The role of semaphorin 3A on chondrogenic differentiation.

Osteoblast-derived semaphorin3A (Sema3A) has been reported to be involved in bone protection, and Sema3A knockout mice have been reported to exhibit chondrodysplasia. From these reports, Sema3A is considered to be involved in chondrogenic differentiation and skeletal formation, but there are many unclear points about its function and mechanism in chondrogenic differentiation. This study investigated the pharmacological effects of Sema3A in chondrogenic differentiation. The amount of Sema3A secreted into the culture supernatant was measured using an enzyme-linked immunosorbent assay. The expression of chondrogenic differentiation-related factors, such as Type II collagen (COL2A1), Aggrecan (ACAN), hyaluronan synthase 2 (HAS2), SRY-box transcription factor 9 (Sox9), Runt-related transcription factor 2 (Runx2), and Type X collagen (COL10A1) in ATDC5 cells treated with Sema3A (1,10 and 100 ng/mL) was examined using real-time reverse transcription polymerase chain reaction. Further, to assess the deposition of total glycosaminoglycans during chondrogenic differentiation, ATDC5 cells were stained with Alcian Blue. Moreover, the amount of hyaluronan in the culture supernatant was measured by enzyme-linked immunosorbent assay. The addition of Sema3A to cultured ATDC5 cells increased the expression of Sox9, Runx2, COL2A1, ACAN, HAS2, and COL10A1 during chondrogenic differentiation. Moreover, it enhanced total proteoglycan and hyaluronan synthesis. Further, Sema3A was upregulated in the early stages of chondrogenic differentiation, and its secretion decreased later. Sema3A increases extracellular matrix production and promotes chondrogenic differentiation. To the best of our knowledge, this is the first study to demonstrate the role of Sema3A on chondrogenic differentiation.

Epidemiological shifts in and impact of COVID-19 on streptococcal toxic shock syndrome in Japan: A genotypic analysis of group A Streptococcus isolates.

OBJECTIVES: Streptococcal toxic shock syndrome (STSS) is caused by group A Streptococcus (GAS; Streptococcus pyogenes) strains. In Japan, the number of STSS cases has decreased; however, the underlying reason remains unclear. Moreover, information on distribution and prevalence of specific emm types in STSS cases is scarce. Hence, we investigated the reason for the decreased number of STSS cases in Japan. METHODS: We genotyped emm of 526 GAS isolates obtained from 526 patients with STSS between 2019 and 2022. The distributions of emm types in each year were compared. RESULTS: The emm1 type was predominant, with the highest proportion in 2019, which decreased after 2020 following the onset of the coronavirus disease 2019 (COVID-19) pandemic. Strains isolated during the pandemic correlated with strains associated with skin infection, whereas those isolated during the prepandemic period correlated with strains associated with both throat and skin infections. The decrease in the annual number of STSS cases during the COVID-19 pandemic could be due to a decreased proportion of strains associated with pharyngeal infections. CONCLUSIONS: Potential associations between pandemic and STSS numbers with respect to public health measures, such as wearing masks and changes in healthcare-seeking behavior, may have affected the number of GAS-induced infections.

Serotype Distribution and Antimicrobial Resistance of Streptococcus agalactiae Isolates in Nonpregnant Adults with Streptococcal Toxic Shock Syndrome in Japan in 2014 to 2021.

The incidence of streptococcal toxic shock syndrome (STSS) due to group B Streptococcus (GBS) has been increasing annually in Japan and is becoming a serious challenge. Furthermore, in recent years, penicillin- or clindamycin-resistant strains used in treating streptococcal toxic shock syndrome have been reported. However, no report analyzed >100 isolates of group B Streptococcus causing streptococcal toxic shock syndrome. Therefore, we aimed to perform serotyping and antimicrobial susceptibility testing of 268 isolated group B Streptococcus strains from streptococcal toxic shock syndrome cases involving nonpregnant adult patients in Japan between 2014 and 2021. The most prevalent serotype was Ib, followed by serotypes V, III, and Ia. Seven isolates were resistant to penicillin G, and 17.9% (48 isolates) were resistant to clindamycin. Of the penicillin-resistant group B Streptococcus isolates, 71.4% (5 isolates) were clindamycin resistant. In addition, group B Streptococcus strains resistant to penicillin and clindamycin were isolated from patients with streptococcal toxic shock syndrome. Therefore, before these strains become prevalent, introduction of the group B Streptococcus vaccine is essential for disease prevention. IMPORTANCE Group B Streptococcus (GBS) has been increasingly associated with invasive disease in nonpregnant adults. Such infections are responsible for substantial morbidity and mortality, particularly in individuals with underlying chronic conditions. Streptococcal toxic shock syndrome (STSS) is a severe invasive infection characterized by the sudden onset of shock, multiorgan failure, and high mortality. In this study, we assessed 268 GBS-related STSS cases in nonpregnant adults in Japan between 2014 and 2021. Serotype Ib was the most prevalent, followed by serotypes V, III, and Ia, which were identified in more than 80% of STSS isolates. We found that 48 clindamycin-resistant strains and 7 penicillin G-resistant strains were isolated between 2014 and 2021. We believe that our study makes a significant contribution to the literature because we show that the GBS vaccine, particularly the hexavalent conjugate vaccine, is important to reduce the number of patients with STSS.

