Prognostic value of serum tartrate­resistant acid phosphatase­5b for bone metastasis in patients with resectable breast cancer.

Bone metastasis significantly affects the quality of life of patients with metastatic breast cancer, and can shorten overall survival. Identifying patients with early-stage breast cancer at high risk for bone metastasis and preventing bone metastasis may lead to a better quality of life and prolonged survival. The present study investigated whether serum tartrate-resistant acid phosphatase-5b (TRACP-5b), a bone turnover marker, can be a prognostic factor for bone metastasis. Female patients who underwent resectable breast surgery between May 2002 and August 2006 were consecutively investigated. A total of 304 patients with a median follow-up of 3,722 days were retrospectively analyzed. TRACP-5b levels in sera prepared from patients' blood drawn preoperatively without any presurgical treatments were measured using an enzyme-linked immunosorbent assay. The cutoff of TRACP-5b levels, in order to separate patients into high and low TRACP-5b groups, was set at median (347 mU/dl). The associations of clinicopathological factors, including TRACP-5b, with bone metastasis-free interval (BMFI), which was defined as the duration between surgery and the diagnosis of bone metastasis at any time point, were examined. Multivariate analysis of various clinicopathological features revealed that lymph node metastasis and histological grade were independent factors associated with BMFI (P=0.017 and 0.030, respectively). In patients with node-positive breast cancer (n=114), a high TRACP-5b level and a high grade were significantly and independently associated with worse BMFI (log-rank P=0.041 and 0.011, respectively). In conclusion, these findings indicated that TRACP-5b may predict bone metastasis in patients with node-positive breast cancer.

High HER2 Intratumoral Heterogeneity Is a Predictive Factor for Poor Prognosis in Early-Stage and Locally Advanced HER2-Positive Breast Cancer.

PURPOSE: Breast cancer tumors frequently have intratumoral heterogeneity (ITH). Tumors with high ITH cause therapeutic resistance and have human epidermal growth factor receptor 2 (HER2) heterogeneity in response to HER2-targeted therapies. This study aimed to investigate whether high HER2 heterogeneity levels were clinically related to a poor prognosis for HER2-targeted adjuvant therapy resistance in primary breast cancers. METHODS: This study included patients with primary breast cancer (n = 251) treated with adjuvant HER2-targeted therapies. HER2 heterogeneity was manifested by the shape of HER2 fluorescence in situ hybridization amplification (FISH) distributed histograms with the HER2 gene copy number within a tumor sample. Each tumor was classified into a biphasic grade graph (high heterogeneity [HH]) group or a monophasic grade graph (low heterogeneity [LH]) group based on heterogeneity. Both groups were evaluated for disease-free survival (DFS) and overall survival (OS) for a median of ten years of annual follow-up. RESULTS: Of 251 patients with HER2-positive breast cancer, 46 (18.3%) and 205 (81.7%) were classified into the HH and LH groups, respectively. The HH group had more distant metastases and a poorer prognosis than the LH group (DFS: p < 0.001 (HH:63% vs. LH:91% at 10 years) and for the OS: p = 0.012 (HH:78% vs. LH:95% at 10 years). CONCLUSIONS: High HER2 heterogeneity is a poor prognostic factor in patients with HER2-positive breast cancer. A novel approach to heterogeneity, which is manifested by the shape of HER2 FISH distributions, might be clinically useful in the prognosis prediction of patients after HER2 adjuvant therapy.

Prognosis of anemic patients with atrial fibrillation undergoing percutaneous coronary intervention.

