Semi-Automated Cell Panning for Efficient Isolation of FGFR3-Targeting Antibody.

Phage display technology is a widely used practical tool for isolating binding molecules against the desired targets in phage libraries. In the case of targeting the membrane protein with its natural conformation, conventional bio-panning has limitations on the efficient screening of the functionally relevant antibodies. To enrich the single-chain variable fragment (scFv) pools for recognizing the natural conformation of the membrane targets, the conventional bio-panning and screening process was modified to include the semi-automated cell panning protocol. Using FGFR3-overexpressing patient-derived cancer cells, biotin-X-DHPE was introduced and coupled to Streptavidin-coated magnetic beads for use in the solution-phage bio-panning procedure. The resulting clones of scFv were compared to the diversity of the binding region, especially on CDR-H3. The clones enriched further by cell-based panning procedure possessed a similar binding site and the CDR-H3 loop structure. The resulting antibodies inhibited cell growth and induced target degradation. This process may be a useful tool for screening biologically related antibodies that recognize natural conformational structure on cell membrane protein. Furthermore, cell-based panning has the potential to further expand to a high-throughput screening (HTS) system and automation process.

Delayed onset filler complication: Two case reports and literature review.

Soft tissue augmentation by fillers has become a popular cosmetic tool to offer rejuvenation and aesthetic improvement. Its results are comparable to those previously achieved only by plastic surgery. However, marked increase of filler procedures is associated with a great number of complications. Complications associated with filler injection might have early onset (within days) or late onset (after weeks to years). Delayed complications include infections, foreign body granulomatous reaction, migration of filler material, persistent discoloration, and scarring. Here we report two cases of delayed complications: one case of foreign body granuloma with migration and one case of acute inflammation involving a quiescent granuloma.

Protective Effect of Botulinum Toxin Type A Against Atopic Dermatitis-Like Skin Lesions in NC/Nga Mice.

BACKGROUND: Botulinum neurotoxin (BTX) A possesses various biological activities, including anti-inflammatory and antipruritic actions. Human and animal studies have shown that BTX is effective in treating histamine-induced itch, lichen simplex chronicus, psoriasis, rosacea, allergic rhinitis, and scar prevention. However, its effect on atopic dermatitis (AD) has not been studied yet. OBJECTIVE: To examine the effect of BTX on AD using a mouse model. The primary outcome was skin thickness and transepidermal water loss (TEWL), and the secondary outcome was the alteration in skin severity scores, histological, and laboratory test results. METHODS: Forty-two NC/Nga mice (a mouse model for AD) were allocated into 6 groups (the untreated, 2-Chloro-1,3,5-trinitrobenzene [TNCB] alone, TNCB + BTX 30 U/kg, TNCB + BTX 60 U/kg, TNCB + vehicle [0.9% saline], TNCB + 0.03% tacrolimus). Those of the BTX group received intradermal injections of BTX on the rostral back once on the day of TNCB sensitization. The effect of BTX in TNCB-treated NC/Nga mice was assessed by measuring skin thickness, TEWL (primary outcome), the skin severity scores, histological changes of test skin including mast cell count, interleukin (IL)-4 mRNA and protein expression, and total serum IgE (secondary outcome). RESULTS: A single intradermal injection of BTX significantly suppressed skin thickness and TEWL in the TNCB-applied skin. The clinical severity scores, acanthosis and mast cell infiltration, were less in the BTX groups. BTX injection also inhibited TNCB-induced increase in IL-4 mRNA and protein expression in mice, but its effect on serum IgE level was not significant. CONCLUSION: The preliminary results suggest that BTX may be a novel approach to the prevention and supplemental treatment of acute AD lesions.

Transient Receptor Potential Vanilloid-1 in Epidermal Keratinocytes May Contribute to Acute Pain in Herpes Zoster.

