Metabolomic Profiling of Lungs from Mice Reveals the Variability of Metabolites in Pneumocystis Infection and the Metabolic Abnormalities in BAFF-R-Deficient Mice.

Purpose: The incidence of Pneumocystis pneumonia (PCP) in patients without human immunodeficiency virus (HIV) has been increasing. In this study, we aimed to investigate the metabolic changes in Pneumocystis infection and the metabolic abnormalities in B-cell-activating factor receptor (BAFF-R)-deficient mice with Pneumocystis infection. Methods: The important function of B cells during Pneumocystis infection is increasingly recognized. In this study, a Pneumocystis-infected mouse model was constructed in BAFF-R-/- mice and wild-type (WT) mice. Lungs of uninfected WT C57BL/6, WT Pneumocystis-infected, and BAFF-R-/- Pneumocystis-infected mice were used for metabolomic analyses to compare the metabolomic profiles among the groups, with the aim of exploring the metabolic influence of Pneumocystis infection and the influence of mature B-cell deficiency during infection. Results: The results indicated that many metabolites, mainly lipids and lipid-like molecules, were dysregulated in Pneumocystis-infected WT mice compared with uninfected WT C57BL/6 mice. The data also demonstrated significant changes in tryptophan metabolism, and the expression levels of key enzymes of tryptophan metabolism, such as indoleamine 2,3-dioxygenase 1 (IDO1), were significantly upregulated. In addition, B-cell development and function might be associated with lipid metabolism. We found a lower level of alitretinoin and the abnormalities of fatty acid metabolism in BAFF-R-/- Pneumocystis-infected mice. The mRNA levels of enzymes associated with fatty acid metabolism in the lung were upregulated in BAFF-R-/- Pneumocystis-infected mice and positively correlated with the level of IL17A, thus suggesting that the abnormalities of fatty acid metabolism may be associated with greater inflammatory cell infiltration in the lung tissue of BAFF-R-/- Pneumocystis-infected mice compared with the WT Pneumocystis-infected mice. Conclusion: Our data revealed the variability of metabolites in Pneumocystis-infected mice, suggesting that the metabolism plays a vital role in the immune response to Pneumocystis infection.

Analysis of lncRNA and mRNA Repertoires in Lung From BAFF-R-Deficient Pneumocystis-Infected Mice.

Background: Pneumocystis pneumonia (PCP) is a common medical issue in immunosuppressive patients. Increasing evidence supports that B cells may play an essential role in PCP individuals. The present study aims to integrate lncRNA and mRNA expression profiles and further investigate the molecular function of mature B cells in PCP. Methods: The lung tissue of wild-type (WT) mice and B-cell-activating factor receptor-deficient (mature B-cell deficiency, BAFF-R-/-) mice were harvested at 3 weeks after being infected with pneumocystis. After total RNAs were extracted, transcriptome profiling was performed following the Illumina HiSeq 3000 protocol. lncRNA-targeted miRNA pairs were predicted using the online databases. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment pathways were analyzed to functionally annotate these differentially expressed genes. Additionally, the immune-related lncRNA-miRNA-mRNA-ceRNA network was subsequently performed. The quantitative real-time PCR (RT-PCR) analysis was conducted to evaluate the lncRNA and mRNA expression profiles in WT-PCP mice and BAFF-R-/- PCP mice. Results: Compared with the control group, 166 mRNAs were observed to be aberrantly expressed (fold change value ≥2; P <0.05) in the BAFF-R-/- PCP group, including 39 upregulated and 127 downregulated genes, while there were 69 lncRNAs differently expressed in the BAFF-R-/- PCP group, including 15 upregulated and 54 downregulated genes. In addition, GO and KEGG pathway analyses showed that BAFF-R deficiency played an important role in the primary and adaptive immune responses in PCP. Furthermore, the lncRNA and mRNA co-expression network was established. We noted that the core network of lncRNA-TF (transcription factor) pairs could be classified into the categories including infection and immunity pathways. Conclusion: In summary, in this study, we further explored the role of mature B cells in the pathogenesis and progression of PCP and the data demonstrated that BAFF-R deficiency could play a significant role in immune regulation in the PCP population.

Proteomic Profiling and Functional Analysis of B Cell-Derived Exosomes upon Pneumocystis Infection.

