Case Report: Technical description and clinical evaluation of three cases of unilateral biportal endoscopic decompression for symptomatic spinal epidural lipomatosis.

Objective: To investigate the clinical characteristics and outcomes of three patients with symptomatic Spinal epidural lipomatosis (SEL) treated using Unilateral Biportal Endoscopic (UBE) surgery. Methods: This report retrospectively analyzed the clinical data of three patients with SEL admitted to our hospital. The analysis covers onset characteristics, clinical manifestations, and the most recent radiologic grading system of neural compression (Manjila classification). Furthermore, it details the decompression accomplished through the application of a minimally invasive UBE surgical technique, specifically targeting the removal of proliferated fat responsible for nerve and spinal cord compression. Results: This technique was performed successfully in 3 patients with SEL. Radiating pain was reduced, and the functional disability and radiologic compression were improved in all three patients. Postoperative spinal instability and surgical complications related to the procedure were not observed. Conclusions: For SEL, timely diagnosis and appropriate intervention can prevent the progression of neurological disability. UBE is a minimally invasive muscle-preserving technique that achieves neural decompression directly by the removal of excessive intraspinal adipose tissue buildup.

Improvement of platelet preservation by inhibition of TRPC6.

BACKGROUND: The preservation of platelets (PLTs) by room temperature (RT) oscillation limits their shelf life to between 4 and 7 days because of the decrease in PLT function. TRPC6 is a non-selective mechanically sensitive cation channel that has been shown to mediate Ca2+ signalling, implying a role in PLT activation during preservation by RT oscillation. OBJECTIVES: This study was designed to investigate whether inhibition of TRPC6 can improve the RT preservation of PLTs and the possible underlying mechanism. METHODS: Human PLTs from whole blood were stored at 22 ± 2°C with oscillation in plasma or M-sol (mixture of solutions). BI-749327, a specific TRPC6 inhibitor, was administered throughout the preservation period. PLT distribution width (PDW), mean platelet volume (MPV), maximum platelet aggregation rate (MAR) and average aggregation rate (AAR) were measured. The MTT method was used to assess the relative viability of PLTs. Flow cytometry was used to measure the changes of Ca2+ concentration in PLTs and phosphatidylserine (PS) exposure on the PLT surface, and western blotting was used to assess the expression changes of platelet TRPC6 and CD62P proteins. RESULTS: Compared with the control group, inhibition of TRPC6 with BI-749327 significantly reduced the PDW, MPV and Ca2+ concentration, the MAR and AAR were significantly increased, the expression of TRPC6 and CD62P protein was significantly down-regulated in PLTs, and the PS exposure was significantly reduced on the PLT surface. However, these effects were all reversed by activation of TRPC6. CONCLUSION: Inhibition of TRPC6 improves the quality of PLT preservation by inhibiting the Ca2+ signal mediated by TRPC6.

Up-regulation of Core 1 Beta 1, 3-Galactosyltransferase Suppresses Osteosarcoma Growth with Induction of IFN-γ Secretion and Proliferation of CD8+ T Cells.

AIM: Abnormal glycosylation often occurs in tumor cells. T-synthase (core 1 beta 1,3- galactosyltransferase, C1GALT1, or T-synthase) is a key enzyme involved in O-glycosylation. Although T-synthase is known to be important in human tumors, the effects of T-synthase and T-antigen on human tumor responses remain poorly defined. METHODS: In this study, a T-synthase-specific short hairpin RNA (shRNA) or T-synthase-specific eukaryotic expression vector(pcDNA3.1(+)) was transfected into murine Osteosarcoma LM8 cells to assess the effects of T-synthase on T cells and cytokines. RESULTS: The up-regulation of T-synthase promoted the proliferation of osteosarcoma cells in vitro, but it promoted the proliferation of tumor initially up to 2-3 weeks but showed significant growth inhibitory effect after 3 weeks post-implantation in vivo. Osteosarcoma cells with high T-synthase expression in vitro promoted the proliferation and inhibited the apoptosis of CD8+ T cells. Further, T-synthase upregulation promoted CD8+ T-cell proliferation and the increased production of CD4+ T cell-derived IFN-γ cytokines to induce the increased tumor lethality of CTLs. CONCLUSION: Our data suggest that high T-synthase expression inhibits tumor growth by improving the body's anti-tumor immunity. Therefore, using this characteristic to prepare tumor cell vaccines with high immunogenicity provides a new idea for clinical immunotherapy of osteosarcoma.