BACKGROUND: There are limited data regarding whether anemia is associated with adverse clinical outcomes in patients with atrial fibrillation (AF) after percutaneous coronary intervention (PCI). METHODS: Patients with AF undergoing PCI at 15 institutions between January 2015 and March 2021 were included in this analysis. Based on the baseline hemoglobin levels, moderate to severe anemia was defined as hemoglobin levels <11 g/dL, and mild anemia was defined as hemoglobin levels 11-12.9 g/dL for men and 11-11.9 g/dL for women. Clinical outcomes within 1 year, including major adverse cardiovascular events (MACE: all-cause death, myocardial infarction, stent thrombosis, and stroke) and major bleeding events (BARC 3 or 5), were compared among patients with moderate/severe anemia, mild anemia, and no anemia. RESULTS: In a total of 746 enrolled patients, 119 (16.0%) and 168 (22.5%) patients presented with moderate/severe and mild anemia. The incidence of MACE (22.5%, 11.0%, and 9.1%, log-rank p < 0.001), all-cause death (20.0%, 7.2%, and 4.8%, log-rank p < 0.001), and major bleeding events (10.7%, 6.5%, and 2.7%, log-rank p < 0.001) were the highest in the moderate/severe anemia group compared with the mild and no anemia groups. Multivariable Cox regression analyses determined moderate/severe anemia as an independent predictor for MACE (p = 0.008), all-cause death (p = 0.005), and major bleeding events (p = 0.031) at 1 year after PCI. CONCLUSION: Moderate/severe anemia was significantly associated with the higher incidence of MACE and all-cause death as well as major bleeding events compared with mild and no anemia in AF patients undergoing PCI.

Evaluation of RASSF1A methylation in the lysate of sentinel lymph nodes for detecting breast cancer metastasis: A diagnostic accuracy study.

The restriction enzyme-based digital methylation-specific polymerase chain reaction (RE-dMSP) assay is useful for diagnosing sentinel lymph node (SN) metastasis in patients with breast cancer, by detecting tumor-derived methylated Ras association domain-containing protein 1 (RASSF1A). In addition, this assay has high concordance (95.0%) with one-step nucleic acid amplification (OSNA). The present study aimed to perform RE-dMSP using OSNA lysate from more patients and to re-evaluate its clinical usage. Overall, 418 SNs from 347 patients were evaluated using both OSNA and RE-dMSP. The concordance rate was 83.3% (348/418). RASSF1A methylation of the primary tumors was negative in 36 patients. When these patients were excluded, the concordance rate improved to 88.2% (330/374). Of the 79 OSNA-negative cases, 19 were RE-dMSP-positive, although all were positive for cytokeratin 19 expression in the primary tumor, suggesting that RE-dMSP can detect tumor-derived DNA with a higher sensitivity. The percent of methylated reference of the breast tumors showed a wide variety in the 16 OSNA-positive/RE-dMSP-negative cases, and such variability of methylation could have affected the results in these patients. In conclusion, although RE-dMSP can diagnose SN metastasis with high sensitivity and accuracy, and can be a supplementary tool to OSNA in breast cancer, RE-dMSP showed certain discordance with OSNA and critically depended on the absence or heterogeneity of DNA methylation in breast tumors. Further research is expected to develop an assay targeting other DNA alterations, such as mutations.

Initial Evaluation of [18F]FAPI-74 PET for Various Histopathologically Confirmed Cancers and Benign Lesions.