Pneumocystis is a life-threatening fungal pathogen that frequently causes fatal pneumonia (PCP) in immunocompromised individuals. Recently, B cells have been reported to play a crucial role in the pathogenesis of PCP through producing antibodies and activating CD4+ T cell response. Exosomes are nanoscale small extracellular vesicles abundant with protein cargo and can mediate immune response during infectious disease. In this study, using tandem mass tag-based quantitative proteomics coupled with bioinformatic analysis, we attempted to characterize exosomes derived from B lymphocytes in response to PCP. Several proteins were verified by parallel reaction monitoring (PRM) analysis. Also, the effects of B cell exosomes on CD4+ T cell response and phagocytic function of macrophages were clarified. Briefly, 1701 proteins were identified from B cell exosomes, and the majority of them were reported in Vesiclepedia. A total of 51 differentially expressed proteins of B cell exosomes were found in response to PCP. They were mainly associated with immune response and transcription regulation. PRM analysis confirmed the significantly changed levels of histone H1.3, vimentin, and tyrosine-protein phosphatase nonreceptor type 6 (PTPN6). Moreover, a functional study revealed the proinflammatory profile of B cell exosomes on CD4+ T cell response in PCP. Taken together, our results suggest the involvement of exosomes derived from B cells in cell-to-cell communication, providing new information on the function of B cells in response to PCP.

Time Efficiency of Immediate Loading of Full-arch Implant Reconstructions Using Prefabricated Prostheses Located by an Anchor Pin: a Pilot Study.

OBJECTIVE: To investigate the time efficiency of prefabricated prostheses located by an anchor pin stereolithographic attachment system for immediate loading implant reconstruction of completely edentulous jaws and compare it with the conventional protocol. METHODS: Edentulous patients were recruited and randomly assigned into two groups: the full digital workflow group (digital group) and the conventional workflow group (conventional group). In the digital group, a provisional prosthesis was fabricated before surgery using a fully digital workflow and delivered immediately after implant placement. The positioning of the provisional prosthesis was guided precisely by the anchor pin attachment system. In the conventional group, the provisional prosthesis was fabricated after implant placement using a conventional procedure. Clinical and laboratory time efficiency were recorded, and clinician and patient satisfaction were evaluated. RESULTS: Six patients were enrolled in this pilot study and 57 implants were placed following the guided surgery protocol. Of these, 54 were immediately loaded. The total clinical chair time in the digital workflow group was significantly less than that in the conventional workflow group (digital 60.0 ± 13.2 minutes; conventional 106.7 ± 24.7 minutes) (P = 0.045). The total post-surgery procedure took significantly less time in the digital group than the conventional group (digital 202.5 ± 22.5 minutes; conventional 403.7 ± 55.4 minutes) (P = 0.004). The patients' and clinicians' satisfaction with the provisional prostheses was similar in both groups. CONCLUSION: Time efficiency in immediate loading of implant-supported full-arch fixed restorations was improved with prefabricated prostheses located by the anchor-pin-attachment system. Less postoperative chair time was required in the digital group than in the conventional group.

PD-1 Deficiency Promotes Macrophage Activation and T-Helper Cell Type 1/T-Helper Cell Type 17 Response in Pneumocystis Pneumonia.

Pneumocystis is an unusual, opportunistic fungal pathogen capable of causing Pneumocystis pneumonia (PCP) in immunocompromised hosts. Although PCP was discovered >100 years ago, its pathogenesis remains unclear. The inhibitory receptor PD-1 (programmed death 1), a negative regulator of activated T cells, has been reported to take part in tumor escape, immune tolerance, and infection immunity. In this study, we examined the role of the PD-1/PD-L1 (programmed death-ligand 1) pathway in patients with PCP and in mice. The expression levels of PD-1/PD-L1 in patients with PCP and in mice were measured by real-time PCR and flow cytometry. The effects of PD-1 deficiency are demonstrated using wild-type and PD-1-/- mice. Our data show that Pneumocystis infection promotes PD-1/PD-L1 expression; PD-1 deficiency enhances the phagocytic function of macrophages and the pulmonary T-helper cell type 1 (Th1)/Th17 response, which might contribute to Pneumocystis clearance; and PD-1 deficiency affects the polarization of macrophages. PCP mice treated with anti-PD-1 antibody showed improved pulmonary clearance of Pneumocystis. Collectively, our results demonstrate that the PD-1/PD-L1 pathway plays a role in regulating the innate and adaptive immune responses, suggesting that manipulation of this pathway may constitute an immunotherapeutic strategy for PCP.

Accuracy of Intraoral Scanning of Edentulous Jaws with and without Resin Markers.

