Discovery of Novel Small Molecule Dual Inhibitor Targeting Toll-Like Receptors 7 and 9.

The aberrant secretion of proinflammatory cytokines by immune cells is the principal cause of inflammatory diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Toll-like receptor 7 (TLR7) and TLR9, sequestered to the endosomal compartment of dendritic cells and macrophages, are closely associated with the initiation and progression of these diseases. Therefore, the development of drugs targeting dysregulated endosomal TLRs is imperative to mitigate systemic inflammation. Here, we applied the principles of computer-aided drug discovery to identify a novel low-molecular-weight compound, TLR inhibitory compound 10 (TIC10), and its potent derivative (TIC10g), which demonstrated dual inhibition of TLR7 and TLR9 signaling pathways. Compared to TIC10, TIC10g exhibited a more pronounced inhibition of the TLR7- and TLR9-mediated secretion of the proinflammatory cytokine tumor necrosis factor-α in a mouse macrophage cell line and mouse bone marrow dendritic cells in a concentration-dependent manner. While TIC10g slightly prevented TLR3 and TLR8 activation, it had no impact on cell surface TLRs (TLR1/2, TLR2/6, TLR4, or TLR5), indicating its selectivity for TLR7 and TLR9. Additionally, mechanistic studies suggested that TIC10g interfered with TLR9 activation by CpG DNA and suppressed downstream pathways by directly binding to TLR9. Western blot analysis revealed that TIC10g downregulated the phosphorylation of the p65 subunit of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinases (MAPKs), including extracellular-signal-regulated kinase, p38-MAPK, and c-Jun N-terminal kinase. These findings indicate that the novel ligand, TIC10g, is a specific dual inhibitor of endosomal TLRs (TLR7 and TLR9), disrupting MAPK- and NF-κB-mediated proinflammatory gene expression.

Measurement of interspinous motion in dynamic cervical radiographs using a deep learning-based segmentation model.

OBJECTIVE: Interspinous motion (ISM) is a representative method for evaluating the functional fusion status following anterior cervical discectomy and fusion (ACDF) surgery, but the associated measuring difficulty and potential errors in the clinical setting remain concerns. The aim of this study was to investigate the feasibility of a deep learning-based segmentation model for measuring ISM in patients who underwent ACDF surgery. METHODS: This study is a retrospective analysis of flexion-extension dynamic cervical radiographs from a single institution and a validation of a convolutional neural network (CNN)-based artificial intelligence (AI) algorithm for measuring ISM. Data from 150 lateral cervical radiographs from the normal adult population were used to train the AI algorithm. A total of 106 pairs of dynamic flexion-extension radiographs from patients who underwent ACDF at a single institution were analyzed and validated for measuring ISM. To evaluate the agreement power between human experts and the AI algorithm, the authors assessed the interrater reliability using the intraclass correlation coefficient and root mean square error (RMSE) and performed a Bland-Altman plot analysis. They processed 106 pairs of radiographs from ACDF patients into the AI algorithm for autosegmenting the spinous process created using 150 normal population radiographs. The algorithm automatically segmented the spinous process and converted it to a binary large object (BLOB) image. The rightmost coordinate value of each spinous process from the BLOB image was extracted, and the pixel distance between the upper and lower spinous process coordinate value was calculated. The AI-measured ISM was calculated by multiplying the pixel distance by the pixel spacing value included in the DICOM tag of each radiograph. RESULTS: The AI algorithm showed a favorable prediction power for detecting spinous processes with an accuracy of 99.2% in the test set radiographs. The interrater reliability between the human and AI algorithm of ISM was 0.88 (95% CI 0.83-0.91), and its RMSE was 0.68. In the Bland-Altman plot analysis, the 95% limit of interrater differences ranged from 0.11 to 1.36 mm, and a few observations were outside the 95% limit. The mean difference between observers was 0.02 ± 0.68 mm. CONCLUSIONS: This novel CNN-based autosegmentation algorithm for measuring ISM in dynamic cervical radiographs showed strong agreement power to expert human raters and could help clinicians to evaluate segmental motion following ACDF surgery in clinical settings.

Changes in Shoulder Trauma during the COVID-19 Pandemic: A South Korean Survey.

