Objective:To establish a method of multi-PCR to amplify the complete DNA sequence (CDS) of TCR ? and ? chain of the antigen-specific T lymphocytes in local pathologic specimen of active pulmonary tuberculosis patients, and to analyze ?/? T cell receptor gene rearrangement and CDR3 repertoire.Methods:The lymphocytes in bronchoalveolar lavage (BAL) of active pulmonary tuberculosis patients were separated. Following total RNA extraction, cDNA synthesis, Multi-PCR, recombinant clones construction, and sequencing, the CDS of TCR ? and ? chains from these lymphocytes were analyzed by using software of DNAstar and internet TCR resources.Results:24 of ? chain CDS and 13 of ? chain CDS from 3 samples of BAL were obtained. As for TCR ? chain, AV1S2 (54%), AV12S3 (41%), and AV12S2(5%) appeared frequently. BV2(38%), BV29S1(46%), BV14(3%), and BV4S2(3%) in TCR ? chain appeared more often. There were CDR3 diversities between samples and even in the same sample by amino acid sequence analysis, but there were a few identical or similar amino acid sequences. There was the same amino acid sequence of SVGTGTLHQETQY in CDR3 region of ? chain of BAL sample No.1 and No.2; The sequence of AVRDWAGNMLT appeared in two ? chains of BAL sample No.2 and No.3; Moreover, the sequence of AV…DNN…RLM appeared in ? chains of BAL sample No.2 and No.3.Conclusion:A method of Multi-PCR is used to amplify TCR ? and ? chain CDS of tuberculosis patients. There are characteristic T cell clones to proliferate,with TCR ? and ? chain repertiore skewing in local infective focus. The sequences of CDR3 in different TCR clones are mostly different but there are a few identical or similar sequences in the same patient or even between different patients. The identical amino acid sequences of CDR3 are possibly specific for recognizing MTB polypeptide.

BackgroundResearch demonstrated that vitamin D3 mediated by its receptor has the potent nonclassical effects,including immunomodulatory,antiinflammatory,and neuroprotective properties,and it can enhance the secretion and sensitivity of insulin and therefore down-regulate hyperglycemia and attenuate the corneal edema.ObjectiveThe present study was to investigate the protective effect of vitamin D3on ocular structure in experimental diabetic rat.Methods Twenty-two healthy SPF C57BL/6 rats were randomly divided into vitamine D3 group (8 rabbits),diabetic control group ( 11 rabbits) and normal control group ( 3 rabbits).2％ streptozotocin ( STZ,175 mg/kg)was intraperitoneally injected to create the diabetic models in the rats of the vitamine D3 group and diabetic control group.Blood glucose was examined for 3 times in the third day after STZ injection,and the rats with the blood glucose concentration ＞16.7 mmol/L was identified as the successful diabetic models.After modeling,the rat tail blood was collected for the monitoring of blood glucose.Two weeks after modeling,vitamine D3 was intraperitoneally injected in each week for 5 times.The fundus was examined using direct ophtalmoscope,and the eyeballs were obtained under the excessive anesthesia for the measurement of thickness of the central cornea,retina and choroids by histopathological examination once a week for 7 weeks after administration of vitamin D3.The administration of the animals complied with the Statement of ARVO.ResultsThe corneal edema appeared with the corneal thickness of (339.14± 11.13) μm in the first week and gradually attenuated with time elapse after modeling in the diabetic group ( F =382.446,P =0.000).The corneal thickness values were significantly decreased from the second week through the seventh week in the vitamin D3 group compared with diabetic control group(P＜0.05).The atrophy of the corneal epithelium was found from the fifth week to the seventh week in diabetic control group,but that in vitamin D3 group was slight (P＜0.05).The gradually thinning of the choroids was seen from the first week to the seventh week in the diabetic control group ( F =437.411,P =0.000 ),however,the thickness values in the vitamin D3 group were significantly increased in comparison with the diabetic control group in various time points (P＜0.05).The retina thickness was gradually reduced during the seven-week duration in the diabetic control group (F =91.859,P =0.000),but no significant change was identified in retina thickness in the vitamin D3 group(P＞0.05).ConclusionsVitamin D3 has prevent and therapeutic effects on experimental diabetic oculopathy.

