I osteotomies and bilateral sagittal split ramus osteotomies (BSSRO) were enrolled in a prospective, randomized, double-blind, placebo-controlled clinical trial. Patients were administered Yunnan Baiyao capsules or placebo capsules, orally for 3 days before surgery. Intraoperative blood loss was estimated and the safety of Yunnan Baiyao capsules was evaluated. The total blood loss in the Yunnan Baiyao group (mean, 330.5+/-134.4 ml) was significantly lower than in the control group (mean, 420.3+/-175.9 ml). No allergic reactions, thromboembolic events or other side effects were recorded in this trial. It can be concluded that the preoperative use of Yunnan Baiyao capsules, in combination with hypotension anaesthesia, results in a reduction in intraoperative blood loss in bimaxillary orthognathic surgery. Yunnan Baiyao capsules are an effective and safe haemostatic Chinese medicine.

Partial molecular cloning, characterization, and analysis of the subcellular localization and expression patterns of the porcine OTUB1 gene.

The product of the OTUB1 gene is a member of the OTU superfamily of predicted cysteine proteases and inhibits cytokine gene transcription via its interaction with a ubiquitin protease and E3 ubiquitin ligase. To further understand the functions of the porcine OTUB1 gene, the subcellular localization of porcine OTUB1 protein was analyzed. We first cloned a partial DNA sequence of porcine OTUB1 which contained an 816 bp ORF encoding 271 amino acids. The deduced protein product was found to contain an OTU domain. The corresponding porcine OTUB1 protein was subsequently demonstrated to localize predominantly in the nucleus by confocal fluorescence microscopy. By spatial expression analysis, we further found that OTUB1 is highly expressed in the brain, liver, spleen, lung, kidney, large intestine, small intestine, stomach, ovary, uterus and thymus. In contrast, only low levels of this gene were evident in the heart, dorsal muscles and leg muscle of the pig. This is the first report to show the subcellular localization of porcine OTUB1, and our current data provides us with an important basis for conducting further studies on the functions and regulatory mechanisms underlying the role of OTUB1 gene in the immune system.

Prolonged gene expression in mouse lung endothelial cells following transfection with Epstein-Barr virus-based episomal plasmid.

The development of a strategy to deliver a gene to pulmonary endothelium will be useful for gene function study and for pulmonary gene therapy. Cationic lipidic vectors are efficient in gene transfer to pulmonary endothelium via the vascular route; however, gene expression is transient and lasts for only a few days. In this study, we show that pulmonary gene transfer via cationic lipidic vectors can be significantly improved using an Epstein-Barr virus (EBV)-based expression plasmid. Systemic administration of cationic liposomes followed by the EBV-based plasmid led to gene expression in the lung that lasted for more than 3 weeks. Prolonged and high levels of gene expression can also be obtained in primary mouse lung endothelial cells (MLEC) following lipofection with an EBV-based plasmid. These results suggest the utility of this gene transfer protocol in studying the expression of cloned genes in lung endothelial cells and in pulmonary gene therapy.

In vitro phosphorylation of caveolin-rich membrane domains: identification of an associated serine kinase activity as a casein kinase II-like enzyme.

Caveolae are flask-shaped micro-invaginations associated with the plasma membrane of a wide variety of cell types. Caveolin, an integral membrane component of caveolae, was first identified as the major phosphoprotein whose phosphorylation was elevated in v-Src transformed cells. As both v-Src transformation and elevated caveolin phosphorylation were dependent on membrane attachment of v-Src, it has been suggested that caveolin is a critical target in v-Src transformation. Although an increase in tyrosine phosphorylation of caveolin was evident, the increase in caveolin phosphorylation was predominantly on serine residues. In accordance with these in vivo observations, isolated caveolin-rich membrane domains undergo phosphorylation in vitro predominantly on serine and contain an unidentified serine kinase activity. Here, we have identified this serine kinase activity as a casein kinase II-like enzyme, since the phosphorylation of caveolin-rich membrane domains is stimulated and inhibited by known effectors of casein kinase II (poly-L-lysine, endogenous polyamines, and a casein kinase II inhibitor peptide), but is unaffected by modulators of other known kinases. In support of these observations, caveolin contains a consensus sequence for casein kinase II phosphorylation in its cytoplasmic N-terminal domain (Ser-88). A peptide containing this sequence inhibits the in vitro phosphorylation of caveolin-rich membrane domains, while many other peptides derived from the N-terminal domain of caveolin do not affect phosphorylation. Caveolin-rich membrane domains were also a substrate for exogenously added purified casein kinase II, but not casein kinase I. Finally, immunoblotting of these domains with an antibody directed against the alpha and alpha' subunits of casein kinase II reveals two bands with apparent molecular weights consistent with the known molecular weights of the alpha and alpha' subunits of casein kinase II. As casein kinase II appears to play a role in mitogenic signalling events and casein kinase II activators (endogenous polyamines) are required for v-Src transformation, our results may have implications for understanding the mechanism of v-Src oncogenesis.