Myelodysplastic syndromes is a heterogeneous group of myeloid neoplastic disorders originating from hematopoietic stem cells and manifesting as pathological bone marrow hematopoiesis and a high risk of transformation to acute myeloid leukemia. In low-risk patients, the therapeutic goal is to improve hematopoiesis and quality of life. Roxadustat is the world's first oral small-molecule hypoxia-inducible factor prolyl hydroxylase inhibitor, which, unlike conventional erythropoietin, corrects anemia through various mechanisms. In this study, we retrospectively analyzed the changes in anemia, iron metabolism, lipids and inflammatory indexes in patients with low-risk myelodysplastic syndromes to evaluate its therapeutic efficacy and safety, and to provide theoretical and practical data for the application of roxadustat in myelodysplastic syndromes.

To investigate the dynamic homing process and characteristics of macrophages in different organs of immune-mediated aplastic anemia (AA) model mice. Macrophages in donor lymph nodes were sorted by magnetic beads and labeled with PKH67. After modeling according to the preparation method of the AA model, peripheral blood rountine analysis, bone marrow biopsy and HE staining results were analyzed to verify the modeling effect. On days 4, 8, and 12 of modeling, the bone marrow, spleen, and lymph node mononuclear cells were collected, and dynamic changes of PKH67-labeled macrophages in donor mice were analyzed by flow cytometry. In this study, dynamic changes in PKH67-labeled macrophages in the pathogenesis of AA model mice were explored. Macrophages in donor mice homed to the lymph nodes, expanding and differentiating in the lymph nodes, and finally transported to the bone marrow and spleen. Through proteomics mass spectrometry analysis, the related immune inflammatory response pathway of macrophages involved in the activation of the AA bone marrow microenvironment was preliminarily revealed, which provides a basis for the pathological macrophages involved in the pathogenesis of AA model mice.

Objective:To investigate the value of cellular immune status before initial 131I treatment for predicting treatment response in young and middle-aged patients with papillary thyroid cancer (PTC). Methods:From March 2018 to April 2019, 150 young and middle-aged patients with PTC (46 males, 104 females, age (40.0±9.8) years) who underwent total thyroidectomy and neck lymph node dissection in the Affiliated Hospital of Qingdao University were enrolled retrospectively. All patients underwent radioablation 1-2 months after operation, and the serum lymphocyte subsets (CD3 + , CD4 + , CD8 + , CD4/CD8) as well as natural killer (NK) cells were detected 1 d before the initial 131I treatment. Patients were divided into excellent response (ER) group and non-ER group according to the response of 6-12 months after 131I treatment. Clinicopathological characteristics, preablative stimulated thyroglobulin (psTg), initial 131I dose and lymphocyte subsets that might affect the response to 131I treatment were analyzed (independent-sample t test, Mann-Whitney U test, χ2 test, multiple logistic regression analysis). ROC curve analysis was used to evaluate the predictive value of significant factors for non-ER. Results:Of 150 patients, 84 cases were in ER group (56.00%), and 66 cases (44.00%) were in non-ER group. Age ( z=-2.86, P=0.004), M stage ( χ2=13.64, P<0.001), psTg ( z=-8.94, P<0.001), initial 131I dose ( z=-7.60, P<0.001), CD4 + ( t=2.50, P=0.014), CD4/CD8 ( z=-2.22, P=0.027) of the two groups were significantly different. Multivariate analysis showed that psTg (odds ratio ( OR)=1.27, 95% CI: 1.16-1.40, P<0.001) and CD4/CD8 ( OR=0.39, 95% CI: 0.15-0.99, P=0.048) were independent factors for predicting 131I treatment response. The cut-off values of psTg and CD4/CD8 for predicting non-ER were 6.78 μg/L and 1.67, respectively. Conclusions:Cellular immune status before initial 131I treatment may predict treatment response in young and middle-aged patients with PTC. It indicates non-ER response when Tg is higher than 6.78 μg/L and CD4/CD8 is lower than 1.67.