Longitudinal effects of estrogen on mandibular growth and changes in cartilage during the growth period in rats.

Estrogen is a steroid hormone that induces skeletal growth and affects endochondral ossification of the long tubular bone growth plate during the growth period. However, the effects of estrogen on endochondral ossification of the mandibular condylar cartilage are unclear. In this study, ovariectomized Wistar/ST rats were used to investigate the longitudinal effects of estrogen on mandibular growth. The rats were administered different doses of estrogen. Longitudinal micro-computed tomographic scanning, histological staining and ELISA on plasma growth hormone were performed to examine the effects of estrogen on mandibular growth. The results showed that mandibular growth was suppressed throughout the growth period by estrogen in a dose-dependent manner. In addition, long-term administration of a high dose of estrogen to the rats resulted in significant increase in growth hormone throughout the growth period, significant circularization of cell nuclei in the proliferative layer, intensely staining cartilage matrix in the subchondral bone, and significant suppression of estrogen receptor (ER) alpha and beta expression in the mandibular cartilage. However, regardless of estrogen concentration, in the posterior part of the mandibular cartilage, ER expression extended to both the hypertrophic and proliferative layers. These results indicate that estrogen suppresses mandibular growth throughout the growth period. Additionally, it influences endochondral ossification via its effect on ERs.

ANGPTL2 Induces Synovial Inflammation via LILRB2.

Angiopoietin-like proteins (ANGPTLs) are circulating proteins that are expressed in various cells and tissues and are thought to be involved in the repair and remodeling of damaged tissues; however, ANGPTL2 hyperfunction has been shown to cause chronic inflammation, leading to the progression of various diseases. ANGPTL2 is known to exert cellular effects via receptors such as integrin α5ß1 and leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2); however, their roles in ANGPTL2-induced inflammation remain unclear. In this study, we investigated the mechanisms underlying ANGPTL2-induced inflammation involving LILRB2 and various signaling pathways in human fibroblast-like synoviocytes (HFLS). The effects of ANGPTL2 and an anti-LILRB2 antibody on the gene expression of various inflammation-related factors were examined using real-time RT-PCR, while their effects on MAPK, NF-κB, and Akt phosphorylation were analyzed by western blotting. We found that the addition of ANGPTL2 enhanced the gene expression of inflammatory factors, whereas pretreatment with the anti-LILRB2 antibody for 12 h decreased the expression of these factors. Similarly, ANGPTL2 addition activated the phosphorylation of ERK, p38, JNK, NF-κB, and Akt in HFLS; however, this effect was significantly inhibited by pretreatment with the anti-LILRB2 antibody. Together, the findings of this study demonstrate that ANGPTL2 induces the expression of inflammatory factors via LILRB2 in synovial cells. Therefore, LILRB2 could be a potential therapeutic agent for treating matrix degradation in osteoarthritis.

ANGPTL2 Promotes Inflammation via Integrin α5ß1 in Chondrocytes.

BACKGROUND: Angiopoietin-like protein 2 (ANGPTL2) is a secreted molecule with numerous physiologic and pathologic functions, for example, in angiogenesis, hematopoiesis, and tumorigenesis. Although recent studies implicated ANGPTL2 in chronic inflammation in mouse peritoneal macrophages, human ligamentum flavum fibroblasts, and human retinal microvascular endothelial cells, the mechanism underlying ANGPTL2-associated inflammation in chondrocytes remains unclear. Therefore, it was investigated whether ANGPTL2 is expressed in or functions in chondrocytes. METHODS: Expression of ANGPTL2 and its receptor, integrin α5ß1 were examined over time in ATDC5 cells using real-time RT-PCR (reverse transcription-polymerase chain reaction) analysis. ATDC5 cells were then incubated with or without ANGPTL2 for 3 hours, and expression of the IL-1ß, TNF-α, COX-2, aggrecanase (ADAMTS)-5, matrix metalloproteinase (MMP)-3, and MMP-13 genes were examined using real-time RT-PCR. Additionally, phosphorylation of ERK, JNK, p38, Akt, and NF-κB was examined by western blotting. Furthermore, it was also investigated for the effect of anti-integrin α5ß1 antibody on the expression of inflammatory markers and intracellular signaling pathways. RESULTS: ANGPTL2 induced the phosphorylation of all 3 MAPKs, Akt, and NF-κB and dramatically upregulated the expression of inflammation-related factor genes. Inhibiting the activation of integrin α5ß1 suppressed these reactions. CONCLUSION: ANGPTL2 may induce inflammatory factors by stimulating the integrin α5ß1/MAPKs, Akt, and NF-κB signaling pathway.