Objective To investigate the safety and clinical response of dendritic cells (DC)vaccine loaded with HLA-A2-restricted peptides MAGE-3、 Tyr、 MART-1 and GP-100 against malignant melanoma. Methods Twenty three HLA-A2 positive patients with malignant melanoma were enrolled.Peripheral blood mononuclear cells were separated into adherent cells for induction of DC loaded with four peptides. DC vaccine was administered subcutaneously once a week in inguinal region. The immunological responses and clinical responses were evaluated. Results Dendritic cell vaccination were well tolerated in all patients and there was no toxicity observed. Serum levels of IL-2, IL-12 and IFN-γincreased significantly after first vaccination and fourth vaccination. The positive rate of DTH test was 50% (5/10), and 2 fold increase of CD8+ IFN-γ+ cells were observed in 6 of 10 patients. Stable disease was observed in 11 of 23 patients, one patient had a complete metastasis regression, four patients had 50% regression of metastasis,four patients had a minor response, and disease progressed in three patients. Conclusion DC vaccines loaded with peptides MAGE-3、 Tyr、 MART-1 and GP-100 can elicit non-specific and specific immune responses, leading disease control. DC vaccine is considered one of safe and effective treatments for malignant melanoma.

In recent years, polysaccharides have received much attention because of their high safety and good immunological activity. The study of polysaccharide in vivo process is a key scientific problem that needs to be solved for polysaccharide drug development. Some progress has been made in the field of polysaccharide pharmacokinetics and immunomodulation. However, due to the lack of both chromogenic and light-absorbing groups and the complex molecular structure of polysaccharides, the in vivo processes and immunomodulatory mechanisms of polysaccharides have been slow to be investigated. The effective combination of multiple techniques can break the bottleneck of difficult tracing and unknown immunomodulatory mechanism of polysaccharides in vivo, and promote the development and utilization of polysaccharides. In this paper, we systematically summarize the key techniques in the study of polysaccharide in vivo processes and immunomodulatory mechanisms in order to provide technical references and research ideas for the study of polysaccharide in vivo processes and immunomodulatory mechanisms.

OBJECTIVETo explore the proliferation-promoting effect of sensory neuropeptide substance P (SP) on the cultured granulation tissue fibroblasts in vitro and its regulative effect on the gene expression of basic fibroblast growth factor (bFGF) mRNA.

METHODSThe proliferation-promoting effect of cultured granulation tissue fibroblasts was observed by means of MTT; the regulative effect of SP on gene expression of fibroblast bFGF by RT-PCR. The time and dose-efficiency relations were also observed.

RESULTSThere was a significant proliferation-promoting effect of SP on the cultured granulation tissue fibroblasts in vitro in a remarkable dose-dependent fashion. However, bFGF antibody only partly exerted its inhibitive effect. SP could induce the bFGF mRNA expression of the fibroblasts at the 3rd and 6th hour (P < 0.01). SP could promote the bFGF mRNA expression of the fibroblasts in the concentration of 10(-9) - 10(-5) mol/L and peaked in the concentration of 10(-7) mol/L.

CONCLUSIONSSP has a significant proliferation-promoting effect on the granulation tissue fibroblasts, which is correlated with SP inducing bFGF mRNA expression of fibroblasts.

Animals ; Cell Division ; drug effects ; Dose-Response Relationship, Drug ; Fibroblast Growth Factor 2 ; genetics ; Fibroblasts ; cytology ; drug effects ; metabolism ; Gene Expression ; drug effects ; Granulation Tissue ; cytology ; drug effects ; metabolism ; Male ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; Substance P ; pharmacology

OBJECTIVETo explore the regulative effects and significance of neuropeptide substance P (SP) on the expression of basic fibroblast growth factor (bFGF) of granulation tissue fibroblasts in vitro.

METHODSA local aseptic inflammation was induced by injection of formaldehyde in rats, and its granulation tissue was cultured. RT-PCR was employed to observe expression of bFGF mRNA after inducement of SP at different concentrations and time points in the granulation tissue, and western blot to assay expression of bFGF protein.

RESULTSThe expression of bFGF mRNA was markedly increased significantly 3 and 6 hours after inducement with SP in 10(-7) mol/L, compared with control group (P < 0.01). The expression of bFGF protein was markedly higher than the control group after 12 hours, and it reached the peak at the 24th hour and declined gradually after 48 hours. SP at concentrations of 10(-9) - 10(-5) mol/L could significantly promote the expression of bFGF mRNA, and that at 10(-8) - 10(-5) mol/L induce the expression of bFGF protein. Both expressions reached the peak when SP concentration was 10(-7) mol/L (P < 0.01).