Objective:This study aims, in addition to characterizing pathogenic T cells trafficking to bone marrow (BM) and other organs, to establish immune-mediated AA C.B10 mouse model by DsRed mouse (B6 background) lymph nodes (LN) cells infusion after a total body irradiation (TBI) .Methods:The C.B10 mice received a 5 Gy TBI and then were infused with DsRed mouse (B6 background) LN cells at 5×10 6/mouse via a tail vein injection. The severity of bone marrow failure (BMF) was observed by mononuclear cell count in bone marrow and peripheral blood cell count. On days 3, 6, 9, and 12, mice were sacrificed and collected BM, spleens, LN, or thymus to analyze the dynamic change and activation status of donor T cells in these organs by a flow cytometry. At day 12, the donor-derived T cells from BM, spleens, and LN were sorted to collect the DsRed +CD3 +CD4 + T cells and DsRed +CD3 +CD8 + T cells for RNA isolation and gene expression analyses by PCR array. Results:Relative to control animals that received 5 Gy TBI without LN cell infusion, AA mice developed severe BMF with dramatic decrease in total BM cells, hemoglobin, white blood cells, and platelets in peripheral blood on days 9 and 12 after the LN cell infusion. The frequencies of DsRed + T cells trafficking to BM, LN, and spleens increased with time. Surprisingly, although the DsRed + T cells in BM increased dramatically at a level much higher than those in the spleens and LN on day 12, there were very few DsRed + T cells in BM on days three and six, which was significantly lower than those in spleens or LN. The frequency of DsRed + T cells in thymus was the lowest during the whole process. On day 12, the DsRed +CD3 +CD4 + T cells of BM, LN, and spleens from AA mice were (91.38±2.10) %, (39.78±6.98) %, and (67.87±12.77) %, respectively. On the contrary, the DsRed +CD3 +CD8 +T cells of BM, LN, and spleens were (98.21±1.49) %, (94.06±4.20) %, and (96.29±1.23) %, respectively. We assessed the donor T cell phenotypes using the CD44 and CD62L markers and found that almost all of the DsRed +CD4 + or DsRed +CD8 + T cells in BM were effector memory T cell phenotype from day 9 to day 12. Meanwhile, transcriptome analyses showed higher expression in CD38, IFN-γ, LAG3, CSF1, SPP1, and TNFSF13B in BM DsRed +CD4 + and DsRed +CD8 + T cells. However, there was a lower expression in FOXP3 and CTLA4 in BM DsRed +CD4 + T cells than those in spleens and LN. Conclusions:The DsRed LN cells infusion to induce BMF in CB10 mice enabled to track the donor-derived pathogenic T cells. Besides previously published findings in this model, we demonstrated that donor CD4 + and CD8 + T cells primarily homed to spleens and LN, expanded and differentiated, then infiltrated in BM with a terminal effector memory phenotype. The T cells infiltrated in BM showed more activation and less immunosuppression characteristics compared to those homing to spleens and LN during the BMF development.