Clinical Features and Outcomes of Spinal Tuberculosis in Central China.

Purpose: The appropriate management of spinal tuberculosis (TB) is challenging for clinicians and the key to treat spinal TB. Surgery and long course anti-TB chemotherapy may not be necessary to all situations. This study aimed to characterize the clinical features and factors affecting treatment outcomes. Patients and Methods: A retrospective study of patients with spinal TB over a 5-year period at a teaching hospital in central China was conducted. Features of patients with spinal TB who received different treatment modalities and factors associated with patient outcomes at the end of chemotherapy were analyzed. Results: Forty-five patients (21 men and 24 women) with spinal TB were available for analysis. The mean age was 55.39 ± 14.94 years. The most common vertebral area involved was the lumbar (42.2%). The mean number of vertebrae involved was 2.20 ± 0.59. 27 patients (60.0%) received surgical treatment, of which 21 (77.8%) received radical surgical treatment. Thirty-five patients (77.8%) had achieved a favorable status. Statistically, there was no significant correlation between favorable status and surgery, but among 27 surgical patients with spinal tuberculosis, patients receiving radical surgery tended to achieve good prognosis (P = 0.010; odds ratio = 0.053; 95% confidence interval 0.006-0.493). Moreover, there was no significant difference between long course and short course of anti-TB chemotherapy in prognosis in different treatment modalities. Conclusion: Although the patients with spinal TB who needed surgical treatment often got a better prognosis when they had radical surgery, surgery was not actually a factor for the favorable outcomes of patients with spinal TB. In different treatment modalities, there was no additional benefit in longer anti-TB chemotherapy periods.

A multicentre double-blind, double-dummy, randomised study of recombinant human thrombopoietin versus eltrombopag in the treatment of immune thrombocytopenia in Chinese adult patients.

We performed a double-blind, double-dummy controlled study to compare the efficacy between recombinant human thrombopoietin (rhTPO) and eltrombopag in rapidly increasing the platelet counts in Chinese patients with immune thrombocytopenia (ITP). A total of 96 patients diagnosed with ITP for ≥6 months who had baseline platelet counts of <30 × 109 /l were randomly assigned (1:1 ratio) to receive eltrombopag 25 mg/day or rhTPO 300 u/kg for 2 weeks. Compared with the eltrombopag group, a significantly higher proportion of patients in the rhTPO group achieved platelet counts of ≥50 × 109 /l [75·00% (36/48) vs. 43·75% (21/48), P = 0·003] or complete response (64·58% vs. 25·00%) on day 15. Moreover, a higher proportion of patients in the rhTPO group either had platelet counts that rapidly increased to twice that of baseline and with platelet counts of ≥30 × 109 /l, or reached ≥50 × 109 /l at least once when analysed on day 9, 12, and 15. However, upon discontinuation of the treatment, the platelet counts reduced to the baseline within 1 week in the rhTPO group, but on the fourth week in the eltrombopag group. Adverse events were similar in patients given rhTPO and eltrombopag. To conclude, rhTPO is superior to eltrombopag at 25 mg/day in rapidly increasing platelet counts in patients with ITP (ClinicalTrials.gov Identifier: NCT03771378).

