The purpose of this study was to explore the clinical and laboratory characteristics of children with refractory Mycoplasma pneumoniae pneumonia (RMPP). Seventy-six children with RMPP and 26 children with non-refractory M. pneumoniae pneumonia (NRMPP), confirmed by both serology and fluorescent quantitation PCR in bronchoalveolar lavage fluid (BALF), were evaluated retrospectively. Compared to those with NRMPP, children with RMPP were older (66.6±39.0 vs. 48.4±35.4 months, p=0.038) and had a longer duration of fever (12.7±2.6 vs. 7.5±1.8 days) and hospital stay (12.1±3.2 vs. 7.4±2.9 days). Children with RMPP presented neutrophil infiltration both in serum and BALF, as well as severe pulmonary lesions with pleural effusion. Children with RMPP had a significantly higher M. pneumoniae DNA load in BALF compared to NRMPP patients, and the M. pneumoniae load in BALF was significantly correlated with neutrophils and inversely correlated with macrophages for both the NRMPP and RMPP groups. The serum concentrations of tumor necrosis factor alpha (median 114.5 pg/ml, range 49.1-897.9 pg/ml) and interferon gamma (median 376.9 pg/ml, range 221.4-1997.6 pg/ml) were significantly higher in children with RMPP compared to children with NRMPP. This study indicates that a direct microbe effect and the subsequent induced excessive host immune response contribute in part to the progression of RMPP.

Early distinction between refractory M. pneumoniae pneumonia (RMPP) and non-RMPP (NRMPP) is still difficult. The community-acquired respiratory distress syndrome (CARDS) toxin can induce inflammatory and histopathological phenotypes associated with M. pneumoniae infection. This study aimed to investigate the clinical significance of CARDS toxin and pro-inflammatory cytokines in children with RMPP and to explore whether CARDS toxin can induce TNF-α expression. Levels of CARDS toxin and cytokines in BALF from control and children with MPP were determined by real-time PCR and ELISA, respectively. A receiver-operating characteristic (ROC) analysis was performed to assess the diagnostic values of CARDS toxin, TNF-α, and IL-6 in RMPP. The recombinant CARDS toxin was constructed and prepared at different concentrations for stimulation of RAW264.7 cells. After co-culture with CARDS toxin, cytokines were detected by ELISA and the mRNA levels were measured by real-time PCR. Effects of CARDS toxin and TNF-α on inflammatory cell infiltration and mucus secretion in mouse lungs were also evaluated. Levels of CARDS toxin, TNF-α and IL-6 in bronchoalveolar lavage fluid (BALF) were significantly higher in RMPP cases compared with NRMPP cases. Furthermore, TNF-α had better diagnostic ability for differentiation of RMPP with AUC of 0.824 and Youden index of 0.692 compared with CARDS toxin and IL-6. Moreover, CARDS toxin was positively correlated with TNF-α level in MPP cases. In vitro assay revealed that CARDS toxin induced RAW264.7 macrophages to secrete TNF-α. Further in vivo assay showed that TNF-α deletion partially abrogated the CARDS toxin-mediated induction of inflammatory cell infiltration and mucus secretion in mouse lungs. The high co-expression of TNF-α and CARDS toxin in BALF is a good diagnostic biomarker for differentiating children with RMPP and NRMPP.

Increasing numbers of refractory or severe, even fatal, cases of Mycoplasma pneumoniae infections have been reported in recent years. Excessive inflammatory responses play a vital role in the pathogenesis of refractory M. pneumoniae pneumonia (RMPP). HMGB1 is an actively secreted cytokine produced by macrophages and other inflammatory cells that participates in various infectious diseases. The present study aimed to explore the role and clinical significance of HMGB1 in children with RMPP and the potential mechanism of HMGB1 expression. Four hundred and fifty-two children diagnosed with M. pneumoniae pneumonia, including 108 children with RMPP, were enrolled from January 2013 to December 2015 at the Children’s Hospital of Soochow University. HMGB1, TNF-α, and IL-6 in peripheral blood from RMPP and non-RMPP (NRMPP) cases were detected by real-time PCR and ELISA. Lipid-associated membrane proteins (LAMPs) were extracted from live M. pneumoniae and prepared at different concentrations for stimulation of THP-1 cells. After coculture with LAMPs, HMGB1, TNF-α, IL-6, RAGE, TLR2, and TLR4 in THP-1 cells were detected by real-time PCR. Occurrences of cough, fever, and abnormal lung signs were more frequent in RMPP cases compared with NRMPP cases (all p < 0.05). Children with RMPP had longer hospital stays than children with NRMPP (p < 0.05). Different distributions of lymphocytes were noted between RMPP and NRMPP cases. HMGB1, TNF-α, and IL-6 levels were significantly higher in RMPP cases compared with NRMPP cases (all p < 0.05). HMGB1 had good diagnostic ability to differentiate RMPP with AUC of 0.876, sensitivity of 0.833, and specificity of 0.824 compared with TNF-α and IL-6. HMGB1 expression in THP-1 cells was increased by stimulation with 10 μg/ml LAMPs. TLR2 expression was increased after stimulation with 6 μg/ml LAMPs. HMGB1 level was positively associated with TNF-α, IL-6, and TLR2 levels. HMGB1 is a good diagnostic biomarker for differentiating RMPP and NRMPP. LAMPs from M. pneumoniae may induce HMGB1 expression in immune cells through the TLR2 pathway. Further in vitro and in vivo studies are needed for the development of a new treatment strategy to inhibit the HMGB1 pathway, thereby preventing the inflammation in RMPP.