Objective:To investigate the preoperative diagnostic value of 99Tc m-methoxyisobutylisonitrile (MIBI) planar imaging and SPECT/CT imaging for primary hyperparathyroidism (PHPT), and analyze the relevant factors affecting the imaging results. Methods:From June 2016 to September 2019, a total of 62 patients (15 males, 47 females, age range: 27-80 years) confirmed as PHPT by postsurgical pathology in Affiliated Hospital of Qingdao University were retrospectively enrolled. The diagnostic efficacies of 99Tc m-MIBI planar imaging and SPECT/CT imaging were compared using χ2 test. The differences of preoperative serum parathyroid hormone (PTH), Ca and the maximum diameter of lesion between the positive and negative groups of planar imaging were analyzed using independent-sample t test and Mann-Whitney U test. The region of interest (ROI) method was applied to calculate the uptake ratio of lesions to normal tissues at the early phase (T/Ne) and delayed phase (T/Nd) in positive cases of planar imaging. Pearson or Spearman correlation analysis was used to evaluate the correlation of T/Ne, T/Nd with preoperative serum PTH, Ca and the maximum diameter of lesion. The receiver operating characteristic (ROC) curves of preoperative serum PTH, Ca and positive planar imaging were drawn and the cut-off values were obtained. Results:The sensitivity of planar imaging and SPECT/CT imaging was 69.35%(43/62) and 87.10%(54/62) respectively ( χ2=5.729, P=0.017). The preoperative serum PTH, Ca levels and the maximum diameter of lesion in patients with positive planar imaging (253.32(107.00, 331.70) ng/L, 2.78(2.51, 2.87) mmol/L, (2.01±0.88) mm) were higher than those with negative planar imaging ((111.86±44.29) ng/L, (2.59±0.21) mmol/L, (1.42±0.55) mm; z values: -2.802, -1.978, t=3.300, all P<0.05). T/Ne was positively correlated with preoperative serum PTH ( rs=0.511, P<0.001) and the maximum diameter of lesion ( r=0.381, P=0.012), and T/Nd was positively correlated with preoperative serum PTH ( rs=0.538, P<0.001), Ca ( rs=0.348, P=0.022) and the maximum diameter of lesion ( r=0.463, P=0.002). The area under the ROC curve between preoperative serum PTH, Ca and planar imaging was 0.725 and 0.646, respectively. Preoperative serum PTH had a better predictive value with the optimal cut-off value of 150.4 ng/L. Conclusions:Preoperative serum PTH, Ca and the maximum diameter of lesion are positively correlated with 99Tc m-MIBI uptake in PHPT patients with positive planar imaging results. When preoperative serum PTH is lower than 150.4 ng/L, planar imaging is prone to false negative. SPECT/CT imaging has a significant value in preoperative diagnosis and the combination of PTH and CT can improve the positive rate.

Objective:To investigate the role of macrophages (M?) in the pathogenesis of modified immune-mediated aplastic anemia (AA) mice model.Methods:Before the establishment of the F1 AA mice model by total-body irradiation combined with allogeneic lymphocyte infusion, the mice of the CLO+AA group were treated with clodronate (CLO) liposomes to remove macrophages, and those of the PBS+AA group were treated with phosphate-buffered saline (PBS) liposomes and used as control. The severity of AA was observed by bone marrow (BM) pathological examination and peripheral blood cell count. Flow cytometry (FCM) was used to detect the CD4 +/CD8 + T lymphocyte subsets in the BM and M? subsets in the BM and spleen of each group. The levels of IFN-γ, TNF-α, G-CSF, GM-CSF, EPO, and TPO in the peripheral blood were detected using enzyme-linked immunosorbent assay. Finally, the relationships between inflammatory factors and M? subsets were analyzed. Results:The BM fatty conversion of mice in the CLO+AA group was significantly alleviated compared with the PBS+AA group. Hemoglobin counts were (91.50±31.63) and (110.65±24.15) g/L, respectively, and the platelet counts were (90.85±121.90) × 10 6/L and (461.13±483.45) ×10 6/L, respectively. The differences were all statistically significant (all P<0.05) . After removing macrophages, the proportions of CD4 + and CD8 + T lymphocytes in BM of mice in the CLO+AA group decreased, but the reduction of CD8 + T cells was more significant. The proportions of CD4 + T cells and CD8 + T cells in BM of the PBS+AA group were (18.5±10.17) % and (36.23±6.40) %, respectively, and in the CLO+AA group were (7.58±8.00) % and (6.67±5.78) %, respectively. Similarly, the percentage of macrophages in the spleen and BM in the CLO+AA group was significantly reduced compared with the PBS+AA group, most of which were M1 macrophages ( P<0.05) . The levels of IFN-γ in peripheral blood of the PBS+AA and CLO+AA groups were (602.37±104.62) ng/L and (303.01±87.22) ng/L, respectively, the levels of TNF-α were (34.46±1.42) ng/L and (23.25±4.21) ng/L, respectively, the levels of GM-CSF were (9.32 ± 2.00) ng/L and (64.85±12.25) ng/L, respectively, the levels of G-CSF were (5 891.78±2 632.39) ng/L and (17 784.16±488.36) ng/L, respectively, the levels of EPO were (9 667.31±4 501.95) ng/L and (2 078.02±897.56) ng/L, respectively, and the levels of TPO were (6.36±2.09) ng/L and (11.67±2.86) ng/L, respectively (all P<0.05) . Conclusions:This study confirmed that macrophages were involved in the pathogenesis of AA, and the degree of BM damage in AA mice was improved by removing macrophages in advance. The imbalance of M1/M2 macrophages and the changes of IFN-γ and TNF-α may be important mechanisms that eventually lead to AA.