Curative or pre-emptive adenovirus-specific T cell transfer from matched unrelated or third party haploidentical donors after HSCT, including UCB transplantations: a successful phase I/II multicenter clinical trial.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT), the most widely used potentially curable cellular immunotherapeutic approach in the treatment of hematological malignancies, is limited by life-threatening complications: graft versus host disease (GVHD) and infections especially viral infections refractory to antiviral drugs. Adoptive transfer of virus-specific T cells is becoming an alternative treatment for infections following HSCT. We report here the results of a phase I/II multicenter study which includes a series of adenovirus-specific T cell (ADV-VST) infusion either from the HSCT donor or from a third party haploidentical donor for patients transplanted with umbilical cord blood (UCB). METHODS: Fourteen patients were eligible and 11 patients received infusions of ADV-VST generated by interferon (IFN)-γ-based immunomagnetic isolation from a leukapheresis from their original donor (42.9%) or a third party haploidentical donor (57.1%). One patient resolved ADV infection before infusion, and ADV-VST could not reach release or infusion criteria for two patients. Two patients received cellular immunotherapy alone without antiviral drugs as a pre-emptive treatment. RESULTS: One patient with adenovirus infection and ten with adenovirus disease were infused with ADV-VST (mean 5.83 ± 8.23 × 103 CD3+IFN-γ+ cells/kg) up to 9 months after transplantation. The 11 patients showed in vivo expansion of specific T cells up to 60 days post-infusion, associated with adenovirus load clearance in ten of the patients (91%). Neither de novo GVHD nor side effects were observed during the first month post-infusion, but GVHD reactivations occurred in three patients, irrespective of the type of leukapheresis donor. For two of these patients, GVHD reactivation was controlled by immunosuppressive treatment. Four patients died during follow-up, one due to refractory ADV disease. CONCLUSIONS: Adoptive transfer of rapidly isolated ADV-VST is an effective therapeutic option for achieving in vivo expansion of specific T cells and clearance of viral load, even as a pre-emptive treatment. Our study highlights that third party haploidentical donors are of great interest for ADV-VST generation in the context of UCB transplantation. (N° Clinical trial.gov: NCT02851576, retrospectively registered).

Usefulness of Flow Cytometric Mepacrine Uptake/Release Combined with CD63 Assay in Diagnosis of Patients with Suspected Platelet Dense Granule Disorder.

Dense granule disorder is one of the most common platelet abnormalities, resulting from dense granule deficiency or secretion defect. This study was aimed to evaluate the clinical usefulness of the flow cytometric combination of mepacrine uptake/release assay and CD63 expression detection in the management of patients with suspected dense granule disorder. Over a period of 5 years, patients with abnormal platelet aggregation and/or reduced adenosine triphosphate (ATP) secretion suggestive of dense granule disorder were consecutively enrolled. The flow cytometric assays were systematically performed to further investigate dense granule functionality. Among the 26 included patients, 18 cases showed impaired mepacrine uptake/release and reduced CD63 expression on activated platelets, consistent with δ-storage pool deficiency (SPD). Another seven patients showed decrease in mepacrine release and CD63 expression but mepacrine uptake was normal, indicating secretion defect rather than δ-SPD. Unfortunately, ATP secretion could not be measured in 7 out of the 26 patients due to insufficient sample and/or severe thrombocytopenia. This test combination provides a rapid and effective method to detect the heterogeneous abnormalities of platelet dense granule by distinguishing between storage and release defects. This combination is particularly advantageous for severely thrombocytopenic patients and pediatric patients in which only minimal sample is required.

Adenovirus-specific T-cell Subsets in Human Peripheral Blood and After IFN-γ Immunomagnetic Selection.

