Application of metagenomic next-generation sequencing in the clinical diagnosis of infectious diseases after allo-HSCT: a single-center analysis.

BACKGROUND: We investigated the value of metagenomic next-generation sequencing (mNGS) in diagnosing infectious diseases in patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: Fifty-four patients who had fever following allo-HSCT from October 2019 to February 2022 were enrolled. Conventional microbiological tests (CMTs) and mNGS, along with imaging and clinical manifestations, were used to diagnose infection following allo-HSCT. The clinical diagnostic value of mNGS was evaluated. RESULTS: A total of 61 mNGS tests were performed, resulting in the diagnosis of 46 cases of infectious diseases. Among these cases, there were 22 cases of viral infection, 13 cases of fungal infection, and 11 cases of bacterial infection. Moreover, 27 cases (58.7%) were classified as bloodstream infections, 15 (32.6%) as respiratory infections, 2 (4.3%) as digestive system infections, and 2 (4.3%) as central nervous system infections. Additionally, there were 8 cases with non-infectious diseases (8/54, 14.81%), including 2 cases of interstitial pneumonia, 2 cases of bronchiolitis obliterans, 2 cases of engraftment syndrome, and 2 cases of acute graft-versus-host disease. The positive detection rates of mNGS and CMT were 88.9% and 33.3%, respectively, with significant differences (P < 0.001). The sensitivity of mNGS was 97.82%, the specificity was 25%, the positive predictive value was 93.75%, and the negative predictive value was 50%. Following treatment, 51 patients showed improvement, and 3 cases succumbed to multidrug-resistant bacterial infections. CONCLUSIONS: mNGS plays an important role in the early clinical diagnosis of infectious diseases after allo-HSCT, which is not affected by immunosuppression status, empiric antibiotic therapy, and multi-microbial mixed infection.

Metagenomic Next-Generation Sequencing (mNGS) of cerebrospinal fluid for diagnosis of human herpesvirus 6B encephalitis following transplantation for severe aplastic anemia.

INTRODUCTION: Human herpesvirus 6B (HHV-6B) encephalitis is common in immunosuppressed patients and presents a diagnostic challenge for physicians. Metagenomic next-generation sequencing (mNGS) may facilitate early diagnosis of HHV-6B encephalitis. Herein, we described a case of HHV-6B encephalitis following transplantation for severe aplastic anemia (SAA) diagnosed by mNGS. CASE SUMMARY: A 31-year-old male underwent myeloablative haploid hematopoietic stem cell transplantation for the treatment of SAA. On day + 21 after transplantation, the patient developed symptoms such as sudden epilepsy, drowsiness, memory dislocation, and memory loss. HHV-6B encephalitis was confirmed based on cranial MRI and mNGS of cerebrospinal fluid. Following antiviral therapy with sodium foscarnet, the symptoms improved and HHV-6B was negative by mNGS. There were no serious sequelae. Currently, the patient is in good health and is still under follow-up. CONCLUSIONS: A case of HHV-6B encephalitis after SAA transplantation was diagnosed by mNGS of cerebrospinal fluid in time and was effectively treated with sodium foscarnet.

The level of Tim-3+CD8+ T cells can serve as a potential marker for evaluating the severity of acute graft-versus-host disease after haplo-PBSCT.

Early and accurate diagnosis of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation is crucial for the prognosis of patients. This study identified a potential biomarker for the severity of aGVHD after human leukocyte antigen (HLA)-haploidentical peripheral blood hematopoietic stem cell transplantation (haplo-PBSCT). We included 20 healthy subjects and 57 patients who underwent haplo-PBSCT. Of these patients, 22 developed aGVHD after haplo-PBSCT. The results showed that patients with aGVHD had significantly increased levels of Tim-3+/Perforin+/Granzyme B+CD8+ T cells, but significantly decreased Galectin-9. The differences in Galectin-9 and Tim-3+/Granzyme B+CD8+ T cells between grade I-II aGVHD and III-IV aGVHD were also significant. In vitro, the apoptosis of CD8+ T cells from aGVHD patients was significantly increased after Tim-3/Galectin-9 pathway activation, which decreased Granzyme B secretion. As revealed by univariate analysis, the level of Tim-3+CD8+ T cells was a risk factor for severe aGVHD. ROC analysis demonstrated that high levels of Tim-3+CD8+ T cells had a significant diagnostic value for severe aGVHD, with an area under the curve of 0.854 and cut-off value of 14.155%. In conclusion, the binding of Tim-3 with exogenous Galectin-9 can promote apoptosis of CD8+ T cells and affect the secretion of Granzyme B. Tim-3+CD8+ T cells have the potential to serve as immunological markers for assessing the severity of aGVHD after haplo-PBSCT and identifying patients at a higher risk for severe aGVHD.

