Development and validation of enzyme-linked immunosorbent assay for anti-mouse pertussis immunoglobulin G using international reference anti-Bordetella pertussis mouse serum NIBSC 97/642.

Purpose: In this study, an in-house enzyme-linked immunosorbent assay (ELISA) was developed and validated. The titer of ELISA was calculated using the reference line (RFL) method based on the standard curve drawn using the international reference anti-mouse serum NIBSC (National Institute for Biological Standards and Control) 97/642. Materials and Methods: In the development step, signal to noise was depicted to select the buffers that showed the most appropriate ratio. In the validation step, standard range, precision, dilution linearity, and specificity were confirmed, and RFL and parallel line (PLL) methods were compared in precision and dilution linearity. Results: Coating concentration for plate was achieved at 0.1 µg/mL for pertussis toxin (PT), 0.15 µg/mL for filamentous hemagglutinin antigen (FHA), and 0.25 µg/mL for pertactin (PRN). The signal to noise ratio was 22.02 for PT, 14.93 for FHA, and 8.02 for PRN with 0.25% goat serum in phosphate-buffered saline (PBS) as a dilution buffer, and 2% skim milk in PBS as a blocking buffer. Based on the precision results, we assessed the lower limit of quantification by 1, 0.2, and 1.5 EU/mL concentration for PT, FHA, and PRN which met the ICH (International Council for Harmonization) M10 criteria of a 25% accuracy and total error of 40%. In specificity, homologous serum was spiked into heterologous serum and the accuracy met the criteria. There was no difference in the results between RFL and PLL calculations (p-value=0.3207 for PT, 0.7394 for FHA, 0.2109 for PRN). Conclusion: ELISA validated with RFL calculation method in this study is a relatively accurate assay for mouse humoral immunogenicity test.

Comparative analysis of organ preservation attempt and radical surgery in clinical T2N0 mid to low rectal cancer.

PURPOSE: Debate persists regarding the feasibility of adopting an organ-preserving strategy as the treatment modality for clinical T2N0 rectal cancer. This study aimed to compare the outcomes of attempting organ-preserving strategies versus radical surgery in patients with clinical T2N0 mid to low rectal cancer. METHODS: Patients diagnosed with clinical T2N0 rectal cancer, with lesions located within 8 cm from the anal verge as determined by pre-treatment magnetic resonance imaging between January 2010 and December 2020 were included. RESULTS: Of 119 patients, 91 and 28 were categorized into the organ-preserving attempt group and the radical surgery group, respectively. The median follow-up duration was 48.8 months (range, 0-134 months). The organ-preserving attempt group exhibited a reduced incidence of stoma formation (44.0% vs. 75.0%; p = 0.004) and a lower occurrence of grade 3 or higher surgical complications (5.8% vs. 21.4%; p = 0.025). Univariate analyses revealed no significant association between treatment strategy and 3-year local recurrence-free survival (organ-preserving attempt 87.9% vs. radical surgery 96.2%; p = 0.129), or 3-year disease-free survival (79.6% vs. 84.9%; p = 0.429). Multivariate analysis did not identify any independent prognostic factors associated with oncologic outcomes. CONCLUSION: Compared with radical surgery, attempted organ preservation resulted in lower incidences of stoma formation and severe surgical complications, whereas oncological outcomes were comparable. Attempting organ preservation may be a safe alternative to radical surgery for clinical T2N0 mid to low rectal cancer.

Macrophage Activation Syndrome in Children: Update on Diagnosis and Treatment.

Macrophage activation syndrome (MAS) is potentially fatal; so, early diagnosis and timely treatment are essential. However, detecting MAS is sometimes challenging because its principal features can be observed in other pediatric diseases that cause severe inflammation. Cytokine storm due to immune dysregulation represents the clinical and laboratory features of MAS that are included in the diagnostic criteria. Most cases of MAS occur as an underlying condition worsens and progresses. Therefore, a patient with autoimmune or autoinflammatory disease who shows unexplained clinical deterioration despite appropriate management should be considered at high risk for MAS (i.e., occult MAS). The basic principles of treatment are control of triggering factors, supportive care, and relief of hyperinflammation. Systemic steroids and cyclosporine A are frequently used as a first-line treatment. For the treatment of refractory MAS, cytokine-specific biologic agents such as anakinra have recently become preferred over traditional immunosuppressive agents such as etoposide. MAS might be underrecognized in pediatric patients with infectious and inflammatory diseases due to its diverse clinical presentations. Clinical suspicion of MAS is of the utmost importance for early recognition of the disease.

RUFY4 deletion prevents pathological bone loss by blocking endo-lysosomal trafficking of osteoclasts.