The 18F-labeled fibroblast activation protein inhibitor (FAPI) [18F]FAPI-74 has the benefit of a higher synthetic yield and better image resolution than 68Ga-labeled FAPI. We preliminarily evaluated the diagnostic performance of [18F]FAPI-74 PET in patients with various histopathologically confirmed cancers or suspected malignancies. Methods: We enrolled 31 patients (17 men and 14 women) with lung cancer (n = 7), breast cancer (n = 5), gastric cancer (n = 5), pancreatic cancer (n = 3), other cancers (n = 5), and benign tumors (n = 6). Twenty-seven of the 31 patients were treatment-naïve or preoperative, whereas recurrence was suspected in the remaining 4 patients. Histopathologic confirmation was obtained for the primary lesions of 29 of the 31 patients. In the remaining 2 patients, the final diagnosis was based on the clinical course. [18F]FAPI-74 PET scanning was performed 60 min after the intravenous injection of [18F]FAPI-74 (240 ± 31 MBq). The [18F]FAPI-74 PET images were compared between the primary or local recurrent lesions of malignant tumors (n = 21) and nonmalignant lesions (n = 8: type-B1 thymomas, granuloma, solitary fibrous tumor, and postoperative or posttherapeutic changes). The uptake and number of detected lesions on [18F]FAPI-74 PET were also compared with those on [18F]FDG PET for available patients (n = 19). Results: [18F]FAPI-74 PET showed higher uptake in primary lesions of various cancers than in nonmalignant lesions (median SUVmax, 9.39 [range, 1.83-25.28] vs. 3.49 [range, 2.21-15.58]; P = 0.053), but some of the nonmalignant lesions showed high uptake. [18F]FAPI-74 PET also showed significantly higher uptake than [18F]FDG PET (median SUVmax, 9.44 [range, 2.50-25.28] vs. 5.45 [range, 1.22-15.06] in primary lesions [P = 0.010], 8.86 [range, 3.51-23.33] vs. 3.84 [range, 1.01-9.75] in lymph node metastases [P = 0.002], and 6.39 [range, 0.55-12.78] vs. 1.88 [range, 0.73-8.35] in other metastases [P = 0.046], respectively). In 6 patients, [18F]FAPI-74 PET detected more metastatic lesions than [18F]FDG PET. Conclusion: [18F]FAPI-74 PET showed higher uptake and detection rates in primary and metastatic lesions than did [18F]FDG PET. [18F]FAPI-74 PET is a promising novel diagnostic modality for various tumors, especially for precise staging before treatment, including characterization of tumor lesions before surgery. Moreover, 18F-labeled FAPI ligand might serve a higher demand in clinical care in the future.

Detection of Ultra-Rare ESR1 Mutations in Primary Breast Cancer Using LNA-Clamp ddPCR.

ESR1 mutations in breast cancer are one of the mechanisms of resistance to aromatase inhibitors. These mutations are common in metastatic breast cancer; however, these are rare in primary breast cancer. However, these data have been analyzed mainly in formalin-fixed, paraffin-embedded tissue; thus, rare mutations that may be present in primary breast cancer may be overlooked. In this study, we developed a highly sensitive mutation detection method called locked nucleic acid (LNA)-clamp droplet digital PCR (ddPCR) and validated it. The mutation detection sensitivity was substantiated to 0.003%. Then, we used this method to analyze ESR1 mutations in fresh-frozen (FF) tissues of primary breast cancer. cDNA extracted from the FF tissues of 212 patients with primary breast cancers were measured. Twenty-eight ESR1 mutations were found in twenty-seven (12.7%) patients. Sixteen (7.5%) patients had Y537S mutations and twelve (5.7%) had D538G mutations. Two mutations with a variant allele frequency (VAF) of ≥0.1% and twenty-six mutations with a VAF of <0.1% were found. By using this LNA-clamp ddPCR, this study demonstrated the presence of minor clones with a VAF of <0.1% in primary breast cancer.

[Usefulness of Simple Dietary Check Sheet as Screening Tool to Assess the Risk of Muscle Mass Reduction in Community-Dwelling Middle-Aged and Older Adults].

OBJECTIVES: The aim of this study was to investigate the usefulness of a simple dietary check sheet to assess the risk of muscle mass reduction in middle-aged and older adults. METHODS: The study participants comprised 1,272 community-dwelling individuals aged 50-89 years (mean age; 68.7 years). Bioelectrical impedance analysis was performed to estimate the appendicular skeletal muscle mass index (SMI, kg/m2). The SMIs were expressed as z-scores and adjusted for age and gender. A simple dietary check sheet was used to assess the daily intake of foods associated with maintaining muscle mass, such as meat, fish, eggs, milk, soybean products, and vegetables. RESULTS: Individuals with reduced muscle masses (SMI z-scores < -1.0) had significantly lower intakes of meat, fish, eggs, milk, and vegetables, and a lower overall dietary intake than individuals without reduced muscle masses (SMI z-scores ≥ -1.0). Food intake score was calculated to obtain quantitative estimates of the daily intake of these foods. The scores ranged from 0 to 14, with higher scores indicating higher intakes of foods that contribute to maintaining the muscle mass. Compared with the reference group with scores of ≥ 10, the groups with lower scores were at a higher risk of muscle mass reduction. The odds ratios (95% confidence interval) of the groups with scores of 9, 8, 7, 6, and ≤ 5 were 1.15 (0.42-3.13), 2.10 (0.89-4.95), 3.64 (1.61-8.23), 4.49 (1.90-10.58), and 7.53 (3.06-18.51), respectively, after adjusting for age, gender, obesity, alcohol intake, smoking, physical inactivity, hypertriglyceridemia, diabetes mellitus, and liver dysfunction. CONCLUSIONS: As the food intake scores were significantly associated with decreased muscle mass, the proposed simple dietary check sheet may help assess the risk of muscle mass reduction in middle-aged and older adults from a nutritional perspective.