Adipose-Derived Mesenchymal Stem Cell Facilitate Hematopoietic Stem Cell Proliferation via the Jagged-1/Notch-1/Hes Signaling Pathway.

Background: Poor graft function (PGF) is a life-threatening complication following hematopoietic stem cell transplantation (HSCT). Current therapies, such as CD34+ cell infusion, have shown limited effectiveness. Conversely, mesenchymal stem cells (MSCs) show potential in addressing PGF. Adipose-derived mesenchymal stem cells (ADSCs) effectively support long-term hematopoietic stem cell proliferation. Therefore, this study aimed to investigate the mechanisms underlying the long-term hematopoietic support provided by ADSCs. Methods: ADSCs were isolated from mice and subsequently identified. In vitro experiments involved coculturing ADSCs as feeders with Lin-Sca-1+c-kit+ (LSK) cells from mice for 2 and 5 weeks. The number of LSK cells was quantified after coculture. Scanning electron microscopy was utilized to observe the interaction between ADSCs and LSK cells. Hes-1 expression was assessed using western blot and real-time quantitative PCR. An γ-secretase inhibitor (GSI) was used to confirm the involvement of the Jagged-1/Notch-1/Hes-1 pathway in LSK cell expansion. Additionally, Jagged-1 was knocked down in ADSCs to demonstrate its significance in ADSC-mediated hematopoietic support. In vivo experiments were conducted to study the hematopoietic support provided by ADSCs through the infusion of LSK, LSK + fibroblasts, and LSK + ADSCs, respectively. Mouse survival, platelet count, leukocyte count, and hemoglobin levels were monitored. Results: ADSCs showed high-Jagged-1 expression and promoted LSK cell proliferation. There was a direct interaction between ADSCs and LSK cells. After coculture, Hes-1 expression increased in LSK cells. Moreover, GSI-reduced LSK cell proliferation and Hes-1 expression. Knockdown of Jagged-1 attenuated ADSCs-mediated promotion of LSK cell proliferation. Furthermore, ADSCs facilitated hematopoietic recovery and promoted the survival of NOD/SCID mice. Conclusion: The hematopoietic support provided by ADSCs both in vivo and in vitro may be mediated, at least in part, through the Jagged-1/Notch-1 signaling pathway. These findings provide valuable insights into the mechanisms underlying ADSCs-mediated hematopoietic support and may have implications for improving the treatment of PGF following HSCT.

APPswe /PS1ΔE9 mice exhibit low oxygen saturation and alterations of erythrocytes preceding the neuropathology and cognitive deficiency during Alzheimer's disease.