Objective: To investigate the response to ¹³¹I therapy and to explore the influence factors in postoperative differentiated thyroid carcinoma (DTC) patients with negative preablative stimulated thyroglobulin (psTg) and iodine-positive lymph node after the first radioablation. Methods: From May 2016 to October 2018, 108 DTC patients (28 males, 80 females, age: (45.7±10.4) years) with negative psTg who underwent ¹³¹I treatment for the first time in the Affiliated Hospital of Qingdao University were retrospectively enrolled. All patients had iodine-positive lymph nodes, which were showed by SPECT/CT imaging 5-6 d after ¹³¹I treatment. The treatment response was evaluated at 6-24 month after ¹³¹I treatment. Patients were divided into excellent response (ER) group and non-excellent response (non-ER) group according to the response to the first ¹³¹I treatment. Independent-sample t test, χ² test and Mann-Whitney U test were used to analyze differences of factors (e.g. age, gender, extraglandular infiltration) between the 2 groups, and then multivariate logistic regression was performed to find the influence factors for treatment response. The receiver operating characteristic (ROC) curve was employed to evaluate the predictive value of the iodine-positive lymph node size in ¹³¹I treatment response. Results: A total of 82 patients (75.93%, 82/108) achieved ER, and 26 (24.07%, 26/108) was non-ER patients. There were significant differences in age (t=-2.540, P=0.016), extraglandular infiltration (χ²=5.764, P=0.016), T stage (χ²=19.857, P<0.001), N stage (χ²=14.145, P=0.001), risk stratification of recurrence (χ²=11.487, P=0.003), ultrasound results before ¹³¹I treatment (χ²=44.819, P<0.001), dose of the first ¹³¹I treatment (U=780.0, P=0.018), size (long diameter) of iodine-positive lymph node (U=184.0, P<0.001), and psTg level (U=776.0, P=0.037) between ER and non-ER groups. Multivariate logistic regression showed that age, size of iodine-positive lymph node and ultrasound results before ¹³¹I treatment were closely related to ¹³¹I treatment response. The odds ratio (OR) values (95% CI) were 1.123 (1.025-1.231), 4.275 (1.893-9.653) and 260.86 (8.123-8 376.764), respectively. The sensitivity, specificity, accuracy, positive predictive value and negative predictive value for non-ER were 100%(26/26), 70.73%(58/82), 77.78%(84/108), 52.00%(26/50) and 100%(58/58) respectively when the cut-off value of iodine-positive lymph node size was 5.5 mm. Conclusion The response of some DTC patients with negative psTg and iodine-positive lymph node after the first radioablation were non-ER. Age, ultrasound results before ¹³¹I treatment and size of iodine-positive lymph node are sensitive indicators for predicting clinical outcome in DTC patients with negative psTg and iodine-positive lymph node after the first radioablation.