Objective: To evaluate the accuracy of digital models obtained from intraoral scanning of edentulous maxilla and mandible models with and without resin markers. Methods: A pair of standard edentulous models were scanned using a laboratory scanner and saved as reference models. The edentulous models were fixed onto a phantom head and scanned with an intraoral scanner (IOS) five times each. Six resin markers were attached on the maxilla model and two on the mandible model, and another five intraoral scans were taken of each model. The scanning time and number of images were recorded. The digital models obtained using the IOS were superimposed on the reference models using image processing software. The trueness and precision of the models made using the IOS were evaluated, and the scanning time and number of images were also compared. Results: The average trueness and precision of the IOS in the maxilla model with resin markers were 135.50 ± 36.28 µm and 254.55 ± 40.62 µm, respectively, while those in the mandible were 161.40 ± 55.45 µm and 368.75 ± 91.03 µm, respectively. Placing resin markers on the edentulous maxilla and mandible did not improve the trueness of the IOS, but placing resin markers on the edentulous maxilla improved the precision and scanning efficiency. However, placing resin markers on the buccal shelf of the edentulous mandible decreased the precision and increased the scanning time. Conclusion: Resin markers placed on the hard palate of edentulous maxillae could improve the precision of the IOS and improve scanning efficiency. However, they did not affect the trueness of the IOS for edentulous maxillae or mandibles.

IL-17 Inversely Correlated with IL-10 via the STAT3 Gene in Pneumocystis-Infected Mice.

BACKGROUND: Pneumocystis pneumonia (PCP) remains a common opportunistic infection in immunosuppressed individuals. Current studies showed that multiple immune cells and cytokines took part in the host defense against Pneumocystis (PC). However, the roles of IL-17 and IL-10 in the development of PCP have not been elucidated. METHODS: IL-10 and IL-17 levels in serum from PCP mice were detected via ELISA. The percentages of B10 cells, IL-10+ macrophages, and IL-10+ T cells in the lung from IL-17-/- PCP mice and Th17 cells and IL-17+ γδT cells in IL-10-/- PCP mice were examined via flow cytometry. Also, antibody neutralization examination was also performed to elucidate the relationship of IL-17 and IL-10 in the PCP model. RESULTS: We noted the increase of IL-17 and IL-10 levels in serum from mice infected with Pneumocystis. Furthermore, deficiency of IL-17 or IL-10 could lead to the delayed clearance of Pneumocystis and more severed lung damage. Our data also demonstrated that IL-17 deficiency enhanced the serum IL-10 level and the percentages of B10 cells, IL-10+ macrophages, and IL-10+ T cells in the lung from PCP mice. Interestingly, we also noted an increase of the IL-17 level in serum and Th17 cell and IL-17+ γδT cell percentages in the lung from IL-10-/- PCP mice. Using antibody neutralization experiments, we found that the STAT3 gene might play a critical role in the interplay of IL-17 and IL-10 in PCP. CONCLUSION: Taken together, our results demonstrated that IL-17 and IL-10 could play the protective roles in the progression of PCP and the inverse correlation of them might be mediated by STAT3.

IL-10-producing B cells regulate Th1/Th17-cell immune responses in Pneumocystis pneumonia.

Pneumocystis pneumonia (PCP) is a common opportunistic infectious disease that is prevalent in immunosuppressed hosts. Accumulating evidence shows that B cells play an important role in infectious diseases. In the present study, the immune regulatory role of mature B cells in host defense to Pneumocystis was evaluated. Pneumocystis infection resulted in a decrease in B cells in patients and mice, and the Pneumocystis burden in B cell-deficient mice also progressively increased from weeks 1 to 7 after infection. The clearance of Pneumocystis was delayed in B cell-activating factor receptor (BAFF-R)-deficient mice (BAFF-R-/- mice), which had few B cells and Pneumocystis-specific IgG and IgM antibodies, compared with clearance in wild-type (WT) mice. There were fewer effector CD4+ T cells and higher percentages of T helper (Th)1/Th17 cells in BAFF-R-/- mice than in WT mice. Adoptive transfer of naive B cells, mRNA sequencing, and IL-1ß neutralization experiments indicated that IL-1ß is a likely determinant of the IL-10-producing B cell-mediated suppression of Th1/Th17-cell immune responses in BAFF-R-/- PCP mice. Our data indicated that B cells play a vital role in the regulation of Th cells in response to Pneumocystis infection.