AIM: The molecular mechanism underlying Alzheimer's disease (AD) pathologies remains unclear. The brain is extremely sensitive to oxygen deprivation, and brief interruptions in oxygen supply may lead to permanent brain damage. The objective here was to access the red blood cell (RBC) physiological alterations and the changes in blood oxygen saturation of an AD model as well as to explore the possible mechanism underlying these pathologies. METHODS: We used female APPswe /PS1ΔE9 mice as AD models. Data were collected at the age of 3, 6, and 9 months. In addition to examining classic features of AD, namely cognitive deficiency and Aß depositions, 24 h blood oxygen saturation was monitored by Plus oximeters in real time. In addition, RBC physiological parameters were measured by blood cell counter using peripheral blood from the epicanthal veins. Furthermore, in the mechanism investigations, the expression of phosphorylated band 3 protein was examined by a series of Western blot analyses, and the levels of soluble Aß40 and Aß42 on the membrane of RBCs were determined by ELISA. RESULTS: Our results showed that the blood oxygen saturation in the AD mice was significantly reduced as early as at 3 months of age, preceding the neuropathological changes and cognitive impairments. Meanwhile, the expression of phosphorylated band 3 protein and levels of soluble Aß40 and Aß42 were all elevated in the erythrocytes of the AD mice. CONCLUSION: APPswe /PS1ΔE9 mice exhibited decreased oxygen saturation together with reduced RBC counts and hemoglobin concentrations at the early stage, which may aid in the development of predictive markers for AD diagnosis. The increased expression of band 3 protein and elevated Aß40 and Aß42 levels may contribute to the deformation of RBCs and, in turn, cause the subsequent AD development.

Unique Reduced-Intensity Conditioning Haploidentical Peripheral Blood Stem Cell Transplantation Protocol for Patients with Hematologic Malignancy.

Reduced-intensity conditioning (RIC) haploidentical (haplo-) hematopoietic stem cell transplantation (HSCT) requires more hematopoietic progenitor and stem cells (HPSCs) to promote engraftment and immune reconstitution and needs a stronger graft-versus-leukemia effect. Peripheral blood stem cells (PBSCs) offer advantages over bone marrow; however, the use of higher-dose non-T cell-depleted (non-TCD) in vitro PBSCs may increase the occurrence of severe graft-versus-host disease (GVHD). This prospective, single-arm clinical study was performed to investigate using high-dose non-TCD in vitro PBSCs as the graft source, using fludarabine/Ara-C/busulfan (FAB) as the conditioning regimen, using rabbit antithymocyte globulin to remove T cells in vivo, and enhancing GVHD prophylaxis with an IL-2 receptor antagonist in RIC-haplo-HSCT in patients with hematologic malignancies age 50 to 70 years or <50 years with comorbidities (Hematopoietic Cell Transplantation Comorbidity Index score ≥2) classified as intermediate to high risk. The primary endpoint was day 100 acute GVHD (aGVHD). A total of 47 patients were enrolled; the median age was 52 years (range, 30 to 68 years), the median duration of follow-up was 34 months (range, 2 to 99 months), and the medium-infused doses of mononuclear cells, CD34+ cells, and CD3+ cells were 15.93 × 108/kg, 8.68 × 106/kg, and 5.57 × 108/kg, respectively. The cumulative incidence of grade II-IV aGVHD at day 100 was 30.3% (95% confidence interval [CI], 15.9% to 44.8%), and that of grade III-IV aGVHD was 10.2% (95% CI, .6% to 19.8%). The 2-year cumulative incidence of chronic GVHD (cGVHD) was 34.9% (95% CI, 19.0% to 50.8%). The 2-year cumulative incidences of localized and extensive cGVHD were 26.1% (95% CI, 11.80% to 40.40%) and 8.7% (95% CI, 3.26% to 20.65%), respectively. The 2-year cumulative incidence of relapse was 17.3% (95% CI, 5.1% to 29.5%), the 2-year overall survival rate was 71.2% (95% CI, 57.9% to 84.5%), and the 2-year disease-free survival rate was 66.2% (95% CI, 52.1% to 80.3%). The incidence of aGVHD was not high, and the overall efficacy was good. This study demonstrates that this unique RIC-haplo-PBSC transplantation protocol was effective in treating hematologic malignancies. Nonetheless, larger prospective multicenter clinical trials and experimental studies should be performed to further confirm our findings.

