A Stratification Approach Based on Salivary Gland Ultrasonography for Assessing Secretory Function in Sjögren's Disease.

OBJECTIVE: Our aim was to develop an ultrasonographic scoring model for staging hypofunction of salivary gland (SG) in patients suffering from Sjögren's disease (SjD). METHODS: The assessment of SG secretory hypofunction was conducted by measuring whole salivary flows. B-mode ultrasonography was performed bilaterally on the parotid and submandibular glands to evaluate the Gland score and OMERACT score quantitatively. The correlation between these scores and SG secretory function in SjD patients was analyzed, leading to the development of an ultrasonographic scoring model for staging SG hypofunction. RESULTS: A one-center derivation cohort comprising 164 SjD patients and a double-center validation cohort consisting of 107 SjD patients were included. Both ultrasonographic scores demonstrated excellent discriminatory ability between SjD patients with hypofunction and those with normal function (both AUC>0.8, p<0.001). A novel ultrasonographic scoring model revealed that low total OMERACT scores (<5) indicated initial-stage SG hypofunction, while high scores (>9) suggested end-stage hypofunction. Conversely, patients with moderate-level total OMERACT scores (5~9) require further stratification using total Gland scores. The incidence of SG hypofunction among all 271 SjD patients was found to be 18% in the initial stage, 58% in the progressive stage, and 100% in the end stage (p<0.001). Furthermore, the incidence of lacrimal gland involvement and hyperglobulinemia (IgG>16 IU/mL) was significantly lower in the initial-stage patients compared to those at other stages (all p<0.001). CONCLUSION: The novel ultrasonographic scoring model incorporates precise definitions for each stage, providing a robust and clinically significant approach to stratify salivary gland secretory hypofunction in SjD.

Forensic bone age estimation of adolescent pelvis X-rays based on two-stage convolutional neural network.

In the forensic estimation of bone age, the pelvis is important for identifying the bone age of teenagers. However, studies on this topic remain insufficient as a result of lower accuracy due to the overlapping of pelvic organs in X-ray images. Segmentation networks have been used to automate the location of key pelvic areas and minimize restrictions like doubling images of pelvic organs to increase the accuracy of estimation. This study conducted a retrospective analysis of 2164 pelvis X-ray images of Chinese Han teenagers ranging from 11 to 21 years old. Key areas of the pelvis were detected with a U-Net segmentation network, and the findings were combined with the original X-ray image for regional augmentation. Bone age estimation was conducted with the enhanced and not enhanced pelvis X-ray images by separately using three convolutional neural networks (CNNs). The root mean square errors (RMSE) of the Inception-V3, Inception-ResNet-V2, and VGG19 convolutional neural networks were 0.93 years, 1.12 years, and 1.14 years, respectively, and the mean absolute errors (MAE) of these networks were 0.67 years, 0.77 years, and 0.88 years, respectively. For comparison, a network without segmentation was employed to conduct the estimation, and it was found that the RMSE of the three CNNs above became 1.22 years, 1.25 years, and 1.63 years, respectively, and the MAE became 0.93 years, 0.96 years, and 1.23 years. Bland-Altman plots and attention maps were also generated to provide a visual comparison. The proposed segmentation network can be used to reduce the influence of restrictions like image overlapping of organs and can thus increase the accuracy of pelvic bone age estimation.

Low expression of estrogen receptor ß in renal tubular epithelial cells may cause hyperuricemia in premenopausal patients with systemic lupus erythematosus.

We investigated the impact of estrogen receptor (ER) expression in renal tubular epithelial cells on serum uric acid (UA) levels in premenopausal patients with systemic lupus erythematosus (SLE). Thirty patients underwent renal biopsy: 18 with SLE (LN group) and 12 with IgA nephritis (IgAN group). ERs (ERα and ERß) in renal tubular epithelial cells were measured using immunohistochemistry. The ER expression levels of the two groups were compared, and the relationship between the expression of ERs and serum UA levels was analyzed. Mean serum UA levels in the LN group were significantly higher than those of the IgA nephropathy group, while the mean creatinine levels and GFRs of the two groups were similar. Pathological changes in the LN group were significantly more severe than those in the IgAN group. ERß was expressed in renal tubular epithelial cells in both groups, but not in the glomeruli. ERß expression in the LN group was significantly lower than that in the IgAN group. ERß expression scores significantly negatively correlated with serum UA levels. These findings suggest that the expression of ERß in premenopausal female SLE patients may cause hyperuricemia, and may subsequently promote glomerular damage, suggesting that ERß may be involved in UA excretion.

Lack of serum- and glucocorticoid-inducible kinase 3 leads to podocyte dysfunction.