Adoptive antiviral cellular immunotherapy by infusion of virus-specific T cells (VSTs) is becoming an alternative treatment for viral infection after hematopoietic stem cell transplantation. The T memory stem cell (TSCM) subset was recently described as exhibiting self-renewal and multipotency properties which are required for sustained efficacy in vivo. We wondered if such a crucial subset for immunotherapy was present in VSTs. We identified, by flow cytometry, TSCM in adenovirus (ADV)-specific interferon (IFN)-γ+ T cells before and after IFN-γ-based immunomagnetic selection, and analyzed the distribution of the main T-cell subsets in VSTs: naive T cells (TN), TSCM, T central memory cells (TCM), T effector memory cell (TEM), and effector T cells (TEFF). In this study all of the different T-cell subsets were observed in the blood sample from healthy donor ADV-VSTs, both before and after IFN-γ-based immunomagnetic selection. As the IFN-γ-based immunomagnetic selection system sorts mainly the most differentiated T-cell subsets, we observed that TEM was always the major T-cell subset of ADV-specific T cells after immunomagnetic isolation and especially after expansion in vitro. Comparing T-cell subpopulation profiles before and after in vitro expansion, we observed that in vitro cell culture with interleukin-2 resulted in a significant expansion of TN-like, TCM, TEM, and TEFF subsets in CD4IFN-γ T cells and of TCM and TEM subsets only in CD8IFN-γ T cells. We demonstrated the presence of all T-cell subsets in IFN-γ VSTs including the TSCM subpopulation, although this was weakly selected by the IFN-γ-based immunomagnetic selection system.

Case Report: Detection and quantification of tumor cells in peripheral blood and ascitic fluid from a metastatic esophageal cancer patient using the CellSearch (®) technology.

Analysis of ascitic fluid should help to identify and characterize malignant cells in gastrointestinal cancer. However, despite a high specificity, the sensitivity of traditional ascitic fluid cytology remains insufficient, at around 60%. Since 2004 the CellSearch (®) technology has shown its advantages in the detection of circulating tumor cells (CTCs) in peripheral blood, which can perform an accurate diagnosis and molecular analysis at the same time. To our knowledge, no previous study has explored the potential utility of this technology for the detection and quantification of tumor cells in ascitic fluid samples. Herein we report a case of metastatic esophageal adenocarcinoma in a 70-year-old man presenting with dysphagia and a large amount of fluid in the peritoneal cavity. Analysis of a peripheral blood sample and ascites sample with the CellSearch (®) technology both revealed the presence of putative tumor cells that were positive for epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK) expression. This study confirmed the hematogenous dissemination of esophageal cancer by the detection of circulating tumor cells in the peripheral blood, and is the first to demonstrate that tumor cells can be identified in ascitic fluid by using CellSearch (®) technology.

Detection and quantification of CSF malignant cells by the CellSearch technology in patients with melanoma leptomeningeal metastasis.

Melanoma is the most frequent solid tumor associated with leptomeningeal metastasis (LM). The usual diagnostic tools, that is, cytomorphological assessment of cerebro-spinal fluid (CSF) and gadolinium-enhanced MRI of the entire neuraxis both lack effectiveness. The CellSearch Veridex technology for the detection of circulating tumor cells (CTC) in blood was designed for the follow-up and prognosis of breast, prostate, colorectal, and lung cancer, which express EpCAM markers. We have previously adapted this technology to detect malignant cells in the CSF of breast cancer LM. Our objective here was to check if this technology would also allow the detection and the enumeration of CTC in the CSF of melanoma patients presenting with LM although melanoma does not express EpCAM markers. On the occasion of the intrathecal treatment of LM in 2 melanoma patients, 5 mL of CSF and 7.5 mL of blood were collected on CellSave Preservative Tubes and analyzed within 3 days after CSF sampling using a melanoma-dedicated kit. The CellSearch Veridex technology was then adapted to direct enrichment, enumeration, and visualization of melanoma cells in the CSF. CD146+, HMW-MAA+, CD34-, and CD45- cells with typical morphology could be observed and enumerated sequentially with reproducible results, corresponding to CSF melanoma cells (CSFMC). In contrast to the current gold standard cytomorphological analysis, this new approach allowed a precise quantification of CSFMC in all samples concomitantly analyzed. This methodology, established on a limited volume of sample and allowing delayed processing, could prove of great interest in the diagnosis and follow-up of melanoma patients with LM.