Hypercalcemia affected in metastatic breast cancer patients without bone metastasis: report of three cases.

BACKGROUND: Since humoral hypercalcemia of malignancy (HHM) in breast cancer patients without bone metastasis is rare, the clinical features of this condition are not fully understood. CASE PRESENTATION: During the recent 12 years, 3602 patients were diagnosed with breast cancer in our institution, and only three patients developed HHM without bone metastasis. They were all recurrent breast cancer patients with visceral metastases including the lung and the liver. It took no more than 2 months since symptomatic onset to hospitalization because of hypercalcemia. The maximum serum calcium concentrations were 15.0 mg/dL or higher. All patients had symptoms related to hypercalcemia. Treatment of hypercalcemia including hydration, calcitonin, bisphosphonate, and diuretics was initially effective in the three patients. However, two of three cases were eventually fatal because of unsuccessful treatment of breast cancer. CONCLUSIONS: The common features of HHM without bone metastasis in breast cancer patients include acute onset, severe symptomatic hypercalcemia, and presence of visceral metastasis. Treatment of hypercalcemia decreased serum calcium level in a short period, while successful treatment of breast cancer was essential for a long-term management of HHM. This report provides a consideration to help elucidate the pathophysiology and medical care of breast cancer patients with HHM without bone metastasis.

Case of a pregnant woman with probable prolonged SARS-CoV-2 viral shedding 221 days after diagnosis.

We describe a case of probable prolonged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Alpha(B.1.1.7) variant shedding for 221 days from the diagnosis, in a healthy 20-year-old Japanese pregnant woman with a normal delivery. To our knowledge, this is the longest duration of SARS-CoV-2 shedding reported in an immunocompetent individual to date.

A case of sternal osteomyelitis during treatment with everolimus for recurrent breast cancer.

BACKGROUND: Everolimus is a mechanistic-target-of-rapamycin (mTOR) inhibitor bearing a potent antitumor effect against hormone receptor-positive breast cancer. Here, we report the case of a patient with recurrent breast cancer who developed osteomyelitis during the treatment with everolimus plus exemestane. CASE PRESENTATION: A 56-year-old woman with early-stage breast cancer underwent right mastectomy and axillary lymph node dissection at the age of 45. Four years after the surgery, she experienced relapse at the chest wall. Radiotherapy was performed on the chest wall, including the sternum, and denosumab was administered. After several regimens of hormonal therapies, everolimus in combination with exemestane was administered. Three months later, the patient visited our clinic because of continuous fever. A computed tomography scan showed an osteolytic change in the sternal bone with pneumomediastinum, which indicated sternal osteomyelitis. Extensive debridement followed by secondary reconstruction of the chest wall was successfully performed. CONCLUSIONS: Everolimus may cause osteomyelitis of the affected bone as a result of tumor necrosis. Everolimus-induced osteomyelitis may be manageable by extensive debridement performed without delay.

Muscle mass reduction, low muscle strength, and their combination are associated with arterial stiffness in community-dwelling elderly population: the Wakayama Study.