Serum- and glucocorticoid-inducible kinase 3 (SGK3) is a downstream mediator of PI3K, which is essential for maintaining the functional integrity of podocytes. However, little is known about the role of SGK3 in podocyte function. Herein, we demonstrated that SGK3 contributes to the maintenance of podocyte integrity. Conditionally immortalized mouse podocyte cells (MPCs) were treated with puromycin aminonucleoside (PAN). PAN treatment inhibited the activity of SGK3 and the expression of podocin. Short hairpin RNA (shRNA)-mediated knockdown of SGK3 also reduced podocin expression in the absence of PAN. Adriamycin (ADR)-treated mice developed proteinuria and had decreased renal glomerular SGK3 expression in comparison to control mice. Consistent with a role for SGK3 in the ADR effect, SGK3 knockout (KO) mice had markedly reduced kidney podocin expression and significantly elevated proteinuria compared with wild-type mice. Electron microscopy revealed that SGK3 KO mice displayed partial effacement of podocyte foot processes. Further, a SGK3 target protein, glycogen synthase kinase-3 (GSK3), was discovered to be dramatically activated in PAN and SGK3 shRNA-treated MPCs and in SGK3 KO mice. Taken together, these data strongly suggest that SGK3 plays a significant role in regulating podocyte function, likely by controlling the expression and activity of GSK3.-Peng, L.-Q., Zhao, H., Liu, S., Yuan, Y.-P., Yuan, C.-Y., Mwamunyi, M.-J., Pearce, D., Yao, L.-J. Lack of serum- and glucocorticoid-inducible kinase 3 leads to podocyte dysfunction.

Can unstimulated whole salivary flow objectively classify salivary gland secretory function in Sjögren's syndrome?

INTRODUCTION: The aim of this study is to investigate whether the testing time for unstimulated whole salivary flow (UWSF) can be shortened to 5 min in patients with suspected Sjögren's syndrome (SjS); and which SjS patients can use UWSF to evaluate salivary gland (SG) secretory function. METHOD: A diagnostic cohort comprising suspected SjS patients was conducted to investigate the correlation between UWSF measurements taken at 10 min (UWSF_10 min) and those taken at 5 min (UWSF_5 min). A group of SjS patients was enrolled for a comparison between UWSF and stimulated whole salivary flow (SWSF). RESULTS: In 734 suspected SjS patients, there was a remarkably high concordance between UWSF_10 min and UWSF_5 min (ICC 0.970, P < 0.001; r 0.973, P < 0.001). Reducing the testing time for UWSF to 5 min resulted in a high PPV of 83.8% and an exceptionally high NPV of 98.7%. In 408 SjS patients, the cut-off values of UWSF_10 min were investigated to classify SG secretory function. Using a threshold of > 0.2 mL/min (36.8%, 150/408) instead of SWSF > 0.7 mL/min (indicating mild secretory hypofunction), the specificity and PPV were found to be 94.2% and 94.0%, respectively; and using a threshold of < 0.05 mL/min (16.9%, 69/408) instead of SWSF ≤ 0.7 mL/min (indicating moderate to severe secretory hypofunction), the specificity was remarkably high at 97.6%, accompanied by a high PPV of 91.3%. CONCLUSIONS: This study supports the possibility of reducing UWSF testing time to 5 min; and the SWSF test may be skipped for SjS patients with USWF > 0.2 mL/min, indicating mild secretory hypofunction, or < 0.05 mL/min, indicating moderate to severe secretory hypofunction. Key Points •A diagnostic cohort of 734 patients with clinical suspicion of SjS provides compelling evidence for the potential to reduce the testing time for UWSF from 10 to 5 min. •Our finding challenges the 2019 treatment recommendation for SjS, which does not require SWSF measurement in SjS patients with UWSF ≥ 0.1 mL/min. •We propose that it may be feasible to consider utilizing UWSF instead of SWSF test for objective classification of SG secretory function in over half of SjS patients.

Mucin 1 aggravates synovitis and joint damage of rheumatoid arthritis by regulating inflammation and aggression of fibroblast-like synoviocytes.