Background: This study aimed to investigate the changes in the incidence of shoulder trauma and surgery 1 year after the outbreak of coronavirus disease 2019 (COVID-19) with social restriction compared with 1 year before the pandemic. Methods: Patients managed in our orthopedic trauma center between February 18, 2020, and February 17, 2021 (COVID-19 period) for shoulder trauma were compared with patients managed for the same duration a year ago (non-COVID-19 period; February 18, 2019, to February 17, 2020). The incidence of shoulder trauma, surgery, and mechanism of injury were compared between these periods. Results: The total number of shoulder trauma cases was lower in the COVID-19 period than in the non-COVID-19 period, although the difference was not significant (160 vs. 180 cases, p = 0.278). In addition, traumatic shoulder surgeries decreased during the COVID-19 period (57 vs. 69 cases, p = 0.285). The incidence of shoulder trauma according to four diagnostic classifications (contusion, sprain/subluxation, fracture, and dislocation) and fracture/dislocation types did not differ between the periods. During the COVID-19 period, accidental falls outdoors (45 vs. 67, p = 0.038) and sports-related injuries (15 vs. 29, p = 0.035) significantly decreased, but accidental falls at home (52 vs. 37, p = 0.112) increased compared with those during the non-COVID-19 period, although the difference was not significant. The monthly incidence of shoulder trauma decreased 2 months after the first outbreak (significant in March, p = 0.019), then steadily increased and significantly decreased during the second outbreak (August, p = 0.012). However, the third outbreak (December, p = 0.077) had little effect on the incidence of shoulder trauma. The number of monthly traumatic shoulder surgeries showed a similar pattern to the monthly incidence of shoulder trauma. Conclusions: During the COVID-19 pandemic, annual shoulder trauma cases and surgeries decreased compared to those in the non-COVID-19 period, even though the difference was insignificant. The incidence of shoulder trauma and surgery was significantly reduced in the early COVID-19 period; however, the effect of the pandemic on orthopedic trauma practice was minimal after approximately half a year. Decreases in falls outdoors and sports-related injuries, but an increase in falls at home, were observed during the COVID-19 pandemic.

Risk factors for hardware-related complications after extra-articular distal humerus fracture fixation using an anatomical locking plate.

PURPOSE: We aimed to identify the incidence and risk factors of hardware-related complications in patients treated with anatomical locking plate fixation for extra-articular distal humerus fractures. METHODS: From 2013 to 2020, patients with extra-articular distal humerus fractures who underwent open reduction and internal fixation with an extra-articular distal humerus locking plate (EADHP) were retrospectively reviewed and categorized according to the presence/absence of hardware-related complications. Hardware-related complications were defined as the occurrence of skin prominence on the plate and discomfort in activities of daily living. Patient demographics, the lateral condylar angle, lateral body length, shaft-condylar angle of the humerus, and plate length were analyzed. RESULTS: Of the 29 patients, 10 (34%) did not develop hardware-related complications (group A), whereas 19 (66%) did (group B). Patient demographics did not differ between the groups. However, the number of patients who underwent hardware removal was significantly greater in group B (16/19) than in group A (4/10; p = 0.032). Radiologic assessment revealed no significant difference in the lateral condylar or shaft-condylar angle. However, the lateral body length was greater in group A than in group B (44.5 ± 4.8 vs. 39.5 ± 3.7, p = 0.007). The plate length significantly differed between the groups. Twelve of 19 (63%) patients in group B received short-hole plates (six holes), while nine of ten (90%) patients in group A received long-hole plates (eight holes). In the multivariable analysis, the lateral body length of the distal humerus (p = 0.047, odds ratio = 0.734, 95% confidence interval: 0.542-0.996) and plate length (p = 0.036, odds ratio = 0.076, 95% confidence interval: 0.542-0.996) were associated with hardware-related complications. CONCLUSIONS: Most patients developed hardware-related complications, particularly with short plates, mainly because of the narrow lateral body length of the distal humerus. Surgeons should be careful to secure EADHP in the appropriate position, especially when short plates are used in patients with narrow lateral body length.

Hyaluronic Acid Nanoparticles as a Topical Agent for Treating Psoriasis.

Although conventional topical approaches for treating psoriasis have been offered as an alternative, there are still unmet medical needs such as low skin-penetrating efficacy and off-target adverse effects. A hyaluronic acid nanoparticle (HA-NP) formed by self-assembly of HA-hydrophobic moiety conjugates has been broadly studied as a nanocarrier for long-term and target-specific delivery of drugs, owing to their excellent physicochemical and biological characteristics. Here, we identify HA-NPs as topical therapeutics for treating psoriasis using in vivo skin penetration studies and psoriasis animal models. Transcutaneously administered HA-NPs were found to be accumulated and associated with pro-inflammatory macrophages in the inflamed dermis of a psoriasis mouse model. Importantly, HA-NP exerted potent therapeutic efficacy against psoriasis-like skin dermatitis in a size-dependent manner by suppressing innate immune responses and restoring skin barrier function without overt toxicity signs. The therapeutic efficacy of HA-NPs on psoriasis-like skin dermatitis was due to the outermost hydrophilic HA shell layer of HA-NPs, independent of the molecular weight of HA and hydrophobic moiety, and comparable with that of other conventional psoriasis therapeutics widely used in the clinical settings. Overall, HA-NPs have the potential as a topical nanomedicine for treating psoriasis effectively and safely.