Age-related loss of skeletal muscle mass and function is associated with some predisposing factors that increase the risk of vascular damage. This study aimed to examine whether muscle mass reduction, low muscle strength, and their combination were related to arterial stiffness in community-dwelling elderly population. Study participants consisted of 1046 elderly individuals (aged 72 ± 5 years) without cardiovascular disease, chronic kidney disease, or liver disease. Bioelectrical impedance analysis was performed to estimate appendicular skeletal muscle mass (ASM). A value for ASM was normalized for height (ASM index, kg/m2). Handgrip strength (HGS) was measured using a Smedley grip dynamometer. Brachial-ankle pulse wave velocity (baPWV) was evaluated as an index of arterial stiffness using a simple automatic oscillometric technique. When participants were stratified based on baPWV cut-off values (< 1800 cm/s, 1800 to 1999 cm/s, ≥ 2000 cm/s), ASM index and HGS progressively decreased with an increase in baPWV levels (P for trend < 0.001). In multiple regression analysis, baPWV was significantly associated with ASM index (ß = -0.270, P < 0.001) and HGS (ß = -0.102, P < 0.001) independent of potential confounding factors. The baPWV of the subgroup with low ASM index and low HGS was significantly higher than that of those with only low ASM index or low HGS (P < 0.001). These results suggest that loss of skeletal muscle mass and function is associated with increased arterial stiffness in the elderly population, and the combination of muscle mass reduction and low muscle strength may lead to greater arterial stiffness than each of the individual conditions.

Vascular Response after Directional Coronary Atherectomy for Left Main Bifurcation Lesion.

AIMS: To evaluate the vascular response after directional coronary atherectomy (DCA) for left main (LM) bifurcation lesion. METHODS: This study was a retrospective, single-center study enrolling 31 patients who underwent stentless therapy using DCA followed by drug-coated balloon (DCB) angioplasty for LM bifurcation lesion. We compared intravascular ultrasound (IVUS) findings before and after DCA. RESULTS: After DCA, the lumen and vessel areas significantly increased, whereas the plaque area (PA) and %PA were significantly reduced. When the lesions were divided into small vessel and large vessel groups using the median value of the vessel area, the maximum balloon pressure of the DCA catheter was greater in the large vessel group. Changes in the lumen and vessel areas were also significantly greater in the large vessel group. On the other hand, the changes in PA and %PA were similar between groups. CONCLUSIONS: The main vascular responses associated with lumen enlargement after DCA were plaque reduction and vessel expansion. Contribution of vessel expansion to lumen enlargement was larger than the effect of plaque reduction in large vessel lesions.

Development of a quantitative, portable, and automated fluorescent blue-ray device-based malaria diagnostic equipment with an on-disc SiO2 nanofiber filter.

There is an urgent need to develop an automated malaria diagnostic system that can easily and rapidly detect malaria parasites and determine the proportion of malaria-infected erythrocytes in the clinical blood samples. In this study, we developed a quantitative, mobile, and fully automated malaria diagnostic system equipped with an on-disc SiO2 nanofiber filter and blue-ray devices. The filter removes the leukocytes and platelets from the blood samples, which interfere with the accurate detection of malaria by the blue-ray devices. We confirmed that the filter, which can be operated automatically by centrifugal force due to the rotation of the disc, achieved a high removal rate of leukocytes (99.7%) and platelets (90.2%) in just 30 s. The automated system exhibited a higher sensitivity (100%) and specificity (92.8%) for detecting Plasmodium falciparum from the blood of 274 asymptomatic individuals in Kenya when compared to the common rapid diagnosis test (sensitivity = 98.1% and specificity = 54.8%). This indicated that this system can be a potential alternative to conventional methods used at local health facilities, which lack basic infrastructure.

High Throughput Screening of Serum γ-Glutamyl Dipeptides for Risk Assessment of Nonalcoholic Steatohepatitis with Impaired Glutathione Salvage Pathway.

