OBJECTIVES(1) To evaluate the prevalence, phenotypes and suppressive function of CD4+CD25+ regulatory T cells (Tregs) among the in peripheral blood mononuclear cells (PBMCs) and tumor-infiltration lymphocytes (TILs) from hepatocellular carcinoma (HCC) patients and patients with chronic hepatitis B. (2) To investigate the correlation between the frequency of CD4+CD25+ Tregs and clinical characteristics of HCC patients.

METHODSPBMCs and TILs in 18 HCC patients, 10 chronic hepatitis B (CHB) patients and 15 healthy donors were evaluated for the phenotypes of CD4+CD25+ Tregs and the proportion of CD4+CD25+ Tregs as a percentage of the total CD4+ cells, by flow cytometric analysis with three or four color staining. The relationship between the frequency of CD4+CD25+ Tregs and tumor TNM stages was analyzed. The CD4+CD25+ Tregs and CD4+CD25- T cells were isolated from PBMC of HCC patients and donors. The suppressive function of CD4+CD25+ Tregs was analyzed.

RESULTSThe percentages of CD4+CD25+ Tregs of the HCC patients (6.38% +/- 6.30%) and CHB patients (4.29% +/- 1.82%) were significantly higher than those of the healthy donors (1.58% +/- 0.55%, P less than 0.01). Among the TILs, the percentage of CD4+CD25+ Tregs was higher (t = 4.39, P < 0.01). There were significant differences in the prevalence of CD4+CD25+ Tregs in early and advanced stage HCCs (stage II vs. III, P less than 0.05; stage II vs. IV P < 0.01). The proliferative capacity of CD4+CD25- T cells was inhibited by the presence of CD4+CD25+ T cells in a dose-dependent manner where the level of suppression was correlated to the ratio of the two-cell populations.

CONCLUSIONThese results suggest that the increase in frequency of CD4+CD25+ Tregs might play a role in the suppression of the immune response against HCC, which may contribute to the HCC cells that escaped from immunological surveillance.

Adult ; Aged ; Carcinoma, Hepatocellular ; metabolism ; Female ; Humans ; Interleukin-2 Receptor alpha Subunit ; Liver Neoplasms ; metabolism ; Male ; Middle Aged ; T-Lymphocytes, Regulatory ; immunology ; Young Adult

BACKGROUNDIt has been found that cardiac protection afforded by ischemic preconditioning (IPC) is significantly reduced in the senescent myocardium. ADAMTS-1 (a disintesrin and metalloprotease with thrombospondin type 1 motifs) has been shown to inhibit angiogenesis in a variety of in vitro and in vivo assays. The aim of this study was to investigate the age-associated differences in ADAMTS-1 protein expression in rat myocardium after ischemic preconditioning.

METHODSSixty-four young (4 months) and old (24 months) male Sprague-Dawley rats were randomly assigned to an IPC group (40 rats) or a sham group (rats). A model of delayed IPC was induced and rats were sacrificed and myocardial samples were harvested from the ischemic-reperfused region for immunohistochemical detection of ADAMTS-1 at serial time points after IPC. A model of myocardial infarction was produced by ligation of the left anterior descending coronary artery in additional sets of young and old rats after sham or IPC procedures, then age-associated myocardial infarction survival after IPC was calculated.

RESULTSADAMTS-1 expression increased significantly in old rats compared to young rats (P < 0.05). The mean densities of ADAMTS-1 protein at 0, 6, 12, and 24 hours in young-IPC group after IPC were 0.05 ± 0.01, 0.13 ± 0.03, 0.16 ± 0.04, and 0.12 ± 0.03 vs. 0.07 ± 0.03, 0.20 ± 0.03, 0.24 ± 0.05, and 0.21 ± 0.04 in old-IPC group. IPC resulted in diminished survival rates (5/35 vs. 6/14, old-IPC group vs. old-sham group, P < 0.05), reduced left ventricular fractional shortening ((13.9 ± 2.8)% vs. (18.3 ± 2.3)%, P < 0.05) and increased the myocardial infarction size ((37.9 ± 3.2)% vs. (32.8 ± 5.1)%, P < 0.05) in the older rats.

CONCLUSIONSCardioprotection with IPC is attenuated in the older heart. ADAMTS-1 expression induced by IPC is greater in old rats. Over-expression of anti-angiogenic factors might be a potential mechanism behind reduced protection after IPC associated with aging.