Mature osteoclasts degrade bone matrix by exocytosis of active proteases from secretory lysosomes through a ruffled border. However, the molecular mechanisms underlying lysosomal trafficking and secretion in osteoclasts remain largely unknown. Here, we show with GeneChip analysis that RUN and FYVE domain-containing protein 4 (RUFY4) is strongly upregulated during osteoclastogenesis. Mice lacking Rufy4 exhibited a high trabecular bone mass phenotype with abnormalities in osteoclast function in vivo. Furthermore, deleting Rufy4 did not affect osteoclast differentiation, but inhibited bone-resorbing activity due to disruption in the acidic maturation of secondary lysosomes, their trafficking to the membrane, and their secretion of cathepsin K into the extracellular space. Mechanistically, RUFY4 promotes late endosome-lysosome fusion by acting as an adaptor protein between Rab7 on late endosomes and LAMP2 on primary lysosomes. Consequently, Rufy4-deficient mice were highly protected from lipopolysaccharide- and ovariectomy-induced bone loss. Thus, RUFY4 plays as a new regulator in osteoclast activity by mediating endo-lysosomal trafficking and have a potential to be specific target for therapies against bone-loss diseases such as osteoporosis.

Characteristics and risk factors of axillary lymph node metastasis of microinvasive breast cancer.

PURPOSE: To select patients who would benefit most from sentinel lymph node biopsy (SLNB) by investigating the characteristics and risk factors of axillary lymph node metastasis (ALNM) in microinvasive breast cancer (MIBC). METHODS: This retrospective study included 1688 patients with MIBC who underwent breast surgery with axillary staging at the Asan Medical Center from 1995 to 2020. RESULTS: Most patients underwent SLNB alone (83.5%). Seventy (4.1%) patients were node-positive, and the majority had positive lymph nodes < 10 mm, with micro-metastases occurring frequently (n = 37; 55%). Node-positive patients underwent total mastectomy and axillary lymph node dissection (ALND) more than breast-conserving surgery (BCS) and SLNB compared with node-negative patients (p < 0.001). In the multivariate analysis, independent predictors of ALNM included young age [odds ratio (OR) 0.959; 95% confidence interval (CI) 0.927-0.993; p = 0.019], ALND (OR 11.486; 95% CI 5.767-22.877; p < 0.001), number of lymph nodes harvested (≥ 5) (OR 3.184; 95% CI 1.555-6.522; p < 0.001), lymphovascular invasion (OR 6.831; 95% CI 2.386-19.557; p < 0.001), presence of multiple microinvasion foci (OR 2.771; 95% CI 1.329-5.779; p = 0.007), prominent lymph nodes in preoperative imaging (OR 2.675; 95% CI 1.362-5.253; p = 0.004), and hormone receptor positivity (OR 2.491; 95% CI 1.230-5.046; p = 0.011). CONCLUSION: Low ALNM rate (4.1%) suggests that routine SLNB for patients with MIBC is unnecessary but can be valuable for patients with specific risk factors. Ongoing trials for omitting SLNB in early breast cancer, and further subanalyses focusing on rare populations with MIBC are necessary.

5-aminosalicylic acid suppresses osteoarthritis through the OSCAR-PPARγ axis.

Osteoarthritis (OA) is a progressive and irreversible degenerative joint disease that is characterized by cartilage destruction, osteophyte formation, subchondral bone remodeling, and synovitis. Despite affecting millions of patients, effective and safe disease-modifying osteoarthritis drugs are lacking. Here we reveal an unexpected role for the small molecule 5-aminosalicylic acid (5-ASA), which is used as an anti-inflammatory drug in ulcerative colitis. We show that 5-ASA competes with extracellular-matrix collagen-II to bind to osteoclast-associated receptor (OSCAR) on chondrocytes. Intra-articular 5-ASA injections ameliorate OA generated by surgery-induced medial-meniscus destabilization in male mice. Significantly, this effect is also observed when 5-ASA was administered well after OA onset. Moreover, mice with DMM-induced OA that are treated with 5-ASA at weeks 8-11 and sacrificed at week 12 have thicker cartilage than untreated mice that were sacrificed at week 8. Mechanistically, 5-ASA reverses OSCAR-mediated transcriptional repression of PPARγ in articular chondrocytes, thereby suppressing COX-2-related inflammation. It also improves chondrogenesis, strongly downregulates ECM catabolism, and promotes ECM anabolism. Our results suggest that 5-ASA could serve as a DMOAD.

Octyl gallate has potent anti-inflammasome activity by directly binding to NLRP3 LRR domain.