FAK inhibition protects condylar cartilage under excessive mechanical stress.

OBJECTIVES: Excessive mechanical stress is assumed to be a major cause of temporomandibular joint (TMJ) osteoarthritis (OA). +Focal adhesion kinase (FAK) is a cytoplasmic non-receptor tyrosine kinase involved in a variety of signaling pathways. Little has been reported on the function of FAK in TMJ-OA. In the present study, we investigated the effect of FAK inhibition on TMJ cartilage under excessive mechanical loading stress. MATERIALS AND METHODS: Articular cartilage explants were harvested from the TMJ of rats and subjected to mechanical loading in the presence of an FAK inhibitor in organ culture. The gene expression of inflammatory cytokines was examined after the application of mechanical loading with or without FAK inhibitor. Paraffin-embedded sections of articular cartilage were stained with hematoxylin and eosin, safranin O and fast Green, toluidine blue, TUNEL staining, and immunohistochemical staining and was performed to investigate the protein expression of IL-1ß and MMP-13. RESULTS: Treatment with FAK inhibitor reduced the gene expression of inflammatory cytokines and inhibited the degradation of articular cartilage, as determined histologically. FAK inhibitor treatment also suppressed the protein expression of IL-1ß and MMP-13 in the hypertrophic zone, as determined immunohistologically. CONCLUSION: Treatment with FAK inhibitor suppresses inflammation and protects condylar cartilage under excessive mechanical loading.

Protective effects of cilengitide on inflammation in chondrocytes under excessive mechanical stress.

Chondrocytes constantly receive external stimuli, which regulates remodeling. An optimal level of mechanical stress is essential for maintaining chondrocyte homeostasis, however, excessive mechanical stress induces inflammatory cytokines and protease, such as matrix metalloproteinases (MMPs). Therefore, excessive mechanical stress is considered to be one of the main causes to cartilage destruction leading to osteoarthritis (OA). Integrins are well-known as cell adhesion molecules and act as receptors for extracellular matrix (ECM), and are believed to control intracellular signaling pathways both physically and chemically as a mechanoreceptor. However, few studies have focused on the roles and functions of integrins in inflammation caused by excessive mechanical stress. In this study, we examined the relationship between integrins (αVß3 and αVß5) and the expression of inflammatory factors under mechanical loading in chondrocytes by using an integrin receptor antagonist (cilengitide). Cilengitide suppressed the gene expression of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), matrix metalloproteinase-3 (MMP-3), and MMP-13 induced by excessive mechanical stress. In addition, the protein expression of IL1-ß and MMP-13 was also inhibited by the addition of cilengitide. Next, we investigated the involvement of intracellular signaling pathways in stress-induced integrin signaling in chondrocytes by using western blotting. The levels of p-FAK, p-ERK, p-JNK, and p-p38 were enhanced by excessive mechanical stress and the enhancement was suppressed by treatment with cilengitide. In conclusion, this study revealed that excessive mechanical stress may activate integrins αVß3 and αVß5 on the surface of chondrocytes and thereby induce an inflammatory reaction by upregulating the expression of IL-1ß, TNF-α, MMP-3, and MMP-13 through phosphorylation of FAK and MAPKs.

Semaphorin 3A Inhibits Inflammation in Chondrocytes under Excessive Mechanical Stress.

BACKGROUND: Excessive mechanical stress causes inflammation and destruction of cartilage and is considered one of the cause of osteoarthritis (OA). Expression of semaphorin 3A (Sema3A), which is an axon guidance molecule, has been confirmed in chondrocytes. However, there are few reports about Sema3A in chondrocytes, and the effects of Sema3A on inflammation in the cartilage are poorly understood. The aim of this study was to examine the role of Sema3A in inflammation caused by high magnitude cyclic tensile strain (CTS). METHODS: Expression of Sema3A and its receptors neuropilin-1 (NRP-1) and plexin-A1 (PLXA1) in ATDC5 cells was examined by Western blot analysis. ATDC5 cells were subjected to CTS of 0.5 Hz, 10% elongation with added Sema3A for 3 h. Gene expression of IL-1ß, TNF-ɑ, COX-2, MMP-3, and MMP-13 was examined by qPCR analysis. Furthermore, the phosphorylation of AKT, ERK, and NF-κB was detected by Western blot analysis. RESULTS: Added Sema3A inhibited the gene expression of inflammatory cytokines upregulated by CTS in a dose-dependent manner. Addition of Sema3A suppressed the activation of AKT, ERK, and NF-κB in a dose-dependent manner. CONCLUSIONS: Sema3A reduces the gene expression of inflammatory cytokines by downregulating the activation of AKT, ERK, and NF-κB pathways in ATDC5 cells under CTS.