ADAM Proteins ; metabolism ; ADAMTS1 Protein ; Aging ; metabolism ; physiology ; Animals ; Immunohistochemistry ; Ischemic Preconditioning, Myocardial ; Male ; Myocardial Infarction ; metabolism ; pathology ; Myocardium ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley

To study the acute Leukemia immunophenotype and its diagnosis value, three color direct immunofluorescece staining methods of flow cytometry and immunophenotype of antibody integration system were used for detection of 180 cases of acute leukemia. The results showed that patients with ALL expressed lymphocyte antigen, and 49.4% patients with ALL accompanied myeloid antigen; all patients with AML expressed myeloid antigen, and 43.2% patients with AML accompanied lymphocyte antigen. In conclusion, leukemia immunophenotyping by three-color direct immunofluore staining methods could define some particular types of leukemia with an important value in diagnosis, treatment and predicting prognosis of acute leukemia.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antigens, CD ; analysis ; CD79 Antigens ; Child ; Child, Preschool ; Female ; Flow Cytometry ; methods ; Humans ; Immunophenotyping ; Infant ; Leukemia, Myeloid, Acute ; immunology ; Leukocyte Common Antigens ; analysis ; Male ; Middle Aged ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; immunology ; Receptors, Antigen, B-Cell ; analysis

To characterize the complete genome sequence of coxsackievirus B1 (CVB1) MSH/KM9/2009 strain isolated from Yunnan, China,2009. Eight overlapping clones covering the whole viral genome (excluding the poly-A tail) were obtained by RT-PCR and sequenced, and their nucleotide and amino acid sequences were compared with other known CVB1 strains. The genome of the CVB1 MSH/KM9/2009 strain had 7384 nucleotides in length, and contained a 741nt non-translated region (NTR) at the 5' end and a 94nt NTR at the 3' end. The entire open reading frame contained 6 549 nt, encoding a 2 183-aa polyprotein. In the coding region, there was no nucleotide deletion or insertion, but some changes of amino acid were unique. The complete genome sequence alignments showed that the CVB1 isolate MSH/KM9/2009 strain shared the highest nucleotide (80.9%, 81.6%, 80.5% and 80%) and amino acid (95.6%, 95.8%, 96.2% and 95.6%) identities to the CVB1 M16560, pmMC, Tucson B1 and CVB1Nm strain, respectively. Phylogenetic tree analysis showed that the MSH/KM9/2009, CVB1 M16560, pmMC, Tucson B1 and CVB1Nm strain clustered into same group. The newly isolated CVB1 strain MSH/KM9/2009 from Yunnan Province belonged to genotype CVB1.
Base Sequence ; Child, Preschool ; China ; Coxsackievirus Infections ; virology ; Enterovirus ; classification ; genetics ; isolation & purification ; Female ; Genome, Viral ; Genotype ; Humans ; Infant ; Male ; Open Reading Frames ; Phylogeny ; Viral Proteins ; genetics

Objective To observe the effect of ischemia preconditioning (IPC) on the expression of pro-angiogenic VEGF, PDGF and anti-angiogenic ADAMTS-1, and arteriogenesis. Methods Rat heart IPC model was made by 4 circles of occluding the LAD for 6 min followed by 6 min of reperfusion. The expression of VEGF, PDGF-B and ADAMTS-1 in the ischemic area was examined with immunohistochemistry at 6, 12 and 24 h after IPC. IPC plus myocardial infarction model was induced by LAD ligation 24 h after IPC, 14 days later, the anti-SM-α-actin antibody was used to detect the mature neovascularization in the border of the infracted area. Results VEGF, PDGF-B and ADAMTS-1 were significantly upregulated in the ischemic area in IPC group compared with the control group ( P < 0. 05 ).Density of mature arteries was also significantly increased in IPC plus MI group than that in MI group (P <0. 05). Conclusion IPC promoted the formation of mature new arteries which may be modulated by upregulating VEGF, PDGF-B, and ADAMTS-1 expressions.

OBJECTIVETo investigate the value of multiparameter flow cytometry (MFC) and flow cytometric scoring system (FCSS) in the diagnosis and prognostic evaluation of childhood myelodysplastic syndrome (MDS).

METHODSA retrospective analysis was performed for the clinical data of 42 children who were diagnosed with MDS. MFC was performed to investigate the phenotype and proportion of each lineage of bone marrow cells. The correlations of FCSS score with MDS type, International Prognostic Scoring System (IPSS) score, and revised IPSS (IPSS-R) score were analyzed.

RESULTSOf all the 42 children, 20 (48%) had an increase in abnormal marrow blasts, 19 (45%) had a lymphoid/myeloid ratio of >1, 14 (33%) had abnormal cross-lineage expression of lymphoid antigens in myeloid cells, 8 (19%) had abnormal CD13/CD16 differentiation antigens, 5 (12%) had abnormal expression of CD56, 3 (7%) had reduced or increased side scatter of granulocytes, 3 (7%) had reduced expression of CD36 in nucleated red blood cells, 2 (5%) had reduced expression of CD71 in nucleated red blood cells, 1 (2%) had absent expression of CD33 in myeloid cells, 1 (2%) had reduced or absent expression of CD11b in granulocytes, and 1 (2%) had absent expression of CD56 and CD14 in monocytes. There were significant differences in the median overall survival time and event-free survival time among the low-, medium-, and high-risk FCSS groups (P<0.05). Among the low-, medium-, and high-risk FCSS groups, the low-risk FCSS group had the highest 2-year overall survival rate, while there was no significant difference between the medium- and high-risk FCSS groups (P>0.05). The three groups had a 2-year event-free survival rate of 95%, 60%, and 46% respectively (P<0.05). FCSS score was positively correlated with MDS type, IPSS score, and IPSS-R score (P<0.05).