The level of Tim-3+CD8+ T cells can serve as a potential marker for evaluating the severity of acute graft-versus-host disease after haplo-PBSCT

Early and accurate diagnosis of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation is crucial for the prognosis of patients. This study identified a potential biomarker for the severity of aGVHD after human leukocyte antigen (HLA)-haploidentical peripheral blood hematopoietic stem cell transplantation (haplo-PBSCT). We included 20 healthy subjects and 57 patients who underwent haplo-PBSCT. Of these patients, 22 developed aGVHD after haplo-PBSCT. The results showed that patients with aGVHD had significantly increased levels of Tim-3+/Perforin+/Granzyme B+CD8+ T cells, but significantly decreased Galectin-9. The differences in Galectin-9 and Tim-3+/Granzyme B+CD8+ T cells between grade I-II aGVHD and III-IV aGVHD were also significant. In vitro, the apoptosis of CD8+ T cells from aGVHD patients was significantly increased after Tim-3/Galectin-9 pathway activation, which decreased Granzyme B secretion. As revealed by univariate analysis, the level of Tim-3+CD8+ T cells was a risk factor for severe aGVHD. ROC analysis demonstrated that high levels of Tim-3+CD8+ T cells had a significant diagnostic value for severe aGVHD, with an area under the curve of 0.854 and cut-off value of 14.155%. In conclusion, the binding of Tim-3 with exogenous Galectin-9 can promote apoptosis of CD8+ T cells and affect the secretion of Granzyme B. Tim-3+CD8+ T cells have the potential to serve as immunological markers for assessing the severity of aGVHD after haplo-PBSCT and identifying patients at a higher risk for severe aGVHD.

Mechanism of Progesterone in Treatment of Traumatic Brain Injury Based on Network Pharmacology and Molecular Docking Technology.

BACKGROUND Previous studies have confirmed that progesterone has a protective effect on traumatic brain injury (TBI). In this paper, network pharmacology and molecular docking technology were used to further explore the potential mechanism of progesterone in the treatment of TBI. MATERIAL AND METHODS Based on network pharmacology, potential targets of progesterone for TBI were obtained. The network diagram of interactions between target proteins was established to screen the key targets of progesterone for TBI. The DAVID database was used to analyze its biological function and enrichment pathway, and to explore and determine the biological pathway of progesterone in treating TBI. Molecular docking technology was used to simulate the interaction between progesterone and key target proteins. RESULTS Progesterone can treat TBI by anti-inflammatory action, repairing damaged cell membranes, stabilizing the structure of the blood-brain barrier, alleviating brain edema, reducing neuronal apoptosis, and improving neurological function. The molecular mechanism involves the PI3K/Akt signaling pathway, MAPK signaling pathway, and Ras signaling pathway. CONCLUSIONS Progesterone is a potential clinical treatment for TBI. Exploring the potential targets and pathways of TBI therapy through network pharmacology can provide a direction for subsequent research.

Incidence of late-onset hemorrhagic cystitis and its effect on PFS in acute leukemia patients after haplo-PBSCT: The 5-year single-center data.

We conducted a single-center 5-year retrospective study on the occurrence of hemorrhagic cystitis (HC) and its effect on survival after haploid high-dose peripheral blood stem cell transplantation (haplo-PBSCT) in patients with acute leukemia. We retrospectively analyzed 153 patients with acute leukemia who were treated with non-in vitro T-cell depleted haplo-PBSCT and myeloablative conditioning regimen. All patients were followed up for more than 180 days after transplantation. HC occurrence and its effect on long-term progression free survival (PFS) were retrospectively analyzed. Totally, 64 out of 153 patients had late onset HC (LOHC). No early onset HC occurred. The median onset time was 38.5 (17-163) days after transplantation. The cumulative incidence of LOHC was 41.8%. The cumulative incidence of LOHC in patients under 27 years old (50.0%) and in ALL patients (54.1%) was significantly higher than that in patients over 27 years old (34.5%) and in AML patients (36.9%), respectively. The cumulative incidence of mild LOHC was 44.2% and that of severe LOHC was 28.6%. However, urine copies of BK virus were not related to LOHC duration. There was no significant difference in 3-year expected PFS between AML and ALL patients with and without LOHC, or between LOHC duration more than and less than 38.5 days (P>0.05). Conclusively, LOHC incidence is higher in patients under 27 years old and in ALL patients. LOHC occurrence is related to urine BK virus copy, but not blood BK virus load. LOHC duration and severity has no significant effect on PFS.