The NOD-, LRR-, and Pyrin domain-containing protein 3 (NLRP3) inflammasome plays key roles in regulating inflammation. Numerous studies show that the abnormal activation of NLRP3 associates with the initiation and progression of various diseases. Hence, the NLRP3 inflammasome may be a promising therapeutic target for these diseases. Octyl gallate (OG) is a small molecule with antioxidant, antimicrobial, antifungal, and anti-inflammatory activities; however, the mechanism underlying its anti-inflammatory activity is still unclear. Here, we developed a screening system for NLRP3-inflammasome inhibitors. A total of 3287 small molecules were screened for inhibitors of nigericin-induced NLRP3 oligomerization. OG was identified as a novel inhibitor. We show that OG directly targets the LRR domain of NLRP3 and thereby blocks the inflammatory cascade of the NLRP3 inflammasome. This contrasts with the mode-of-action of other direct NLRP3 inhibitors, which all bind to the NACHT domain of NLRP3. Interestingly, OG also inhibits the priming step by downregulating the Raf-MEK1/2-ERK1/2 axis. Thus, OG inhibits the NLRP3 inflammasome by two distinct mechanisms. Importantly, OG injection ameliorated the inflammation in mouse models of foot gout and sepsis. Our study identifies OG as a potential therapeutic agent for NLRP3-associated diseases.

Seroprevalence of varicella-zoster virus as measured by fluorescent antibody to membrane antigen assay and glycoprotein enzyme-linked immunosorbent assay more than 10 years after initiation of a universal vaccination program: An observational study.

Universal varicella vaccination (UVV), as a single dose to children aged 12 to 15 months, was introduced in Korea in 2005. A seroprevalence study is required to upgrade this UVV strategy. The fluorescent antibody to membrane antigen (FAMA) assay is the gold standard for varicella-zoster virus (VZV) immunity testing. However, no standard operating procedure (SOP) has been developed for the FAMA assay, in which either glutaraldehyde or acetone may be used for VZV-infected cell fixation. In this observational study, we aimed to investigate the age-specific seroprevalence in Korean children and adults. Additionally, with glycoprotein enzyme-linked immunosorbent assay (gpELISA) as the reference, we evaluated the performance of the FAMA assay using acetone-fixed cells. Four hundred sera were analyzed using the FAMA assay (acetone-fixed cells) and gpELISA, comprising 50 subjects from each age category. In the FAMA assay, the seropositivity rate decreased from 82.0% in the 1 to 4-year-old group to 58.0% in the 5 to 9-year-old group (95% confidence interval [CI]: 69.2-90.2 and 44.2-70.6, respectively; P = .009), while that in the gpELISA decreased from 80.0% to 52.0% (95% CI: 67.0-88.8 and 38.5-65.2, respectively; P = .003). In both methods, the seropositivity rates ranged from 95% to 100% in the population aged ≥ 20 years. We observed a significant correlation between the 2 methods, with a correlation coefficient of 0.795 (P < .001). In receiver operating characteristic analysis using the gpELISA results as a reference, the area under the curve for the FAMA assay was very high at 0.995 (95% CI: 0.990-1.000; P < .001). Compared to the gpELISA, the sensitivity, specificity, and kappa value of the FAMA assay were 99.4%, 79.3%, and 0.84 (nearly perfect), respectively. The seropositivity rate of the 5 to 9-year-old group indicated waning immunity over time and supported implementation of a second dose in the UVV program. The results of the FAMA assay were comparable to those of the gpELISA. Although further study is needed to standardize procedures, our results suggest that the FAMA assay using acetone-fixed cells can be used widely and can be included in a universal FAMA assay SOP.

Comparative Evaluation of Recombinant and Acellular Pertussis Vaccines in a Murine Model.

Since the 2000s, sporadic outbreaks of whooping cough have been reported in advanced countries, where the acellular pertussis vaccination rate is relatively high, and in developing countries. Small-scale whooping cough has also continued in many countries, due in part to the waning of immune protection after childhood vaccination, necessitating the development of an improved pertussis vaccine and vaccination program. Currently, two different production platforms are being actively pursued in Korea; one is based on the aP (acellular pertussis) vaccine purified from B. pertussis containing pertussis toxoid (PT), filamentous hemagglutin (FHA) and pertactin (PRN), and the other is based on the recombinant aP (raP), containing genetically detoxified pertussis toxin ADP-ribosyltransferase subunit 1 (PtxS1), FHA, and PRN domain, expressed and purified from recombinant E. coli. aP components were further combined with diphtheria and tetanus vaccine components as a prototype DTaP vaccine by GC Pharma (GC DTaP vaccine). We evaluated and compared the immunogenicity and the protective efficacy of aP and raP vaccines in an experimental murine challenge model: humoral immunity in serum, IgA secretion in nasal lavage, bacterial clearance after challenge, PTx (pertussis toxin) CHO cell neutralization titer, cytokine secretion in spleen single cell, and tissue resident memory CD4+ T cell (CD4+ TRM cell) in lung tissues. In humoral immunogenicity, GC DTaP vaccines showed high titers for PT and PRN and showed similar patterns in nasal lavage and IL-5 cytokine secretions. The GC DTaP vaccine and the control vaccine showed equivalent results in bacterial clearance after challenge, PTx CHO cell neutralization assay, and CD4+ TRM cell. In contrast, the recombinant raP vaccine exhibited strong antibody responses for FHA and PRN, albeit with low antibody level of PT and low titer in PTx CHO neutralization assay, as compared to control and GC DTaP vaccines. The raP vaccine provided a sterile lung bacterial clearance comparable to a commercial control vaccine after the experimental challenge in murine model. Moreover, raP exhibited a strong cytokine response and CD4+ TRM cell in lung tissue, comparable or superior to the experimental and commercial DTaP vaccinated groups. Contingent on improving the biophysical stability and humoral response to PT, the raP vaccine warrants further development as an effective alternative to aP vaccines for the control of a pertussis outbreak.