Objective: To explore the effects and possible mechanism of rapamycin (RAPA) on apoptosis of CD4⁺CD25⁺ Tregs from the mouse severe aplastic anemia (SAA) model. Methods: The BALB/c female SAA model mice were induced by interferon-gamma in combination with busulphan. The SAA model mice were intraperitoneal injection with RAPA at daily dose of 0.5 mg/kg for 5 days (the RAPA-treated group, n=15) in the SAA group (n=15) and the un-treated group (n=15) were control. Bone marrow hematopoiesis changes were observed by the patho-morphological examination of femurs. The mononuclear cells of the peripheral blood and spleen were subjected to assess the intracellular Foxp3 expression in CD4⁺CD25⁺ Tregs by flow cytometry (FCM). In addition, after being pured by immunomagnetic beads, the splenic CD4⁺CD25⁺ Tregs was subjected to assess apoptosis by FCM and the Akt and Stat3 phosphorylation by using of western blot. Results: The patho-morphological examination of femurs showed normal marrow cell proliferation in un-treated group and hypocellularity in both SAA group and RAPA-treat group, with an increase in the number of fat cells. The bone marrow hematopoietic tissue ratio in RAPA-treat group was higher than SAA group [(9.75±1.83)% vs (7.00±2.00)%, Δx=2.15% (95%CI 0.15%-5.35%), P=0.037]. In the SAA group, FCM analysis showed down-expression of Foxp3 in CD4⁺CD25⁺ Tregs compared with the un-treated group. However, after treatment with RAPA, the expression of Foxp3 in CD4⁺CD25⁺ Tregs was increased (P<0.017). Compared with the un-treated group, increased CD4⁺CD25⁺ Tregs apoptosis [(19.84±1.39)% vs (29.85±2.72)%] with increased Akt phosphorylation accompanied by increased Stat3 phosphorylation was found in SAA group (P<0.05, respectively). On the contrary, RAPA-treated group exhibited CD4⁺ CD25⁺ Tregs with a reduction in apoptosis rate [(22.39±3.71)%], Akt phosphorylation and Stat3 phosphorylation compared with the SAA group (P<0.05, respectively). Conclusion These results indicate that RAPA may increase the expression of Foxp3 by down-regulation the levels of Akt and Stat3 phosphorylation and reduce apoptosis in splenic CD4⁺CD25⁺ Tregs from the mice model of SAA.

Objective To investigate the inhibitory effects of RNA interference targeting GFI?1 on growth and proliferation of atypical chronic myelogenous leukemia ( aCML) NT1 cells. Methods NT1 cells were transfected with PBS and liposome complex ( vehicle group) , scrambled siRNA and liposome complex ( negative control, NC group ) , and GFI?1 siRNA and liposome complex ( GFI?1 siRNA group ) , respectively. Real?time quantitative RT?PCR ( qRT?PCR) and Western blot were performed to examine the expression levels of GFI?1 mRNA and protein, respectively. The proliferation abilities of NT1 cells of the three groups were evaluated by MTT assay. The cell cycle in cells of the three groups was analyzed by flow cytometry. Moreover, nude mouse xenograft model was used to detect the tumor formation ability in the three group cells. Results Quantitative real?time PCR data showed that the expression level of GFI?1 mRNA in GFI?1 siRNA group was significantly lower than those of NC group and vehicle group [(0.367±0.017) vs. (0.918±0.006) and (1.010±0.005), respectively, (P<0.05)]. Western blot results showed that the GFI?1 protein expression level in the GFI?1 siRNA group was also significantly reduced, compared with those of the NC group and vehicle group ( P<0.05 for both) . From MTT assay data, the absorbance value of NT1 cells in the GFI?1 siRNA group (0.667±0.059) was significantly lower than those of the NC group (1.096±0.049) and vehicle group (1.193±0.064, P=0.023). Flow cytometry data showed that sub?G1 and G0/G1 phase proportions of the GFI?1 siRNA group were significantly higher than those of the NC and vehicle groups [ sub?G1: (8.2±2.5)% vs. (1.9±1.3)% and (2.0±3.6)%, respectively, (P<0.05);G0/G1:(66.7±3.8)% vs. (53.3±4.5)% and (48.6±3.2)%, respectively, (P<0.05)]. Furthermore, the tumor weight in the GFI?1 siRNA group [(0.37±0.02) g] was significantly lower than those in the NC group [(0.83±0.06) g] and vehicle group [(0.92±0.04) g] (P<0.05). Conclusions RNA interference targeting GFI?1 inhibits the growth and proliferation of NT1 cells, which may provide a new therapeutic target for atypical chronic myelogenous leukemia.