New Understanding on the Pathophysiology and Treatment of Constipation in Parkinson's Disease.

Constipation, one of the most common prodromal non-motor symptoms of Parkinson's disease (PD), usually occurs several years earlier than the onset of motor symptoms. Previous studies have shown that constipation occurrence increases as the disease progresses. However, the mechanism underlying this pathologic disorder is not clear yet. Moreover, chronic constipation causes slowness in gastric emptying and, therefore, may lead to a delay in the absorption of medications for PD, including levodopa and dopamine agonists. Accordingly, it is necessary to understand how the pathophysiological factors contribute to constipation during PD as well as pursue precise and effective treatment strategies. In this review, we encapsulate the molecular mechanism of constipation underlying PD and update the progress in the treatments of PD-associated constipation.

Non-surgical management of tubal ectopic pregnancy: A systematic review and meta-analysis.

BACKGROUND: Ectopic pregnancy (EP) is a common cause of acute abdominal pain in the field of gynecology. Because the majority of women with EP are hemodynamically stable, non-surgical therapy is a viable option. The goal of this study was to determine the most effective non-surgical therapy for hemodynamically stable EP. METHODS: We performed a systematic review and meta-analysis. We searched PubMed, LILACS, SciELO, CINAHL, Embase, and the Cochrane library in May 2020, with no starting date restrictions.Studies were restricted to randomized controlled trials, which were included if the target population contained women with tubal EP and the intervention was non-surgical management. The primary outcome measure was treatment success defined by a decrease in serum hCG to a level ranging from five mIU/mL to 50 mIU/mL. Secondary outcome measures were side effects, time needed to treat, number of injections and operative rate. RESULTS: We conducted a meta-analysis of 15 studies that included 1573 women who were diagnosed with EP and managed non-surgically. There was no significant difference in treatment success in the matched groups; however, single-dose MTX was associated with fewer side effects than multiple-dose (relative risk 0.48, 95% confidence interval 0.28-0.80, P = .006) and two-dose therapies (relative risk 0.74, 95% confidence interval 0.55-1.00, P = .05). CONCLUSIONS: We highly recommend that single-dose MTX without mifepristone be used first-line in patients who require conservative therapy due to the inherent negative effects of mifepristone. An EP woman with a low -hCG level that is falling or plateauing should receive expectant treatment to reduce adverse effects.

Peripheral blood Th9 cells reconstitution and its relationship with acute graft-versus-host disease after matched-sibling peripheral blood hematopoietic stem cell transplantation.

T helper type 9 (Th9) cells have recently been identified as a new effector T cell subset. This study is to analyze the reconstitution of Th9 cell after matched sibling peripheral blood hematopoietic stem cell transplantation (MS-PBSCT) and the relationship between Th9 cell and acute graft-versus-host disease (aGVHD). Flow cytometry and ELISA were used to analyze the percentages of Th9 cell, levels of IL-9, TGF-ß, IFN-γ, and IL-4. The results showed that for patients without aGVHD, Th9 cells recovery started from day 60 after transplantation and reached normal level on day 90. Serum TGF-ß, IL-4, and IFN-γ reached normal levels on day 60, 60, and 90 post transplantation respectively. The serum IL-9 recovery is slower than that of IFN-γ and IL-4. For patients suffering from aGVHD, they had declined Th9 cell numbers, lower IL-9 and TGF-ß levels, but higher serum IFN-γ level when compared with those without aGVHD after transplantation. Serum IFN-γ/IL-9 ratios increased linearly with grades of aGVHD. In conclusion, Th9 cells recovery is delayed after MS-PBSCT in patients with aGVHD but early in patients without aGVHD, indicating quick immune reconstitution of Th9 cells and IL-9 after MS-PBSCT may promote the immune tolerance.

Reconstitution and clinical significance of T cell subsets in the early stage after related HLA-mismatched peripheral blood hematopoietic SCT without T-cell depletion in vitro.