The role of transient receptor potential vanilloid-1 (TRPV1) in the initiation of neurogenic inflammation and transduction of pain is well established. In this study 33 patients with herpes zoster (HZ) were recruited from a single centre and underwent a questionnaire interview at their first visit. Punch biopsies from the HZ lesions and the contralateral unaffected skin were performed to localize and quantify the expression of TRPV1. Immunofluorescent staining for TRPV1 was most prominent in the epidermal keratinocytes. Both TRPV1 mRNA and protein levels were significantly higher in the HZ epidermis than in control epidermis (relative ratio 1.62 ± 0.27, p = 0.033 and 2.55 ± 0.51, p = 0.005, respectively). Protein TRPV1 ratio (HZ lesion/control) correlated with the degree of pain (measured on a visual analogue scale; VAS) (p = 0.017) and was significantly lower in patients who had taken either HZ medication or painkillers prior to their visit. These results suggest that non-neuronal TRPV1 may contribute to acute pain in herpes zoster.

Corrigendum: Macrophage SREBP1 regulates skeletal muscle regeneration.

[This corrects the article DOI: 10.3389/fimmu.2023.1251784.].

descSPIM: an affordable and easy-to-build light-sheet microscope optimized for tissue clearing techniques.

Despite widespread adoption of tissue clearing techniques in recent years, poor access to suitable light-sheet fluorescence microscopes remains a major obstacle for biomedical end-users. Here, we present descSPIM (desktop-equipped SPIM for cleared specimens), a low-cost ($20,000-50,000), low-expertise (one-day installation by a non-expert), yet practical do-it-yourself light-sheet microscope as a solution for this bottleneck. Even the most fundamental configuration of descSPIM enables multi-color imaging of whole mouse brains and a cancer cell line-derived xenograft tumor mass for the visualization of neurocircuitry, assessment of drug distribution, and pathological examination by false-colored hematoxylin and eosin staining in a three-dimensional manner. Academically open-sourced ( https://github.com/dbsb-juntendo/descSPIM ), descSPIM allows routine three-dimensional imaging of cleared samples in minutes. Thus, the dissemination of descSPIM will accelerate biomedical discoveries driven by tissue clearing technologies.

Transgenic viral expression of PH-20, IL-12, and sPD1-Fc enhances immune cell infiltration and anti-tumor efficacy of an oncolytic virus.

Oncolytic viruses are of significant clinical interest due to their ability to directly infect and kill tumors and enhance the anti-tumor immune response. Previously, we developed KLS-3010, a novel oncolytic virus derived from the International Health Department-White (IHD-W) strain vaccinia virus, which has robust tumoricidal effects. In the present study, we generated a recombinant oncolytic virus, KLS-3020, by inserting three transgenes (hyaluronidase [PH-20], interleukin-12 [IL-12], and soluble programmed cell death 1 fused to the Fc domain [sPD1-Fc]) into KLS-3010 and investigated its anti-tumor efficacy and ability to induce anti-tumor immune responses in CT26.WT and B16F10 mouse tumor models. A single injection of KLS-3020 significantly decreased tumor growth. The roles of the transgenes were investigated using viruses expressing each single transgene alone and KLS-3020. PH-20 promoted virus spread and tumor immune cell infiltration, IL-12 activated and reprogrammed T cells to inflammatory phenotypes, and sPD1-Fc increased intra-tumoral populations of activated T cells. The tumor-specific systemic immune response and the abscopal tumor control elicited by KLS-3020 were demonstrated in the CT26.WT tumor model. The insertion of transgenes into KLS-3020 increased its anti-tumor efficacy, supporting further clinical investigation of KLS-3020 as a novel oncolytic immunotherapy.

Macrophage SREBP1 regulates skeletal muscle regeneration.