Cyclic Tensile Strain Upregulates Pro-Inflammatory Cytokine Expression Via FAK-MAPK Signaling in Chondrocytes.

Excessive mechanical stimulation is considered an important factor in the destruction of chondrocytes. Focal adhesion kinase (FAK) is non-receptor tyrosine kinase related to a number of different signaling proteins. Little is known about the function of FAK in chondrocytes under mechanical stimulation. In the present study, we investigated the function of FAK in mechanical signal transduction and the mechanism through which cyclic tensile strain (CTS) induces expression of inflammation-related factors. Mouse ATDC5 chondrogenic cells were subjected to CTS of 0.5 Hz to 10% cell elongation with an FAK inhibitor. The expression of genes encoding COX-2, IL-1ß, and TNF-α was examined using real-time RT-PCR after CTS application with FAK inhibitor. Phosphorylation of p-38, ERK, and JNK was analyzed by Western blotting. Differences in COX-2 expression following pretreatment with FAK, p-38, ERK, and JNK inhibitors were compared by Western blotting. We found that CTS increased the expression of genes encoding COX-2, IL-1ß, and TNF-α and activated the phosphorylation of FAK, p-38, ERK, and JNK. Pretreatment with an FAK inhibitor for 2 h reduced the expression of genes encoding COX-2, IL-1ß, and TNF-α induced by CTS-associated inflammation and decreased phosphorylation of FAK, p-38, ERK, and JNK. Pretreatment with FAK, p-38, ERK, and JNK inhibitors markedly suppressed COX-2 and IL-1ß protein expression. In conclusion, FAK appears to regulate inflammation in chondrocytes under CTS via MAPK pathways.

Propofol drip infusion anesthesia for MRI scanning: two case reports.

The magnetic resonance imaging (MRI) room is a special environment. The required intense magnetic fields create unique problems with the use of standard anesthesia machines, syringe pumps, and physiologic monitors. We have recently experienced 2 oral maxillofacial surgery cases requiring MRI: a 15-year-old boy with developmental disability and a healthy 5-year-old boy. The patients required complete immobilization during the scanning for obtaining high-quality images for the best diagnosis. Anesthesia was started in the MRI scanning room. An endotracheal intubation was performed after induction with intravenous administration of muscle relaxant. Total intravenous anesthesia via propofol drip infusion (4-7 mg/kg/h) was used during the scanning. Standard physiologic monitors were used during scan pauses, but special monitors were used during scanning. In MRI scanning for oral maxillofacial surgery, general anesthesia, with the added advantage of having a secured airway, is recommended as a safe alternative to sedation especially in cases of patients with disability and precooperative chidren.

Zinc bioavailability is improved by the micronised dispersion of zinc oxide with the addition of L-histidine in zinc-deficient rats.

Zinc fortification of milk or soft drinks is usually used to combat zinc deficiencies in developing countries. Water-soluble zinc compounds, such as zinc sulfate or zinc citrate, are better absorbed but have an unacceptable taste. A micronised, dispersible zinc oxide (MDZnO), which does not have such a problem concerning taste, had higher solubility compared to ZnO (zinc oxide) in an artificial gastric solution. MDZnO was tested for its bioavailability using zinc-deficient Wistar rats. Prior to the experiment, rats were fed zinc-deficient diet for 3 wk and were orally administered control (distilled water) or zinc solutions (ZnO, ZnO+L-histidine (His), MDZnO, MDZnO+His, 1 mg zinc/kg or 3.2 mg His/kg body weight). Compared to ZnO, MDZnO showed a lag in peak time and a lengthy period of continued high plasma zinc concentration after the single oral administration of zinc compounds. Addition of His to MDZnO elevated serum zinc concentration. Serum zinc concentration (area under the curve) in rats administered MDZnO with His was significantly higher than in rats administered distilled water (p<0.05). Liver zinc level was significantly higher in rats administered MDZnO with His compared with control rats (p<0.05), although the level was not affected by the administration with ZnO alone, ZnO+His, or MDZnO alone. In conclusion, the solubility of ZnO was elevated by the micronised dispersion tecnique and an in vivo study using zinc-deficient rats confirmed that its bioavailability was significantly improved compared to ZnO and the coadministration of His additively enhanced the bioavailability of MDZnO.