IL-9 Deficiency Promotes Pulmonary Th17 Response in Murine Model of Pneumocystis Infection.

Introduction: Pneumocystis pneumonia (PCP) remains a severe complication with high mortality in immunocompromised patients. It has been well accepted that CD4+ T cells play a major role in controlling Pneumocystis infection. Th9 cells were the main source of IL-9 with multifaced roles depending on specific diseases. It is unclear whether IL-9/Th9 contributes to the immune response against PCP. The current study aims to explore the role of IL-9 and the effect of IL-9 on Th17 cells in murine model of PCP. Materials and methods: Mice were intratracheally injected with 1 × 106Pneumocystis organisms to establish the murine model of Pneumocystis infection. Pneumocystis burden was detected by TaqMan real-time PCR. Using IL-9-deficient (IL-9-/-) mice, flow cytometry, real-time PCR and enzyme-linked immunosorbent assay (ELISA) were conducted to investigate the immune function related to Th17 response in defense against Pneumocystis infection. Results: Reduced Pneumocystis burden was observed in lungs in IL-9-/- mice compared with WT mice at 3-week postinfection. IL-9-/-mice exhibited stronger Th17 immune responses than WT PCP mice through flow cytometer and real-time PCR. ELISA revealed higher levels of IL-17 and IL-23 in bronchoalveolar lavage fluid from IL-9-/- mice than WT mice. And IL-9 deficiency promoted Th17 differentiation from CD4+ naive T cells. IL-17A neutralization increased Pneumocystis burden in IL-9-/- mice. Conclusion: Although similar basic clearance of Pneumocystis organisms was achieved in both WT and IL-9-/- PCP mice, IL-9 deficiency could lower Pneumocystis organism burden and promote pulmonary Th17 cells response in the early stage of infection.

Diagnosis of Pneumocystis jirovecii pneumonia with serum cell-free DNA in non-HIV-infected immunocompromised patients.

Conventional respiratory tract specimens, such as bronchoalveolar lavage (BAL) fluid and induced sputum for diagnosing Pneumocystis jirovecii pneumonia (PCP) in immunocompromised patients are difficult to obtain. Besides, bronchoscopy is an invasive procedure that carries the risk of causing rapidly progressive respiratory insufficiency. By contrast, serum cell-free DNA (cfDNA) is easy to obtain and has been proven useful in diagnosing cancer, pregnancy associated complications, parasite infection and sepsis. In this study, we performed quantitative polymerase chain reaction (qPCR) to assess the diagnostic efficiency of using serum cfDNA, BAL fluid, and sputum DNA for PCP. Seventy-one patients (35 PCP patients and 36 non-PCP patients) were enrolled according to the clinical PCP diagnostic criteria. The sensitivity, specificity, positive predictive value, and negative predictive value of PCR using serum cfDNA were 68.6% (95% CI, 50.7-83.1), 97.2% (95% CI, 85.5-99.9), 96.0%, and 76.1%, respectively. PCR using BAL fluid and sputum had a high sensitivity (97.1% and 91.4%, respectively) but relatively low specificity (86.1% and 86.1%, respectively). The combination of the sputum PCR OR serum cfDNA PCR yielded a sensitivity of 97.1%.These results indicated that serum cfDNA might be a valuable method in PCP diagnosis.

Expression,roles and therapy target values of CD24 in oral squamous cell carcinoma / 北京大学学报(医学版)

Objective:To determine the expression profile and potential roles of CD24 in oral squamous cell carcinoma and explore the values of CD24 function as a potential target of clinical therapy.Me-thods:Semi-quantitative immunohistochemistry was used to construct the expression profile of CD24 in 78 human oral tissues and 59 Hamster buccal pouch tissues.Real-time RT-PCR and Western blot were used to analyze the CD24 expression levels in oral DOK4 cells,oral cancer CAL-27 and WSU-HN6 cells. Then these two cancer cell lines were selected to evaluate the effect of all-trans retinoic acid (ATRA)and CD24 antibody on CD24 expression,and the proliferation and tumorsphere formation capacity of these two cell lines.Results:CD24 expression was found significantly elevated in both human and animal tissues compared with normal and benign tissues (P<0.05),as well as in oral cancer CAL-27 and WSU-HN6 cells compared with DOK cells (P<0.05).CAL-27 and WSU-HN6 cells possess increased proliferative and specific tumorsphere formation capability compared with DOK cells (P<0.05 ).Both ATRA and CD24 antibody were able to effectively inhibit the proliferation and tumorsphere formation of CAL-27 and WSU-HN6 cells (P<0.05).Among them ATRA at least involved partially in the proliferation by down-regulating the CD24 expression (P<0.05 ),while CD24 antibody blocking had no effect on the CD24 expression.Conclusion:CD24 was upregulated in oral cancer and functioned as a potential factor that promoted the proliferation and tumorsphere formation of CAL-27 and WSU-HN6 cells.Both ATRA and CD24 antibody might effectively inhibit the proliferation and tumorsphere formation of CAL-27 and WSU-HN6 cells and function as a potential therapy target.