Macrophage activation syndrome in neonatal lupus presenting with fever and rash.

Neonatal lupus can occur in infants born to mother with autoimmune disorders through transplacental auto-antibodies. Clinical manifestations in neonatal lupus include cutaneous lesions and hematologic or hepatobiliary findings resembling those seen in systemic lupus erythematosus. In autoimmune state, macrophage activation syndrome (MAS) represent a critical and potentially fatal complication that can result in mortality if not immediately identified and managed with the appropriate care. Here we present a 33-day-old girl diagnosed with neonatal lupus and serious MAS. She was delivered by a primipara mother who did not exhibit any autoimmune symptoms. The patient visited the hospital due to fever and pancytopenia. Laboratory data were compatible with MAS, including pancytopenia, high level of ferritin, soluble interleukin-2, and decreased natural killer cell activity. In addition, autoimmune study showed positive results for anti-nuclear antibody (ANA), anti-Sjogren syndrome antigen A (SSA), and SSB, The autoimmune study for mother also showed positive results for ANA, anti-SSA, and SSB. The patient recovered after she received high dose steroid and supportive care. Our case indicates that neonatal lupus should be taken into consideration when fever, erythematous skin rash, and pancytopenia are observed in infants, even if their mothers have no prior history of autoimmune conditions.

Relationships among sensory analysis, isoflavone and hexanal contents of soymilk powder

The objective of this work was to study the sensory attributes and their relationships with isoflavones and hexanal contents of soymilks made in laboratory and commercial samples. The laboratory soymilk samples showed cooked grain and cotton candy aroma and cooked grain, malty and sweetness flavor (a mild flavor). The commercial samples presented stronger roasted soy, rancid, sesame seeds and fishy aroma and roasted soy, sesame seeds and bitterness flavor, and bitter taste (closed nose) and starchy texture. No differences were noted among laboratory soymilks, denoting inactivation of lipoxygenases enzymes in the soymilks process. There were differences between the samples prepared in laboratory and commercial soymilks, which was due to several factors related to processing techniques. The hexanal average content was positively associated with the cooked grain aroma and isoflavones was positively associated with the cooked grain and cotton candy aroma, cooked grain, malty and sweetness flavor and starchy texture.

O trabalho foi conduzido com o objetivo de descrever os atributos sensoriais de bebidas de extratos de soja em pó produzidos a partir de cultivares desprovida de lipoxigenases, convencional e tipo hortaliça e dois extratos comerciais e correlacioná-los com as quantidades de isoflavonas e n-hexanal nos extratos em pó. Os extratos de soja em pó obtidos em laboratório foram caracterizados pelos atributos: aroma de feijão cozido e de algodão doce; sabor de grão cozido, de malte e gosto doce sugerindo, sabor suave. Os extratos comerciais apresentaram forte aroma de soja tostada, de ranço, de sementes de gergelim e de peixe; sabor de soja tostada, gosto amargo e de sementes de gergelim; gosto amargo (com nariz preso) e consistência de amido. Não foram observadas diferenças sensoriais entre os extratos produzidos em laboratório, indicando a ocorrência da inativação das enzimas lipoxigenases durante o processamento dos produtos. Contudo, diferenças marcantes entre os extratos obtidos em laboratório e os comerciais quanto aos atributos sensoriais estudados foram encontradas provavelmente, devido às técnicas desconhecidas de processamento utilizadas pelos fabricantes dos produtos comerciais. O teor médio de n-hexanal presente nos extratos de soja comerciais e produzidos em laboratório apresentou correlação positiva e significativa com o atributo aroma de grão cozido, já a quantidade de isoflavonas com aroma de grão cozido e de algodão doce, sabor de grão cozido, de malte, gosto doce e consistência de amido.