[Effect of baicalein on proliferation and migration in multiple myeloma cell lines RPMI 8226 and U266 cells].

OBJECTIVE: To investigate the effect of baicalein on proliferation and migration of multiple myeloma (MM) cell lines and its molecular mechanism. METHODS: The MM cell line RPMI-8226 and U266 cells were used as the model, and treated with different concentration and time of baicalein the effect of baicalein on the MM cells proliferation was assessed by MTT assay. With or without baicalein or Interleukin-6 (IL-6) treatment, the ß-catenin protein level was analyzed by immunofluorescence assay and western blot assay and mRNA levels of ß-catenin, c-myc, cyclin D1 and integrin 7 gene by RT-PCR. Transwell chamber migration assay was used to detect the cells migration ability with different concentration of baicalein cultured. RESULTS: Baicalein inhibited the MM cell line RPMI 8226 and U266 cell proliferation in a dose- and time-dependent manner. It simultaneously inhibited ß-catenin protein level to resist the effect of IL-6 on inducing MM cell proliferation, and resulted in decrease of ß-catenin, c-myc, cyclinD1 and integrin ß7 mRNA levels. Baicalein also decreased migration ability of MM cells in a dose-dependent manner by SDF-1. CONCLUSION: Baicalein can inhibit MM cells proliferation and migration, and its molecular mechanisms are associated with inhibition of proliferation related genes ß-catenin, c-myc, cyclin D1 and integrin ß7 expression.

N-glycan-defective breast cancer cells induce a phenotypic switch in polarization of bone marrow-derived macrophages.

PURPOSE: To investigate the effect of N-glycan-defective mammary adenocarcinoma cells on the polarization of macrophages. METHODS: N-glycan-defective breast cancer cells (MA782 cells) were prepared by swainsonine (SW) treatment and the cytotoxicity of SW to MA782 cells was evaluated using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The N-glycan-defective MA782 cells were co-cultured with bone marrow-derived macrophages (BMDMs) for 48 h in vitro, and then the BMDMs and the co-cultured supernatant were analyzed for macrophage phenotypic using FQRT-PCR, FCM and ELISA. RESULTS: SW-treated MA782 cells expressed defective N-glycan on the cell surface in a dose-dependent manner (*p < 0.05). MTT assays showed that neither the 1 µg/mL nor 5 µg/mL SW treatments showed significant inhibition of MA782 cell growth in vitro. The expression of iNOS and agr-1 in the 5 µg/mL SW-treated group were 4.75-fold higher and 3.7-fold lower than that in the untreated group, respectively (*p < 0.05). Mean fluorescence intensity of CD16/32 expressed in the cells treated with 5 µg/mL SW was significantly higher in comparison with the untreated group (65 vs. 7, *p < 0.05), though the percentage of CD16/32-positive cells were not significantly different. Furthermore, the expression of CD206 and dectin-1 in the 5 µg/mL SW-treated group was significantly decreased (3.1±0.3% and 4.1±1.1%, respectively) in comparison with the untreated group (40±3% and 8.9±1.2%, respectively, both p < 0.05). In addition, the 5 µg/mL SW-treated group secreted more TNF-alpha (350 ±25 pg/mL) and less IL-10 (89±7.2 pg/mL) than the untreated group (80 ±3 pg/mL and 150 ±10 pg/mL, respectively, both p < 0.05). CONCLUSION: N-glycan-defective MA782 cells can induce the differentiation of BMDM into proinflammatory M1 macrophages in vitro.

[Relationship between CD45 molecule expression and apoptotic sensitivity of myeloma cells].