Related HLA-haploidentical HSCT has been applied more and more recently, but the reconstitution of T lymphocyte subsets and its clinical significance in patients received related HLA-haploidentical non T-cell depleted in vitro high-dose peripheral blood hematopoietic SCT (RHNT-PSCT) are incompletely defined. In the present study of our RHNT-PSCT, we found that in non-aGVHD group, CD3(+) T lymphocyte recovered to normal levels gradually between 60 and 90 days, and the recovery of CD4(+) T lymphocyte was retarded significantly, CD4(+)/CD8(+) ratio was apparently inverted. Whereas, the ratio of CD4(+) CD25(+) Foxp3(+) Treg cells was significantly lower in aGVHD group than in healthy control group and non-aGVHD group, and also in grade III-IV aGVHD patients than in grade I-II aGVHD patients. Meanwhile, we observed the level of interleukin-10 (IL-10) gradually increased in serum of patients without aGVHD, but decreased in III-IV aGVHD patients significantly. Spearman correlation analysis showed that serum IL-10 level was negatively correlated with the grade of aGVHD. These results suggest that the reconstitution of peripheral blood T lymphocyte subsets is good, and dynamic detection of Treg cells and serum IL-10 level might predict aGVHD in the early stage after our RHNT-PSCT.

[Clinical features and risk factors analysis of acute graft-versus-host disease in patients with related HLA-haploidentical non T cell-depleted in vitro peripheral hematopoietic stem cell transplantation].

OBJECTIVE: To study the clinical features of acute graft-versus-host disease (aGVHD) and its risk factors for the related HLA-haploidentical non T cell-depleted in vitro peripheral hematopoietic stem cell transplantation (RHNT-PBSCT). METHODS: From July 2002 to December 2012, 104 patients who underwent the RHNT-PBSCT were enrolled to analyze the incidences, location and its risk factors of aGVHD, compared with those of the 103 patients who received the HLA-matched sibling non T cell-depleted in vitro PBSCT (MSNT-PBSCT) in the same period. RESULTS: (1)The cumulative incidence of aGVHD in the RHNT-PBSCT group was significantly higher than the MSNT-PBSCT group [(56.2±4.7)% vs (34±3.6)%, P<0.05], but the cumulative incidences of II-IV and III-IVgrade aGVHD had no significant difference between the two groups[(39.5±2.9)% vs (21.2±5.4)%, P>0.05; (12.6±4.1)% vs (10.8±2.4)%, P>0.05]. (2)The cumulative incidence of cutaneous aGVHD was significantly higher in RHNT-PBSCT group than that in MSNT-PBSCT group [(42.3±3.2)% vs (17.5±2.3)%, P<0.05]. The cumulative incidences of liver and gastrointestinal aGVHD between the two groups had no significant difference [(7.7±2.1)% vs (12.6±3.4)%, P>0.05; (16.3±4.5)% vs (10.3±2.5)%, P>0.05]. (3)The 3-year disease free survival (DFS) and overall survival(OS) of RHNT-PBSCT group and MSNT-PBSCT group were (63±5.5)%, (65.2±4.7)% and (74.2±5.4)%, (77.4±5)% respectively, without significance (P=0.078, P=0.052). (4)aGVHD occurrence with HLA haplotype (P=0.003) and matched loci (P=0.002) were significantly correlated by univariate analysis. Multivariate analysis showed that only the HLA typing is a risk factor for aGVHD (HR=1.891, P=0.03). CONCLUSION: Although the incidence of total aGVHD in RHNT-PBSCT protocol is higher than that in MSNT-PBSCT, but there was no significance in severe aGVHD and cutaneous aGVHD was the common type, which indicates that RHNT-PBSCT protocol is feasible.

[Analysis of the therapeutic efficacy and prognosis for acute myeloid leukemia M2a patients treated by IA and DA regimens].