The aim of this study was to investigate the mechanisms of OX40L in regulating helper T (Th) cells differentiation through phosphoinositide 3-kinase (PI3K)/AKT and p38 mitogen-activated protein kinase signaling pathway in vitro and in vivo experiments. Serum samples of patients with asthma and healthy controls were used to explore the association between OX40L and Th cells. Enzyme-linked immunosorbent assay (ELISA) was used to measure the serum concentrations of OX40L, IL-4, IFN-γ, IL-17 and TGF-β. Flow cytometry method was used to analyze Th1, Th2, Th17 and Treg cells. 3H-thymidine was used to determine the proliferation of T cells. Western Blot was used to detect protein expression and phosphorylation. Immunohistochemistry was used to detect the expression of OX40L in lung tissues. OX40L, IL-4, IL-17 increased in patient serum compared to healthy control and in the ovalbumin (OVA)-primed mononuclear cells compared to normal cells, while IFN-γ and TGF-β were decreased. Besides, the OVA-primed CD4+ T cells treated with OX40L-Ig fusion protein promoted the proliferation of T cells and Th2 and Th17 cells differentiation as well as PI3K/AKT and p38 MAPK signaling pathway, but suppressed Th1 and Treg cells differentiation. Moreover, helper T cells differentiation in OVA-primed CD4+ T cells could be markedly reversed by the addition of PI3K/AKT inhibition, p38 MAPK inhibition and anti-OX40L monoclonal antibody. In this study, we revealed that OX40L could regulate differentiation of helper T cells via PI3K/AKT and p38 MAPK signaling pathway in asthma. Besides, blockade of OX40/OX40L could inhibit the proliferation of CD4+ T cells and regulate polarization of helper T cells.

Background: Recently, many cases of pneumonia in children with Mycoplasma pneumoniae infection have been shown to have varying degrees of intrabronchial mucus plug formation. The clinical, laboratory, radiological characteristics, and treatment of patients with Mycoplasma infection are analyzed in this study. The risk factors for M. pneumoniae pneumonia (MPP) mucus plug formation in children are explored, and a risk factor scoring system is established. Methods: MPP patients treated with bronchoscopy were retrospectively enrolled in the study from February 2015 to December 2019. The children were divided into a mucus plug group and a control group according to the presence or absence of mucus plug formation. The clinical, laboratory, radiological characteristics, and treatment of the two groups of children were compared. Univariate and multivariate logistic regression models were used to identify the risk factors for MPP mucus plug formation. The receiver operating characteristic (ROC) curve was drawn to evaluate the regression model and establish the MPP mucous plug risk factor scoring system. Results: A univariate analysis showed that the children in the mucous group were older and had a longer fever duration, longer hospital stay, higher fever peak, more cases of wheezing symptoms and allergies, and azithromycin or corticosteroids were administered later. In addition, neutrophil, C-reactive protein (CRP), lactate dehydrogenase (LDH), D-dimer (DD), sputum MP-DNA copy number, and total immunoglobulin A (IgA) levels were higher, while prealbumin (PA) levels were lower. The ROC curve analysis showed that children with MPP had PA ≤144.5 mg/L, had used corticosteroids during the course of the illness of ≥4.5 days, CRP ≥12.27 mg/L, an LDH ≥ 462.65 U/L, and there was a possibility of intra-airway mucus formation. The independent risk factors were scored according to their odds ratio (OR) value. Among the 255 children with MPP, the high-risk group had 44 (83.02%) mucus plugs out of 53; the middle-risk group had 35 (34.3%) mucus plugs out of 102; and the low-risk group had 11 (11%) mucus plugs out of 100. Conclusions: PA levels, timing of corticosteroid use (use in the first few days), CRP levels, and LDH levels were independent risk factors for MPP mucus plug formation. This provides a basis for the early identification of MPP in children combined with mucus plug formation.