OBJECTIVE: To investigate the effects of CD45 expression on induction of apoptosis in multiple myeloma cells. METHODS: Melphalan was used to induce myeloma cell line U266 apoptosis. Serum-free culture was used to induce CD45RB gene or empty plasmid transfected U266 apoptosis. The glucose-free culture was used to induce high CD45 (CD45(hi)) or low CD45 (CD45(low)) expression AMO1 apoptosis. Intraperitoneal inoculation was used to compare the survival of CD45(-) or CD45(+) U266 cells in mice. The number of apoptotic cells and mitochondrial membrane potential (MMP) was detected by flow cytometry. Western blotting was used to detect the cytochrome C release from mitochondrial and caspase-9 activation. RESULTS: Melphalan treatment induced 45% of CD45(+) and 30% of CD45(-) U266 cells apoptosis. Compared with the CD45(low) AMO1 cells, CD45(hi) cells were more susceptible to apoptosis. In serum-free culture for five days, 60% of CD45RB transferred U266 cells underwent apoptosis, while in the empty plasmid transfected ones, apoptotic cell number was not significantly increased. The survival time of CD45(-) U266 cells in the SCID-hIL-6 mice was 5 times that of CD45(+) cells. After melphalan treatment, 60% of the CD45(+) U266 cells lost MMP, while only 30% of CD45(-) U266 cells, and 10% of control cells did so. After UV irradiation, CD45(+) U266 cells mitochondria released more cytochrome C, leading to more caspase-9 activation. CONCLUSION: CD45 expression is involved in mitochondria-mediated apoptotic process and increases apoptotic sensitivity of myeloma cells under a variety of stimulation.

Inhibitory effect of baicalein on IL-6-mediated signaling cascades in human myeloma cells.

Interleukin-6 (IL-6) is an important growth factor for myeloma cells. IL-6 promotes the survival and proliferation of multiple myeloma (MM) cells through the phosphorylated proteins, including STAT3, MAPK, and Akt. Chemical components that suppress the signaling proteins' phosphorylation have a potential role for MM therapy. We recently identified that baicalein, a component of Scutellaria radix, suppressed proliferation and induced apoptosis of myeloma cells by blocking IkappaB-alpha degradation followed by down-regulating IL-6 and XIAP gene expression. In the present study of four myeloma cell lines, namely U266, NOP2, AMO1, and ILKM2, we demonstrated that baicalein not only inhibited IL-6-mediated phosphorylation of signaling proteins, such as Jak, STAT3, MAPK, and Akt, but also inhibited the expression of their target genes, such as bcl-xl. Finally, baicalein facilitated myeloma cell proliferation inhibited by dexamethasone. In contrast, baicalin, another major flavonoid derived from Scutellaria radix, had no significant effect on IL-6-mediated protein phosphorylation. Baicalein had no effect on Akt phosphorylation induced by the insulin-like growth factor-1 (IGF-1) in NOP2 cells. Compared with PD98059, an MAPK inhibitor, baicalein exhibited a stronger inhibitory effect on Erk(1/2) phosphorylation. Our results demonstrate that baicalein is a potent inhibitor of protein phosphorylation induced by IL-6, and thus may be a useful agent for the treatment of MM.

[Cigarette smoke affects sexual function of male rats].

OBJECTIVE: To study the effect of cigarette smoke on the sexual function of male rats. METHODS: Based on Ozyurt's smoking model, we equally divided 30 male adult Sprague-Dawley rats into a control and a smoking group, and exposed the latter to cigarette smoke for 60 days. A week before the end of the experiment, we added 5 female rats to each group and observed their mating through 24-hour video surveillance. Sixty days later, all the rats were killed for the determination of the level of testosterone (T) in the plasma and the activity of nitric oxide synthase (NOS) in the corpus cavernosum, and Masson-dyeing image analysis of the penile tissue. RESULTS: Compared with the controls, the rats in the smoking group showed a significant reduction in the times of mating, the level of plasma T (P < 0.05) and the activity of NOS in the penile cavernous tissue (P < 0.05), but a slight increase in the collagen fibers and obvious changes in the blood sinuses. CONCLUSION: Cigarette smoke seriously affected penile erection in the experimental rats. The decrease in plasma T, NOS activity and the area of smooth muscle may be an important mechanism underlying their erectile dysfunction.