This study was purposed to compare the therapeutic efficacy and prognosis of acute myeloid leukemia M2a (AML-M2a) patients treated by idarubicin (IDA) combined with cytarabine (Ara-C) (IA) and daunorubicin (DNR) combined cytarabine (Ara-C) (DA) regimens. The clinical data of 65 patients with AML-M2a in our hospital were collected from May 2009 to May 2013 and analyzed. The results indicated the complete remission in IA group was slightly higher than that in DA group, there was no statistically significant difference(P > 0.05); leukocyte minimum value in IA group [(0.58 ± 0.40)×10(9)/L] was obviously lower than that in DA group [(0.99 ± 0.67)×10(9)/L] (P < 0.05); neutrophil minimum value in IA group [(0.19 ± 0.09)×10(9)/L] was significantly lower than that in DA group [(0.21 ± 0.16)×10(9)/L] (P < 0.05); the neutropenia duration in IA group (12.59 ± 5.31)d was much longer than that in DA group (9.17 ± 7.04)d (P < 0.05). The median survival time of patients in IA group was 36.67 months, which was obviously longer than that of patients in DA group (21.45 months) (P < 0.05). The lactate dehydrogenase (LDH) value and chemotherapy regimens were the independently risk factor affecting the prognosis of AML-M2a patients. It is concluded that as compared with DA regimen, the IA regimen can prolong the median survival time and has better long-term therapeutic efficacy, thus it can be used as the first chemotherapy regimen for treatment of AML-M2a.

Impact of fibroblast activation protein on osteosarcoma cell lines in vitro.

Fibroblast activation protein (FAP) or seprase, which belongs to the group type II integral serine proteases, is an integral membrane serine peptidase. Previous studies have demonstrated that FAP has an effect on tumor growth, proliferation and invasion. However, the cellular functional role that FAP plays in osteosarcoma (OS) remains unknown. The aim of the present study was to investigate the activities of FAP in OS cell lines. The gene expression of FAP was knocked down through a hammerhead ribozyme transgene, and the various functions between the knockdown cells and their control cells were tested using a series of functional assays in vitro. The results indicated that knockdown of FAP markedly reduced the ability of cellular growth, matrix adhesion, migration and invasion in MG-63 and HOS cell lines compared with the control cells (P<0.05). In conclusion, FAP influences OS cells and may play a role in OS tumor progression and metastasis.

MiR-223/Ect2/p21 signaling regulates osteosarcoma cell cycle progression and proliferation.

Osteosarcoma is one of the most common tumors. The mechanisms of formation and development of osteosarcoma have been studied for a long time. Recently, more and more evidence showed that miRNAs play important roles in regulating tumor growth. In this study we found that miRNA-223 was downregulated in both osteosarcoma patients' tumor tissues and osteosarcoma cell lines. Overexpression of miRNA-233 greatly inhibited the proliferation of Saos-2 cells. Cell cycle analysis by flow cytometry showed the arrest of cell cycle progression at the G1 phase. Further mechanistic study indicated that Ect2 was directly targeted by miR-223. Downregulation of Ect2 by miR-223 induces the expression of p21, p27 and the phospharylation of retinoblastoma, which are involved in the G1 block. We concluded that miR-223 functions as a tumor suppresser in osteosarcoma and miR-223/Ect2/p21 signaling is an important pathway that regulates the osteosarcoma cell cycle progression and proliferaion.

Antagonism of miR-21 reverses epithelial-mesenchymal transition and cancer stem cell phenotype through AKT/ERK1/2 inactivation by targeting PTEN.

BACKGROUND: Accumulating evidence suggested that epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) characteristics, both of which contribute to tumor invasion and metastasis, are interrelated with miR-21. MiR-21 is one of the important microRNAs associated with tumor progression and metastasis, but the molecular mechanisms underlying EMT and CSC phenotype during miR-21 contributes to migration and invasion of breast cancer cells remain to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: In this study, MDA-MB-231/anti-miR-21 cells were established by transfected hsa-miR-21 antagomir into breast cancer MDA-MB-231 cells. EMT was evaluated by the changes of mesenchymal cell markers (N-cadherin, Vimentin, and alpha-SMA), epithelial cell marker (E-cadherin), as well as capacities of cell migration and invasion; CSC phenotype was measured using the changes of CSC surface markers (ALDH1 and CD44), and the capacity of sphereforming (mammospheres). We found that antagonism of miR-21 reversed EMT and CSC phenotype, accompanied with PTEN up-regulation and AKT/ERK1/2 inactivation. Interestingly, down-regulation of PTEN by siPTEN suppressed the effects of miR-21 antagomir on EMT and CSC phenotype, confirming that PTEN is a target of miR-21 in reversing EMT and CSC phenotype. The inhibitors of PI3K-AKT and ERK1/2 pathways, LY294002 and U0126, both significantly suppressed EMT and CSC phenotype, indicating that AKT and ERK1/2 pathways are required for miR-21 mediating EMT and CSC phenotype. CONCLUSIONS/SIGNIFICANCE: In conclusion, our results demonstrated that antagonism of miR-21 reverses EMT and CSC phenotype through targeting PTEN, via inactivation of AKT and ERK1/2 pathways, and showed a novel mechanism of which might relieve the malignant biological behaviors of breast cancer.