Nonalcoholic fatty liver disease (NAFLD) is the most common preventable chronic liver disorder in developed countries, the prevalence of which is increasing worldwide due to its association with obesity and type 2 diabetes. However, the exact mechanisms of NAFLD pathophysiology remain poorly understood including its progression to the more severe nonalcoholic steatohepatitis (NASH). New advances for early detection and monitoring of NASH progression are limited due to the lack of specific blood biomarkers, thus requiring invasive liver biopsies for histopathology. Herein, multisegment injection-capillary electrophoresis-tandem mass spectrometry (MSI-CE-MS/MS) is validated as a high throughput, robust, and quantitative platform for targeted analysis of a panel of 16 serum γ-glutamyl dipeptides from a cohort of NASH adult patients from Japan (median age = 53 years, median BMI = 27 kg/m2, n = 116). Multiplexed separations based on MSI-CE-MS/MS enable the design of unique data workflows that rely on customizable serial sample injection formats for accurate determination of γ-glutamyl dipeptides with quality control. Also, the introduction of a liquid coolant device to the capillary outlet improves long-term migration time stability in CE. Unsupervised pattern recognition methods revealed two distinctive NASH subgroups based on their contrasting γ-glutamyl dipeptide status despite patients having similar clinical phenotypes and NASH activity scores (median NAS ≈ 6.0). There was an inverse correlation between serum γ-glutamyl dipeptide concentrations and γ-glutamyltransferease (GGT) enzyme activity (r = -0.46; p = 2.5 × 10-7), which was indicative of a low-risk (n = 64) as compared to a high-risk (n = 52) patient subgroup with impaired glutathione salvage pathway and likely poor clinical prognosis. Our findings highlight the key role of defects in the γ-glutamyl cycle for differentiation of NASH patients, which may enable better risk assessment of long-term survivorship as a complement to standard liver enzyme screens and histopathology.

Development of a highly sensitive, quantitative, and rapid detection system for Plasmodium falciparum-infected red blood cells using a fluorescent blue-ray optical system.

A highly sensitive diagnostic system for determining low-density infections that are missed by conventional methods is necessary to detect the carriers of Plasmodium falciparum. A fluorescent blue-ray optical system with a polycarbonate scan disc was developed to detect P. falciparum-infected red blood cells (Pf-iRBCs), and nine samples could be analyzed simultaneously. The cultured P. falciparum strain 3D7 was used to examine the potential of the system for diagnosing malaria. After an RBC suspension had been applied to the disc, the cells were dispersed on the disc by rotation. During the 10 min standing period to allow the RBCs to settle on the disc surface, the cells were simultaneously stained with nuclear fluorescence staining dye Hoechst 34580, which was previously adsorbed on the disc surface. RBCs were arranged on the disc surface as a monolayer by removing excess cells through momentary rotation. Over 1.1 million RBCs remained on the disc for fluorescence analysis. A portable, battery-driven fluorescence image reader was employed to detect fluorescence-positive RBCs for approximately 40 min. A good correlation between examination of Giemsa-stained RBCs by light microscopy and the developed system was demonstrated in the parasitemia range of 0.0001-1.0% by linear regression analysis (R2 = 0.99993). The limit of detection of 0.00020% and good reproducibility for parasitemia determination were observed. The ability of the developed system to detect sub-microscopic low-density Pf-iRBCs and provide accurate quantitative evaluation with easy operation was demonstrated.

[Asynchronous Bilateral Neuroendocrine Breast Carcinoma - A Case Report].

We report a case of asynchronous bilateral neuroendocrine breast carcinoma. The patient was a 49-year-old woman presenting with a bloody nipple discharge from the right breast. We suspected intraductal papilloma and performed a microdochectomy. A pathological analysis of the resected specimen confirmed the diagnosis as neuroendocrine carcinoma. The tumor was positive for estrogen receptor, progesterone receptor, chromogranin A, and synaptophysin, but negative for the HER2/neu marker. The Ki-67 labeling-index was 40%. As the tumor margin was positive, breast-conserving surgery plus level II axillary lymph node dissection was performed. After surgery, radiotherapy(total dose of 50 Gy)was administered for treating residual breast involvement. Adjuvant hormonal therapy was performed for 5 years. Ten years after surgery, ultrasonography revealed a 12mm irregular hypoechoic mass in the left breast. The mass was diagnosed as a solid tubular carcinoma based on core needle biopsy findings. Subsequently, we performed breast-conserving surgery. The pathological diagnosis was a neuroendocrine carcinoma, and the tumor was positive for estrogen receptor, progesterone receptor, chromogranin A, synaptophysin, and CD56, but negative for the HER2/neu marker. The Ki-67 labeling-index was 50%. We report our experiences with a rare case of asynchronous bilateral neuroendocrine breast carcinoma. In this case, ultrasonography was a useful modality for detecting both the lesions.