Macrophages are essential for the proper inflammatory and reparative processes that lead to regeneration of skeletal muscle after injury. Recent studies have demonstrated close links between the function of activated macrophages and their cellular metabolism. Sterol regulatory element-binding protein 1 (SREBP1) is a key regulator of lipid metabolism and has been shown to affect the activated states of macrophages. However, its role in tissue repair and regeneration is poorly understood. Here we show that systemic deletion of Srebf1, encoding SREBP1, or macrophage-specific deletion of Srebf1a, encoding SREBP1a, delays resolution of inflammation and impairs skeletal muscle regeneration after injury. Srebf1 deficiency impairs mitochondrial function in macrophages and suppresses the accumulation of macrophages at sites of muscle injury. Lipidomic analyses showed the reduction of major phospholipid species in Srebf1 -/- muscle myeloid cells. Moreover, diet supplementation with eicosapentaenoic acid restored the accumulation of macrophages and their mitochondrial gene expression and improved muscle regeneration. Collectively, our results demonstrate that SREBP1 in macrophages is essential for repair and regeneration of skeletal muscle after injury and suggest that SREBP1-mediated fatty acid metabolism and phospholipid remodeling are critical for proper macrophage function in tissue repair.

Impact of height difference between coronary ostium and location of intracoronary pressure sensor on fractional flow reserve measurements.

BACKGROUND: During fractional flow reserve (FFR) measurements, distal coronary pressure (Pd) can be influenced by hydrostatic pressure changes resulting from the height difference (HD) between the coronary ostium and the location of the distal pressure sensor. AIMS: We investigated the effect of aortocoronary HD on the FFR measurements in each coronary artery. METHODS: In this retrospective cohort study, we analyzed 257 patients who underwent FFR measurements and coronary computed tomography (CCTA) within a year. Using CCTA, we measured HD as the vertical distance between the coronary ostium and a matched point of the distal coronary pressure sensor identified on coronary angiography. RESULTS: The location of the Pd sensor was higher than the coronary ostium in the left anterior descending artery (LAD) (-4.64 ± 1.15 cm) and lower than the coronary ostium in the left circumflex artery (LCX) (2.54 ± 1.05 cm) and right coronary artery (RCA) (2.03 ± 1.28 cm). The corrected FFR values by HD were higher in the LAD (0.78 ± 0.09 to 0.82 ± 0.09, P<0.01) and lower in the LCX and RCA than the original FFR values (0.87 ± 0.07 to 0.85 ± 0.08, P<0.01; 0.87 ± 0.10 to 0.86 ± 0.10, P<0.01, respectively). Using an FFR cut-off value of 0.8, the concordance rates between the FFR and corrected FFR values were 77.8%, 95.2%, and 100% in the LAD, LCX, and RCA, respectively. CONCLUSION: HD between the coronary ostium and the distal coronary pressure sensor may affect FFR measurements and FFR-guided treatment decisions for coronary artery disease.

Native Aortic Valve Thrombus Complicating Intermittent Occlusion of a Left Main Coronary Artery.

Intermittent left main coronary artery ostium obstruction (LMOO) caused by native aortic valve thrombus (NAVT) is an extremely rare condition. It may therefore be challenging to identify the cause using only coronary angiography, even though the clinical presentation and electrocardiography (ECG) strongly suggest myocardial infarction. We herein report a 53-year-old man with NAVT complicating intermittent occlusion of left main disease in preexisting coronary artery stenosis.

Characterization of Gut Microbiome in Korean Patients with Metabolic Associated Fatty Liver Disease.

Metabolic associated fatty liver disease (MAFLD) is a new concept where the presence of both fatty liver and metabolic abnormality are necessary for diagnosis. Several studies have reported that altered gut microbiome is closely associated with metabolic diseases and non-alcoholic fatty liver disease. However, the studies on MAFLD population are scarce. This prospective study aimed to identify differences in gut microbiome between patients with MAFLD and healthy controls in Korean population. In this study, patients with MAFLD and age, sex-matched healthy controls were included, and their stool samples were collected. Taxonomic composition of gut microbiota was analyzed using 16S ribosomal ribonucleic acid pyrosequencing. Twenty-two MAFLD patients and 44 healthy controls were included. Taxonomic diversity was lower in patients with MAFLD in the aspect of alpha and beta diversity. The differences were also found at phylum, class, family, and genus levels between the two groups. Phylum Proteobacteria, family Enterobactereriaceae, genus Citrobacter abundance was significantly increased and genus Faecalibacterium was significantly decreased in patients with MAFLD. In addition, butyrate-producing bacteria were decreased and ethanol-producing bacteria were increased in patients with MAFLD. The composition of gut microbiome was different between MAFLD and healthy controls in Korean population. This could offer potential targets for therapeutic intervention in MAFLD.