Reconstruction of epidural fat with engineered adipose tissue from adipose derived stem cells and PLGA in the rabbit dorsal laminectomy model.

Epidural fibrosis resulted from epidural fat destruction following laminectomy operation is regarded as a main cause of failed back surgery syndrome, which represents one of the most common complications in spine surgery. Up to now, the effectiveness of currently available treatments to prevent such a syndrome is quite limited. In the present study, we aimed to restore epidural fat using adipose tissue engineered from adipose derived stem cells (ASCs) in a rabbit dorsal laminectomy model. ASCs isolated from subcutaneous fat were first expanded to passage 3, seeded on porous poly(lactic-co-glycolic acid, PLGA) scaffold and then adipogenically induced for 7 days in vitro to form cell-scaffold complex. Laminectomy sites were created at T13-L1 level in each animal. The laminectomy defect was implanted either with cell-scaffold complex or PLGA scaffold alone. Non-treated defect was also included as a control. The animals were subjected to MRI evaluation at 1, 12 and 24 weeks post-surgery, and sacrificed at 24 weeks for gross and histological observation. It was demonstrated by MRI evaluation that scar tissue of coarse and high density was formed within laminectomy site in PLGA alone and non-treated groups as early as 12 weeks. However, the defect implanted with engineered adipose had formed a continuous linear adipose tissue regenerated along the spinal cord at 24 weeks. Histologically, a distinct area of adipose tissue just overlaying the dura mater could be identified in cell-scaffold complex treated group at 24 weeks post-operation. Regeneration of epidural fat was further confirmed by positive Oil Red O staining. As to the defect treated with PLGA alone or left untreated, either fine or dense scar tissue adhering to the dura mater was observed. Moreover, we could track the implanted ASCs labeled by magnetic nanoparticles within epidural area for as long as four weeks by MRI detection. Thus, adipose tissue engineered from ASCs exhibited great potential in restoration of epidural fat to prevent formation of epidural fibrosis.

Characterization of patients and its relationship with medical complaints.

BACKGROUND: Patient characteristics may be an internal factor influencing patient complaints, but in China patient characterization is restricted to patient satisfaction surveys, and few studies have considered the relationship between patient characteristics and patient complaints. The aim of this research was to determine the reasons for complaints. METHODS: In this study, we analyzed the characteristics of hospitalized patients and explored their relationship with medical complaints. RESULTS: The significant factors were age (P = 0.045), hospital cost (P = 0.003), household nature (P < 0.001), and education (P < 0.001). The complaint rate decreased when the patients' age increased (regression coefficient, -0.606; OR, 0.545, and 95%CI, 0.301 - 0.987). The complaint rate increased with an increase in hospital cost (regression coefficient, 0.818; OR, 2.266; and 95%CI, 1.320 - 3.889). Patients from non-agricultural households had a higher complaint rate (regression coefficient, 1.051; OR, 2.861; and 95%CI, 1.611 - 5.082). Patients with higher education levels had lower complaint rates (regression coefficient, -0.944; OR, 0.389; and 95%CI, 0.234 - 0.647). CONCLUSION: The survey confirms that older patients and patients with higher education levels had lower complaint rates, while non-agricultural population and patients with higher hospital expenses had higher complaint rates.