Supplemental Material - Using combination of albumin to fibrinogen ratio and prognostic nutritional index model for predicting disease activity in patients with systemic lupus erythematosus

Supplemental Material for What Do We Know About people’s Politics? Testing a New Framework for Understanding Different Conceptions of Politics by Hongshuai Zhao, Zikun Huang, Shiqian Wang, Peng Fu, Biqi Fu, Yang Guo, Junming Li, and Qing Luo in Lupus</p

Characteristics of <i>in vitro</i> tuberculous granuloma model and the expression of granulomatous macrophage markers.

<p>A. Uninfected PBMCs at day 6 (a, ×200) and the morphology and structure of in vitro tuberculous granuloma model at day 3 (b, ×100), day 6 (c, ×100; d, ×400) and day 9 (e, ×400) in light microscopy. The actin filament in cells was labeled with rhodamine-phalloidin and the bacteria between and possibly within the cells was shown in green (f), (×1000). There were mainly macrophages and some lymphocytes (arrows) in granuloma observed by HE (g), (×400), and May-Grünwald-Giemsa staining (h), (×400). Size scale is indicated. B. Supernatant was collected and Macrophages were isolated from in vitro tuberculous granuloma as described in Materials and Methods at indicated times. The phenotypic markers on macrophages were determined by FCM. MFI was measured and depicted as relative expression as compared to isotype control. Represented is the mean of three donors ±SD. (a). Effector molecules were determined by real-time RT-PCR. Represented is mean ±SD of relative level of mRNA in activated MDM compared to M0 macrophage (b). Cytokines in the supernatant were measured by ELISA. Represented is the mean of three donors ±SD (c). Significance was determined as compared to untreated group. *P < 0.05, **P < 0.01, ***P < 0.001. Significance in corresponding marker expression between 9d, 6d and 3d was also calculated and represented by the following symbols: ^#P < 0.05, ^{# #}P < 0.01.</p

Determination of polarization markers of differently activated MDM.

<p>MDM were activated by IFN-γ/LPS, IL-4 or left untreated for 2 days. Markers reported for M1 and M2 macrophages were determined. A. Phenotypic markers were measured by FCM. Mean fluorescent intensity (MFI) was measured and depicted as relative expression as compared to isotype control. Represented is the mean of three donors ±SD. Significance was determined as compared to untreated group. *P < 0.05, **P < 0.01, ***P < 0.001. B. Effector molecules of differently activated macrophages were determined by real-time RT-PCR. Represented is mean ±SD of relevant level of mRNA in activated MDM compared to M0 macrophage. C. Cytokines in the supernatant were measured by ELISA. Represented is the mean of three donors ±SD. Significance was determined as compared to untreated group. *P < 0.05, **P < 0.01, ***P < 0.001.</p

Macrophage polarization modulates granuloma formation and the bactericidal activity of macrophages.

<p><b>A.</b> PBMC were left untreated, or infected with <i>M</i>.<i>tuberculosis</i>, or pretreated with IFN-γ+LPS following infection with <i>M</i>.<i>tuberculosis</i>, or pretreated with IL-4 following infection with <i>M</i>.<i>tuberculosis</i> (×100). The aggregate of cells was observed at indicated times in light microscopy. Size scale is indicated. <b>B</b>. The number of granuloma-like structures in each group was counted in 20 randomly selected low-power field (LPF, ×100). *P < 0.05. <b>C</b>. The <i>in vitro</i> model of tuberculous granuloma was generated as described in Materials and Methods. At 48h before <i>M</i>.<i>tuberculosis</i> infection, PBMC were pretreated with IFN-γ+LPS, or IL-4, or left untreated. Supernatant were collected and cells were lysed at indicated time points. Bacteria in supernatant and cell lysate were collected, mixed and inoculated onto the Middlebrook 7H10 solid medium containing 10% OADC. The bacterial load was determined by CFU counting. Represented is the mean of at least three tests±SD. Significance was determined as compared to untreated group. *P < 0.05, **P < 0.01. <b>D</b>. MDM, pretreated with IFN-γ+LPS, or IL-4, or left untreated for 2 day, were infected with <i>M</i>.<i>tb</i> and cultured as described in Materials and Methods. Supernatant were collected and cells were lysed at indicated time points. Bacteria in supernatant and cell lysate were collected, mixed and inoculated onto the Middlebrook 7H10 solid medium containing 10% OADC. The bacterial load was determined by CFU counting. Represented is the mean of at least three tests±SD. Significance was determined as compared to untreated group. *P < 0.05, **P < 0.01.</p

Primers for target genes.

<p>Primers for target genes.</p

Determination of polarization markers of <i>M</i>.<i>tuberculosis</i> infected MDM.

<p>MDM were infected with <i>M</i>.<i>tuberculosis</i> H37Rv at MOI of 5 and polarization markers were determined at indicated times. A. Phenotypic markers were measured by FCM. MFI was measured and depicted as relative expression as compared to isotype control. Represented is the mean of three donors ±SD. Significance was determined as compared to uninfected group. *P < 0.05, **P < 0.01, ***P < 0.001. B. Effector molecules of macrophages were determined by real-time RT-PCR. Represented is mean ±SD of fold change of mRNA level in infected macrophages to uninfected MDM. C. Cytokines in the supernatant were measured by ELISA. Represented is the mean of three donors ±SD. Significance was determined as compared to uninfected group. *P < 0.05, **P < 0.01, ***P < 0.001.Significance in corresponding marker expression between 6d and 3d was also calculated and represented by the following symbols: ^#P < 0.05.</p

Activation status of macrophages in human tuberculous granuloma.

<p><b>A.</b> HE and acid-fast staining of tuberculous granuloma from the human lung tissue of tuberculosis (TB) patients. The structure of tuberculous granuloma with caseous necrosis was obviously observed by HE staining (<b><i>a</i></b>), (×200). Macrophages with irregular shapes were frequently observed (<b><i>b</i></b>), (×1000). Bacteria (stained in red, arrows) between and possibly within the granuloma cells were shown by the acid-fast staining (<b><i>c</i></b>), (×1000). <b>B.</b> The immunostaining of aggregate of CD68-positive macrophages in granuloma with caseous necrosis (<b><i>a</i></b>) or without caseous necrosis (<b><i>b</i></b>), (×100). CD206-positive multinucleated giant cells were frequently observed (c), (×400). <b>C.</b> The immunostaining of serial sections of the same tissue zone showed the expression levels of CD68, CD206 and iNOS in granulomas (×100) and nongranulomatous tissue (×400). D. The levels of CXCL10, CXCL11, CCR7, CCL17 and CCL18 mRNA in granulomatous lung tissues of tuberculosis patients were determined by real-time RT-PCR, in comparison with lung tissues containing no granuloma. The bars represent means ± SD of fold changes of mRNA expression levels in granulomatous tissues (G, showed as black boxes) compared to tissues containing no granuloma (NG, showed as blank boxes). (**P <0.01, ***P <0.001).</p