Comparative analysis of three nutrition scores in predicting mortality after acute myocardial infarction.

OBJECTIVE: We investigated the utility of nutrition scores in predicting mortality and prognostic importance of nutrition status using three different scoring systems in patients with acute myocardial infarction (AMI). METHODS: In total, 1147 patients with AMI were enrolled in this study (72.5 % men; mean age 65.6 years). Patients were divided into three groups according to the geriatric nutritional risk index (GNRI); prognostic nutritional index (PNI); and triglycerides, total cholesterol, and body weight index(TCBI) scores as tertile: low (GNRI ≤ 103.8, n = 382), intermediate (103.8 < GNRI ≤ 112.3, n = 383), and high (GNRI > 112.3, n = 382) GNRI groups; low (PNI ≤ 50.0, n = 382), intermediate (50.0 < PNI ≤ 56.1, n = 383), and high (PNI > 56.1, n = 382) PNI groups; and low (TCBI ≤ 1086.4, n = 382), intermediate (1086.3 < GNRI ≤ 2139.1, n = 383), and high (TCBI > 2139.1, n = 382) TCBI groups. RESULTS: In the GNRI, TCBI, and PNI groups, the cumulative incidence of all-cause death and major adverse cardiovascular events (MACEs) was significantly higher in the low score group, followed by the intermediate and high score groups. Moreover, both intermediate and low PNI groups had a similar cumulative incidence of all-cause death and MACE. The GNRI score (AUC 0.753, 95% CI 0.608~0.745, P = 0.009) had significantly higher areas under the curve (AUCs) than the TCBI (AUC 0.659, 95% CI 0.600~0.719, reference) and PNI (AUC 0.676, 95% CI 0.608~0.745, P = 0.669) scores. CONCLUSIONS: Patients with low nutrition scores were at a higher risk of MACE and all-cause death than patients with high nutrition scores. Additionally, the GNRI had the greatest incremental value in predicting risks among the three different scoring systems used in this study.

cIRCR201-dPBD, a Novel Pyrrolobenzodiazepine Dimer-Containing Site-Specific Antibody-Drug Conjugate Targeting c-Met Overexpression Tumors.

c-Met, as a receptor expressed on the cell membrane, contributes to the growth and metastasis of tumors, as well as angiogenesis, mainly through the hepatocyte growth factor (HGF)/c-Met axis during tumor progression. Although several c-Met inhibitors, including small molecules and monoclonal antibody inhibitors, are currently being investigated, their clinical outcomes have not been promising. Development of an antibody-drug conjugate (ADC) against c-Met could be an attractive therapeutic strategy that would provide superior antitumor efficacy with broad-spectrum c-Met expression levels. In the present study, site-specific drug-conjugate technology was applied to develop an ADC using the human-mouse cross-reactive c-Met antibody and a prodrug pyrrolobenzodiazepine (PBD). The toxin payload was uniformly conjugated to the light-chain C-terminus of the native cIRCR201 antibody (drug-to-antibody ratio = 2), as confirmed using LC-MS. Using a high-throughput screening system, we found that cIRCR201-dPBD exhibited varying sensitivities depending on the expression levels of c-Met, and it induced receptor-mediated endocytosis and toxin-mediated apoptosis in 47 different cancer cell lines. cIRCR201-dPBD also showed significant antitumor activity on the MET-amplified cancer cells using in vivo xenograft models. Therefore, cIRCR201-dPBD could be a promising therapeutic strategy for tumors with c-Met expression.