Advanced glycation end-products and Porphyromonas gingivalis lipopolysaccharide increase calprotectin expression in human gingival epithelial cells.

Accumulation of advanced glycation end-products (AGEs) in periodontal tissues of patients with diabetes mellitus aggravates periodontitis, but the mechanisms are unknown. Calprotectin, a heterocomplex of S100A8 and S100A9 proteins, is a constitutive cytoplasmic component of healthy gingival epithelial cells. This study aimed at investigating the effects of AGE and Porphyromonas gingivalis lipopolysaccharide (PgLPS) on calprotectin expression in the human gingival epithelial cell line OBA-9. AGE and PgLPS increased the expression of S100A8 and S100A9 mRNAs, and AGE+PgLPS co-stimulation amplified their expression in OBA-9 cells. A higher concentration of calprotectin in cell lysates was also induced by stimulation with AGE and/or PgLPS. S100A8 was mainly translocated from the nucleus to the cytoplasm by AGE stimulation, while cytoplasmic localization of S100A9 was not altered following stimulation with AGE and/or PgLPS. Calprotectin was found in the cytoplasm of BSA-treated cells, but cytoplasmic and nuclear localization was observed following stimulation with AGE and/or PgLPS. AGE-induced S100A8, and S100A9 mRNA expression was partially suppressed by RAGE-specific siRNA. In contrast, PgLPS-induced S100A8 and S100A9 mRNA expression was strongly suppressed by TLR2-specific siRNA. Furthermore, the inhibition of p38, JNK MAPK, and NF-κB attenuated AGE- and PgLPS-induced S100A8 and S100A9 mRNA expression. Taken together, these results demonstrate that AGE acts in synergy with PgLPS to stimulate RAGE and TLR2 expression and activate p38, JNK MAPK, and NF-κB signaling pathways, resulting in increased activation of calprotectin (S100A8/S100A9) in human gingival epithelial cells. Our results suggest that calprotectin may be involved in the pathogenesis of diabetic periodontitis.

PKR induces the expression of NLRP3 by regulating the NF-κB pathway in Porphyromonas gingivalis-infected osteoblasts.

The double-stranded RNA-dependent kinase (PKR), which is activated by double stranded RNA, induces inflammation by regulating NF-κB signaling. The NLR family pyrin domain-containing 3 (NLRP3) inflammasome also modulates inflammation in response to infection. Porphyromonas gingivalis (P.gingivalis) is an oral bacterium which is implicated in the pathogenesis of periodontal diseases. We previously reported that PKR is a key modulator of bone metabolism and inflammation in the periodontal tissue. PKR was also reported to induce inflammation in response to microbes by regulating the NLRP3 inflammasome, suggesting that PKR could affect inflammation along with NLRP3 in periodontal diseases. In this study, we investigated the effects of PKR on NLRP3 expression and NF-κB activity in P. gingivalis infected osteoblasts. We first constructed a SNAP26b-tagged P.gingivalis (SNAP-P. g.) and traced its internalization into the cell. SNAP-P. g. increased the activity of PKR and NF-κB and also induced NLRP3 expression in osteoblasts. Inhibition of NF-κB attenuated SNAP-P. g.-induced NLRP3 expression. The knockdown of PKR using shRNA decreased both the activity of NF-κB and the expression of NLRP3 induced by SNAP-P.g.. We therefore concluded that in osteoblasts, P. gingivalis activated PKR, which in turn increased NLRP3 expression by activating NF-κB. Our results suggest that PKR modulates inflammation by regulating the expression of the NLRP3 inflammasome through the NF-κB pathway in periodontal diseases.