Effects of Propranolol on the Left Ventricular Volume of Normal Subjects During CT Coronary Angiography

OBJECTIVE: The purpose of this study is to determine the effects of propranolol on the left ventricular (LV) volume during CT coronary angiography. MATERIALS AND METHODS: The LV volume of 252 normal Chinese subjects (126 subjects with propranolol medication and 126 age- and gender-matched Chinese subjects without medication) was estimated using 64 slices multi-detector CT (MDCT). The heart rate difference was analyzed by the logistic linear regression model with variables that included gender, age, body height, body weight, systolic blood pressure (SBP), diastolic blood pressure (DBP) and the dosage of propranolol. The following global LV functional parameters were calculated: the real-end diastolic volume (EDV), the real-end systolic volume (ESV) and the real-ejection fraction (EF). RESULTS: The female subjects had a greater decrease of heart rate after taking propranolol. The difference of heart rate was negatively correlated with the dosage of propranolol. The real-EDV, the real-ESV and the real-EF ranged from 48.1 to 109 mL/m2, 6.1 to 57.1 mL/m2 and 41% to 88%, respectively. There was no significant difference in the SBP and DBP between the groups without and with propranolol medication (123 +/- 17 and 80 +/- 10 mmHg; 120 +/- 14 and 80 +/- 11 mmHg, respectively). The real-EDV showed no significant difference between these two groups, but the real-ESV and real-EF showed significant differences between these two groups (69.4 +/- 9.3 and 70.6 +/- 8.9 mL/m2; 23.5 +/- 5.7 and 25.6 +/- 3.7 mL/m2, 66.5 +/- 5.1% and 63.5 +/- 4.6%, respectively). CONCLUSION: The difference of heart rate is significantly influenced by gender and the dosage of propranolol. Propranolol will also increase the ESV, which contributes to a decreased EF, while the SBP, DBP and EDV are not statistically changed.

Expression of nuclear transcription factor kappaB in hamster buccal pouch carcinogenesis / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To study the expression and significance of p65 which is an important subtype of nuclear transcription factor kappaB (NF-kappaB) and its inhibitory protein IkappaBalpha in malignant transformation of hamster buccal mucosa.</p><p><b>METHODS</b>The animal model of malignant transformation in hamster buccal mucosa induced by DMBA (0.5%) was established. Twelve paired specimens including normal buccal mucosa, epithelial hyperplasia, epithelial dysplasia and squamous cell carcinoma (SCC) were subjected to Western blot for the analysis of p65. The expression of IkappaBalpha was observed in 22 normal buccal epithelia, 20 hyperplasia epithelia, 35 dysplasia epithelia and 23 SCC by immunohistochemical evaluation.</p><p><b>RESULTS</b>In normal buccal epithelia and hyperplasia epithelia, the expression of p65 was not obvious, and there was no significant difference between them (P > 0.05). The expression of IkappaBalpha existed at large, but mostly localizing in cell cytoplasmic staining in the basal cell layer and bottom of spinocelluar layer. With the occurrence of epithelia dysplasia, the expression of p65 gradually increased, comparing with normal buccal epithelia and hyperplasia epithelia (P < 0.01). But the positive intensity of IkappaBalpha was dramatically decreased (P < 0.05). In SCC, p65 expressed at a higher level comparing with normal buccal mucosa and dysplasia (P < 0.01), while the staining of IkappaBalpha was feedbackly higher comparing with dysplasia (P < 0.01) and even with normal buccal mucosa (P < 0.01).</p><p><b>CONCLUSION</b>NF-kappaB p65 is strongly activated in malignant transformation of hamster buccal mucosa. The abnormal expression of p65 and IkappaBalpha, especially the ascending expression of p65 as well as the descending expression of IkappaBalpha in dysplasia may be an early event during oral carcinogenesis, and can be used as biomarkers for supervising oral malignancy.</p>