Predictive value of the combination of brachial-ankle pulse wave velocity and ankle-brachial index for cardiovascular outcomes in patients with acute myocardial infarction.

BACKGROUND: Although ankle-brachial index and brachial-ankle pulse wave velocity measurement are well-established modalities for assessing peripheral artery disease and arterial stiffness and predicting cardiovascular events, it is unclear which one is more important or if a combination of the two is more effective for determining prognosis among patients with acute myocardial infarction. METHODS: Patients with acute myocardial infarction (n = 889) were stratified into four groups according to a brachial-ankle pulse wave velocity (cut-off value: 1684 cm/s) and ankle-brachial index (cut-off value: 0.98): group I (high ankle-brachial index and low brachial-ankle pulse wave velocity, n = 389), group II (high ankle-brachial index and high brachial-ankle pulse wave velocity, n = 281), group III (low ankle-brachial index and low brachial-ankle pulse wave velocity, n = 103), group IV (low ankle-brachial index and high brachial-ankle pulse wave velocity, n = 116). The mean follow-up duration was 348 days. RESULTS: Major adverse cardiovascular events or cardiac death occurred in 64 (7.2%) and 26 patients (2.9%), respectively. In multivariable analysis, group III and IV had a significant high hazard ratio for major adverse cardiovascular events (5.93, 5.43) and cardiac death (13.51, 19.06). Additionally, ankle-brachial index had a higher hazard ratio than brachial-ankle pulse wave velocity for major adverse cardiovascular events (3.38 vs. 1.40) and cardiac death (6.21 vs. 2.40). When comparing receiver operating characteristic curves of the combined models of risk factors, brachial-ankle pulse wave velocity, and ankle-brachial index, pulse wave velocity plus ankle-brachial index or pulse wave velocity plus ankle-brachial index plus risk factors were significantly more predictive of major adverse cardiovascular events than risk factors. CONCLUSION: Our findings indicate that ankle-brachial index is a strong independent prognostic factor and adding a brachial-ankle pulse wave velocity measurement to ankle-brachial index increases the prognostic power for cardiac events in patients with acute myocardial infarction, while ankle-brachial index and pulse wave velocity showed additive value to risk factors.

The Beneficial Effect of Korean Red Ginseng Extract on Atopic Dermatitis Patients: An 8 Weeks Open, Noncomparative Clinical Study.

BACKGROUND: Atopic dermatitis (AD) is a chronic or chronically relapsing, eczematous, severely pruritic inflammatory skin disorder. Korean red ginseng (KRG) has been shown previously to exhibit diverse biological effects including anti-inflammatory and antipruritic effects in a murine model. OBJECTIVE: We aimed to investigate the beneficial effects of KRG on AD patients, to determine whether there was improvement in disease severity, skin barrier function, pruritus and sleep disturbance relief. METHODS: An open, noncomparative clinical study that utilized KRG tablets (500 mg/tablet) was conducted. This study included 41 patients with mild to moderate AD diagnosed by the Korean atopic dermatitis guidelines. Three visits to the hospital at days 1, 28±7, and 56±7 for evaluation were made. The effects of KRG were assessed by measuring eczema area and severity index (EASI) score, transepidermal water loss (TEWL), the visual analogue scale (VAS), total amount of topical agents used in recent 8 weeks and investigator global assessment (IGA). RESULTS: Patients taking KRG tablets showed significant decreases in EASI score and TEWL, and the VAS of pruritus and sleep disturbance were significantly reduced. The amount of topical agents used was reduced but not by a statistically significant amount. IGA at the third visit showed improvement of AD compared to the second visit, but the difference was not statistically significant. CONCLUSION: KRG can be safely used as a health food to achieve clinical improvement of AD as well as improving overall quality of life, and has potential for further development.

Infundibulocystic Squamous Cell Carcinoma.