CONCLUSIONSMFC and FCSS help with the diagnosis and prognostic evaluation of childhood MDS.

Objective:To apply contrast-enhanced ultrasound in renal function evaluation for patients with spinal cord injury complicated with hydronephrosis. Methods:From October, 2015 to November, 2018, 23 patients with spinal cord injury complicated with hydronephrosis and renal disfunction (spinal cord injury group) and 19 cases of normal kidneys (control group) accepted contrast-enhanced ultrasonography and the image was analyzed with software. The region of interest (ROI) in the renal cortex, and the time intensity curve was drawn. Logistic regression was performed with time to initial peak (TTP), peak intensity (DPI), slope of ascending time (A), area under the curve (AUC) as the independent variable and renography as the dependent variable. The data was analyzed with ROC. Results:There was no significant difference in serum creatinine and ureophil between two groups (P > 0.05). TTP was longer (t = 5.068, P < 0.001), and A and AUC were lower (t > 3.784, P < 0.01) in the spinal cord injury group than in the control group. AUC was the factor related to renography (P < 0.01). The smaller the AUC was, the greater the likelihood of kidney damage was. The sum of sensitivity and specificity was 1.759 and the corresponding AUC was 982.518 dBS. Conclusion:Contrast-enhanced ultrasound can evaluate renal function of patients with spinal cord injury complicated with hydronephrosis. The decrease in AUC of the time-intensity curve indicates that the renal function is impaired.

Objective To evaluate the amplification rate and the lowestlower detection limit of an in-house HIV-1 Drug resistant (HIVDR) genotyping test.Methods A total of 30 plasma samples were selected,which covered allmajor HIV-1 subtypes predominating prevailing in china (B',CRF07_BC,CRF01 _AE ).The viral loads of the 30 selected samples were detected in triplicate by Easy Q method and the average values were taken as the viral loads of the samples.Each sample was diluted to the concentration of ＞ 1000copies/ml,401-1000 copies/ml,101-400 copies/ml,50-100 copies/ml and ＜ 50 copies/ml with HIV-negative plasma. After extraction of nucleic acids, RT-PCR and nested PCR amplification were performed,the efficiency of amplification of each subtype and the minimum detection limit were determined statistically based on the PCR results.Results The viral loads of the selected samples ranged from 2.03 × 102 - 5.92 × 104 copies/ml.The sample of 50 - 1000 copies/ml have a high amplification rate (86％).Conclusion The In-house method for HIV-1 drug resistance genotyping has a high sensitivity with a high successful amplification rate,especially in the samples with low viral load.This method can be used to the detectionof drug-resistant virus and to provide scientific data to treatment options for patients.

Objective To explore a new long-term efficient embedding technique of tumor spheres.Methods Tumor spheres,mainly composed of cancer stem cells,were cultured from glioblastoma tissues.The fifth generation of tumor spheres was chosen for egg-white-paraffin embedding and section.Then,those tumor sphere slices were observed by HE staining,immunohistochemistry and immunofluorescence staining.And the immunofluorescence results of these tumor sphere slices were compared with those tumor spheres kept with traditional methods.Results Immunofluorescence results showed that tumor spheres kept with traditional methods looked blurry,and the positive cells and the positive protein expression sites in the cells could not be displayed.HE staining demonstrated that the tumor sphere slices had well-distributed intact spheres and coloring cells with high karyoplasm contrast.Immunohistochemistry and immunofluorescence staining of tumor sphere slices showed clear background,from which positive cells and positive locus could be easily displayed; therefore,semi-quantitative analysis of the positive cells could be performed.Conclusion Egg-white-paraffin embedding technique of the tumor sphere slices can reduce experimental errors and cut down the costs,which enjoys its advantage as compared with traditional embedding technique of the tumor sphere slices.

Melatonin is mainly an endogenous indoleamine hormone with many physiological functions. Melatonin not only plays an important role in the treatment of sleep disorders, but also plays an important role in the treatment of nervous system diseases, cancer, cardiovascular diseases, and bone diseases. In this paper, the human Melatonin is mainly an endogenous indoleamine hormone with many physiological functions. Melatonin not only plays an important role in the treatment of sleep disorders, but also plays an important role in the treatment of nervous system diseases, cancer, cardiovascular diseases, and bone diseases. In this paper, the human body networks mechanisms and the clinical applications of melatonin were summarized to provide reference for exploring the focus and direction of further clinical application research.