AIMS: To explore the synovial expression of mucin 1 (MUC1) and its role in rheumatoid arthritis (RA), as well as the possible downstream mechanisms. METHODS: Patients with qualified synovium samples were recruited from a RA cohort. Synovium from patients diagnosed as non-inflammatory orthopaedic arthropathies was obtained as control. The expression and localization of MUC1 in synovium and fibroblast-like synoviocytes were assessed by immunohistochemistry and immunofluorescence. Small interfering RNA and MUC1 inhibitor GO-203 were adopted for inhibition of MUC1. Lysophosphatidic acid (LPA) was used as an activator of Rho-associated pathway. Expression of inflammatory cytokines, cell migration, and invasion were evaluated using quantitative real-time polymerase chain reaction (PCR) and Transwell chamber assay. RESULTS: A total of 63 RA patients and ten controls were included. Expression of MUC1 was observed in both the synovial lining and sublining layer. The percentage of MUC1+ cells in the lining layer of synovium was significantly higher in RA than that in control, and positively correlated to joint destruction scores of RA. Meanwhile, MUC1+ cells in the sublining layer were positively correlated to the Krenn subscore of inflammatory infiltration. Knockdown of MUC1, rather than GO-203 treatment, ameliorated the expression of proinflammatory cytokines, cell migration, and invasion of rheumatoid synoviocytes. Knockdown of MUC1 decreased expression of RhoA, Cdc42, and Rac1. Treatment with LPA compromised the inhibition of migration and invasion, but not inflammation, of synoviocytes by MUC1 knockdown. CONCLUSION: Upregulated MUC1 promotes the aggression of rheumatoid synoviocytes via Rho guanosine triphosphatases (GTPases), thereby facilitating synovitis and joint destruction during the pathological process of RA.Cite this article: Bone Joint Res 2022;11(9):639-651.

Interaction of glycyrol with calcineurin A studied by spectroscopic methods and docking.

We have shown previously that glycyrol has an inhibitory effect on the immune response in mice by reducing calcineurin activity (Li et al., 2010, Pharm Biol 48:1177­1184). Here, we investigated the interaction of glycyrol with calcineurin A (CNA, catalytic A subunit of calcineurin) by spectroscopic methods and docking. We showed that glycyrol binds to CNA via hydrophobic interactions in a ratio of 1:1, and the main binding site is in the catalytic domain of CNA close to the calcineurin B subunit-binding domain. Binding of glycyrol changes the secondary structure of CNA, and this effect may possibly inhibit CN activity.

Quercetin binds to calcineurin at a similar region to cyclosporin A and tacrolimus.

Quercetin, the primary dietary flavonol, exerts a strong inhibitory effect on calcineurin (CN), a unique Ca(2+)/calmodulin-dependent serine/threonine protein phosphatase. Using fluorescence spectroscopy (FS) we showed quercetin strongly bound to calcineurin catalytic subunit (CNA) with a ratio of 1:1; we also showed that calcineurin regulatory subunit (CNB) weakened this binding. In addition, the secondary structure of CNA was much tighter in the presence of quercetin. An FS study with CNA truncated mutant CNAa showed that the binding area for quercetin was reduced to the catalytic domain of CNA. Furthermore, fluorescence resonance energy transfer (FRET) results and molecular docking indicated three potential binding sites for quercetin, which were located at a region between the active centre of CNA and the CNB binding domain, a similar binding area to that of cyclosporin A and tacrolimus. Interestingly, this region was also important for CN substrate recognition.

The SGK3-triggered ubiquitin-proteasome degradation of podocalyxin (PC) and ezrin in podocytes was associated with the stability of the PC/ezrin complex.

Podocyte damage is commonly accompanied by destabilization of the podocalyxin (PC)/ezrin complex. Serum- and glucocorticoid-inducible kinase 3 (SGK3) plays a role in the maintenance of podocyte function, but the details of this role are poorly understood. Herein we demonstrated that SGK3 and its downstream target protein neural precursor cell expressed developmentally downregulated protein 4 subtype 2 (Nedd4-2) triggered PC and ezrin interaction. In adriamycin (ADR)-induced nephritic mice, and after puromycin aminonucleoside (PAN)-induced podocyte damage in vitro, PC and ezrin protein expression levels decreased significantly, while Nedd4-2 activity increased. Moreover, PAN treatment increased PC and ezrin ubiquitination and decreased PC/ezrin interaction in cultured mouse podocytes. The downregulation of SGK3 activity in mouse podocytes resulted in decreased PC and ezrin protein expression and increased the ubiquitin-proteasome degradation of PC and ezrin. Furthermore, upregulation of SGK3 activity mostly reversed the PAN-induced decrease in PC and ezrin protein expression. Overexpression of Nedd4-2 led to decreased ezrin protein expression via the upregulation of ezrin ubiquitination. In contrast, Nedd4-2 knockdown resulted in increased ezrin protein expression but decreased ezrin ubiquitination. In PC-transfected human embryonic kidney (HEK293T) cells, SGK3 activity downregulation and Nedd4-2 overexpression resulted in decreased PC/ezrin interaction. These results suggested that SGK3 triggers the ubiquitin-proteasome degradation of PC and ezrin, while the SGK3/Nedd4-2 signaling pathway regulates ezrin, but not PC, ubiquitination. Thus SGK3 helps to regulate podocyte function by maintaining the stability of the PC/ezrin complex.