Prevalence of sarcopenia and mortality rate in older adults with hip fracture.

BACKGROUND: Several international study groups adopted appendicular skeletal muscle mass (ASM) index adjusted by (1) height squared, (2) weight, and (3) body mass index (BMI) in the diagnosis of sarcopenia. However, different prevalence rates of sarcopenia by each index and clinical implications were not well known. The purpose of this study was to compare the differences in (1) the percentage of sarcopenia in hip fracture patients and (2) the relative mortality rate according to the sarcopenia criteria of three ASM indices. METHODS: Between January 2009 and December 2020, 1003 older adult hip fracture patients at a tertiary institution were eligible and retrospectively reviewed for this study. Based on the ASM measured on dual-energy X-ray absorptiometry, three indices were calculated, and sarcopenia was diagnosed. The proportion of sarcopenia was evaluated according to each index. One, two, and five-year mortality rates were compared between each sarcopenia group and a normal musculature group, based on ASM criteria. RESULTS: The proportion of sarcopenia patients differed according to three ASM indices. The proportion of sarcopenic patients by ASM/height2 index was higher than those of the other two indices in both male and female hip fracture patients. In male patients, 61% were sarcopenic by ASM/height2 index, 37% by ASM/weight index, and 44% by ASM/BMI index. In female patients, 26%, 11%, and 14% were sarcopenic, respectively. Among the three indices, only ASM/height2 had significant correlations with all 1-, 2-, and 5-year mortality rates. CONCLUSIONS AND IMPLICATIONS: The prevalence of sarcopenia in hip fracture patients differed substantially according to ASM indices. Sarcopenic hip fracture patients had a higher mortality rate than those with normal musculature. The 1-year, 2-year, and 5-year mortality rates were discriminated by ASM/height2 criteria in both men and women. Future prospective studies in a larger cohort are warranted.

Positivity of Rapid Antigen Testing for SARS-CoV-2 With Serial Followed-up Nasopharyngeal Swabs in Hospitalized Patients due to COVID-19.

Despite the accuracy of nucleic acid amplification tests (NAATs), rapid antigen tests (RATs) for severe acute respiratory syndrome coronavirus-2 are widely used as point-of-care tests. A total of 282 pairs of reverse transcription-polymerase chain reaction and Standard Q COVID-19 Ag tests were serially conducted for 68 patients every 3-4 days until their discharge. Through a field evaluation of RATs using direct nasopharyngeal swabs, the sensitivities were 84.6% and 87.3% for E and RNA-dependent RNA polymerase (RdRp) genes, respectively, for specimens with cycle thresholds (Cts) < 25. The Ct values of E and RdRp genes for 95% detection rates by RATs were 16.9 and 18.1, respectively. The sensitivity of RAT was 48.4% after the onset of symptoms, which was not sufficient. RAT positivity gradually decreased with increased time after symptom onset and had continuously lower sensitivity than NAATs.

Toll-like Receptor Signaling Inhibitory Peptide Improves Inflammation in Animal Model and Human Systemic Lupus Erythematosus.

Toll-like receptors (TLRs) play a major role in the innate immune system. Several studies have shown the regulatory effects of TLR-mediated pathways on immune and inflammatory diseases. Dysregulated functions of TLRs within the endosomal compartment, including TLR7/9 trafficking, may cause systemic lupus erythematosus (SLE). TLR signaling pathways are fine-tuned by Toll/interleukin-1 receptor (TIR) domain-containing adapters, leading to interferon (IFN)-α production. This study describes a TLR inhibitor peptide 1 (TIP1) that primarily suppresses the downstream signaling mediated by TIR domain-containing adapters in an animal model of lupus and patients with SLE. The expression of most downstream proteins of the TLR7/9/myeloid differentiation factor 88 (MyD88)/IFN regulatory factor 7 signaling was downregulated in major tissues such as the kidney, spleen, and lymph nodes of treated mice. Furthermore, the pathological analysis of the kidney tissue confirmed that TIP1 could improve inflammation in MRL/lpr mice. TIP1 treatment downregulated many downstream proteins associated with TLR signaling, such as MyD88, interleukin-1 receptor-associated kinase, tumor necrosis factor receptor-associated factor 6, and IFN-α, in the peripheral blood mononuclear cells of patients with SLE. In conclusion, our data suggest that TIP1 can serve as a potential candidate for the treatment of SLE.