Infundibulocystic squamous cell carcinoma was first reported in 2008 as a subset of squamous cell carcinoma arising from the infundibulum of the hair follicle and exhibiting infundibular differentiation. It has well-differentiated, less-differentiated, and infiltrative forms. It was thoroughly analyzed in a series of cases in 2011 by Misago et al. and has been redefined to include only the infiltrative form owing to its unique clinical and histological characteristics. Here, we report an interesting case of infundibulocystic squamous cell carcinoma in a 72-year-old man presenting with a mass on the left helix of the ear.

NDRG2-mediated Modulation of SOCS3 and STAT3 Activity Inhibits IL-10 Production.

BACKGROUND: N-myc downstream regulated gene 2 (NDRG2) is a member of the NDRG gene family. Our previous report indicated a possible role for NDRG2 in regulating the cytokine, interleukin-10 (IL-10), which is an important immunosuppressive cytokine. Several pathways, including p38-MAPK, NF-κB, and JAK/STAT, are used for IL-10 production, and the JAK/STAT pathway can be inhibited in a negative feedback loop by the inducible protein, SOCS3. In the present study, we investigated the effect of NDRG2 gene expression on IL-10 signaling pathway that is modulated via SOCS3 and STAT3. METHODS: We generated NDRG2-overexpressing U937 cell line (U937-NDRG2) and treated the cells with PMA to investigate the role of NDRG2 in IL-10 production. U937 cells were also transfected with SOCS3- or NDRG2-specific siRNAs to examine whether the knockdown of SOCS3 or NDRG2 influenced IL-10 expression. Lastly, STAT3 and SOCS3 induction was measured to identify the signaling pathway that was associated with IL-10 production. RESULTS: RT-PCR and ELISA assays showed that IL-10 was increased in U937-mock cells upon stimulation with PMA, but IL-10 was inhibited by overexpression NDRG2. After PMA treatment, STAT3 phosphorylation was decreased in a time-dependent manner in U937-mock cells, whereas it was maintained in U937-NDRG2 cells. SOCS3 was markedly reduced in U937-NDRG2 cells compared with U937-mock cells. IL-10 production after PMA stimulation was reduced in U937 cells when SOCS3 was inhibited, but this effect was less severe when NDRG2 was inhibited. CONCLUSION: NDRG2 expression modulates SOCS3 and STAT3 activity, eventually leading to the inhibition of IL-10 production.

SK-126, a synthetic compound, regulates the production of inflammatory cytokines induced by LPS in antigen-presenting cells.

A variety of mediators released by immune cells triggers or enhances specific aspects of the inflammatory response. Dendritic cells (DCs) play an essential role in the innate immune system by shaping the adaptive immune responses and by controlling the production of cytokines in response to inflammatory stimuli. In the present study, we investigated whether SK-126, a pyridine derivative based on gentianine originated from a natural product, can affect the LPS-induced inflammatory cytokine production in DC. Interestingly, treatment of mouse bone marrow-derived dendritic cells (BMDCs) and the murine dendritic cell line, DC 2.4, with SK-126 completely suppressed LPS-induced TNF-alpha expression at both transcriptional and protein levels. In contrast to TNF-alpha, SK-126 enhanced IL-10 expression at both transcriptional and protein levels. To determine signaling pathways involved in the regulation of inflammatory cytokines, we examined the involvement of MAPK and the transcription factor, NF-kappaB. SK-126 enhanced ERK1/2 and p38 activation following LPS stimulation, but it did not induce phosphorylation of SAPK/JNK and NF-kappaB. Also, STAT3 phosphorylation after LPS stimulation was increased by SK-126 to a large extent. Using specific inhibitors, we confirmed that SK-126 has dual effects in which it suppresses TNF-alpha production and enhances IL-10 production via the up-regulation of ERK1/2 and p38. Finally, LPS-induced inflammatory responses such as TNF-alpha production in vivo were significantly reduced by treatment with SK-126. Therefore, our findings suggest that SK-126 may be a useful drug candidate to treat inflammatory diseases in which pro- or anti-inflammatory cytokines play a significant role in their pathogenesis.