The αC helix of TIRAP holds therapeutic potential in TLR-mediated autoimmune diseases.

Despite being crucial for combating microbes, paradoxical Toll-like receptors (TLRs) signaling have been associated with the aggravation of multiple immune disorders such as systemic lupus erythematosus, psoriasis, rheumatoid arthritis, and nonalcoholic steatohepatitis. The stoichiometry and precise arrangement of the interaction of adapters (via their Toll/interleukin-1 receptor [TIR] domains) are indispensable for the activation of TLRs and of downstream signaling cascades. Among adapters, plasma membrane-anchored MyD88 adaptor-like (MAL) has the potential for BB-loop-mediated self-oligomerization and interacts with other TIR domain-containing adaptors through αC and αD helices. Here, we used information on the MAL-αC interface to exploit its pharmacophores and to design a decoy peptide (MIP2) with broad-range TLR-inhibitory abilities. MIP2 abrogated MyD88- and TRIF-dependent lipopolysaccharide (LPS)-induced TLR4 signaling in murine and human cell lines and manifested a therapeutic potential in models of psoriasis, systemic lupus erythematosus, nonalcoholic steatohepatitis, and sepsis. Levels of hallmark serological and histological biomarkers were significantly restored and the disease symptoms were substantially ameliorated by MIP2 treatment of the animals. Collectively, our biophysical, in vitro, and in vivo findings suggest that MIP2 has broad specificity for TLRs and may be effective in modulating autoimmune complications caused by microbial or environmental factors.

A peptide derived from the core ß-sheet region of TIRAP decoys TLR4 and reduces inflammatory and autoimmune symptoms in murine models.

BACKGROUND: TLRs are some of the actively pursued drug-targets in immune disorders. Owing to a recent surge in the cognizance of TLR structural biology and signalling pathways, numerous therapeutic modulators, ranging from low-molecular-weight organic compounds to polypeptides and nucleic acid agents have been developed. METHODS: A penetratin-conjugated small peptide (TIP3), derived from the core ß-sheet of TIRAP, was evaluated in vitro by monitoring the TLR-mediated cytokine induction and quantifying the protein expression using western blot. The therapeutic potential of TIP3 was further evaluated in TLR-dependent in vivo disease models. FINDINGS: TIP3 blocks the TLR4-mediated cytokine production through both the MyD88- and TRIF-dependent pathways. A similar inhibitory-effect was exhibited for TLR3 but not on other TLRs. A profound therapeutic effect was observed in vivo, where TIP3 successfully alleviated the inflammatory response in mice model of collagen-induced arthritis and ameliorated the disease symptoms in psoriasis and SLE models. INTERPRETATION: Our data suggest that TIP3 may be a potential lead candidate for the development of effective therapeutics against TLR-mediated autoimmune disorders. FUNDING: This work was supported by the National Research Foundation of Korea (NRF-2019M3A9A8065098, 2019M3D1A1078940 and 2019R1A6A1A11051471). The funders did not have any role in the design of the present study, data collection, data analysis, interpretation, or the writing of the manuscript.

Proteomic profiling of serum from patients with tuberculosis.

BACKGROUND: Effective treatment and monitoring of tuberculosis (TB) requires biomarkers that can be easily evaluated in blood samples. The aim of this study was to analyze the serum proteome of patients with TB and to identify protein biomarkers for TB. METHODS: Serum samples from 26 TB patients and 31 controls were analyzed by using nano-flow ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry in data-independent mode, and protein and peptide amounts were calculated by using a label-free quantitative approach. The generated data were analyzed by using principal component analysis and partial least squares discriminant analysis, a multivariate statistical method. RESULTS: Of more than 500 proteins identified, alpha-1-antitrypsin was the most discriminative, which was 4.4 times higher in TB patients than in controls. Peptides from alpha-1-antitrypsin and antithrombin III increased in TB patients and showed a high variable importance in the projection scores and coefficient in partial least square discriminant analysis. CONCLUSIONS: Sera from patients with TB had higher alpha-1-antitrypsin levels than sera from control participants. Alpha-1-antitrypsin levels may aid in the diagnosis of TB.