CONCLUSIONSP can induce the expressions of bFGF mRNA and bFGF protein of granulation tissue fibroblasts in vitro, which may possess an important significance in wound healing.

Animals ; Cells, Cultured ; Fibroblast Growth Factor 2 ; metabolism ; Fibroblasts ; drug effects ; metabolism ; Granulation Tissue ; drug effects ; metabolism ; Male ; RNA, Messenger ; metabolism ; Rats ; Rats, Wistar ; Substance P ; pharmacology ; Wound Healing ; drug effects

OBJECTIVE: By observing the cerebral beta-amyloid precursor protein (beta-APP) expression in the chronic alcoholism rats with slight cerebral injury, to discuss the correlation of chronic alcoholism and death caused by traumatic subarachnoid haemorrhage (TSAH). METHODS: Sixty male SD rats were randomly divided into watering group, watering group with strike, alcoholism group and alcoholism group with strike. Among them, the alcohol was used for continuous 4 weeks in alcoholism groups and the concussion was made in groups with strike. In each group, HE staining and immunohistochemical staining of the cerebral tissues were done and the results were analyzed by the histopathologic image system. RESULTS: In watering group, there was no abnormal. In watering group with strike, mild neuronic congestion was found. In alcoholism group, vascular texture on cerebral surface was found. And the neurons arranged in disorder with dilated intercellular space. In alcoholism group with strike, diffuse congestion on cerebral surface was found. And there was TSAH with thick-layer patches around brainstem following irregular axonotmesis. The quantity of beta-APP IOD in alcoholism group was significantly higher in the frontal lobe, hippocampus, cerebellum, brainstem than those in watering group with strike and alcoholism group with strike. CONCLUSION The cerebral tissues with chronic alcoholism, due to the decreasing tolerance, could cause fatal TSAH and pathological changes in cerebral tissues of rats under slight cerebral injury.
Alcoholism/pathology* ; Amyloid beta-Protein Precursor/metabolism* ; Animals ; Brain/pathology* ; Brain Concussion/pathology* ; Disease Models, Animal ; Ethanol/adverse effects* ; Male ; Neurons/pathology* ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Subarachnoid Hemorrhage, Traumatic/pathology*

OBJECTIVETo assess the effectiveness of acupuncture for treatment of herpes zoster.

METHODSAccording to the requirement of evidence-based medicine, acupuncture, body acupuncture, electroacupuncture, head acupuncture, three edged needle, plum-blossom needle, fire needle, elongated needle, encircling needling, herpes zoster, etc. were selected as subject words to retrieve the relative medical database at home, and clinically randomized controlled trials were used as enrolled criteria, the treatment group were treated with acupuncture or acupuncture plus other therapies, and the control group with medicine, the cured rate and the time of killing pain for herpes zoster were used as assessment indexes. Altogether 43 papers were enrolled. Among them 10 papers were conducted for Meta-analysis by RevMan 4.2.9.

RESULTSThe total OR was 4.27 with 95% CI [2.90, 6.29] of the clinically cured rate in the 10 studies, and the total OR was -7.64 with 95% CI [-8.12, -7.15] of the time of killing pain in the 4 studies. The therapeutic effect in the treatment group on herpes zoster was superior to that of the western medicine (P < 0.01).

CONCLUSIONAcupuncture therapy for herpes zoster is effective, but more high-quality studies are required to prove this view point.

Acupuncture Therapy ; methods ; Herpes Zoster ; therapy ; Humans

Adult ; Aged ; Cell Cycle Proteins ; analysis ; Coal Mining ; Cyclin-Dependent Kinase Inhibitor p27 ; Female ; Humans ; Immunohistochemistry ; Lung ; chemistry ; pathology ; Male ; Middle Aged ; Occupational Diseases ; metabolism ; Pneumoconiosis ; metabolism ; Tumor Suppressor Proteins ; analysis

Objective To evaluate the accuracy of serum pro-adrenomedullin (pro-ADM) concentration in predicting sepsis at different degrees of severity.Methods A total of 145 patients of both sexes,aged 18-64 yr,who were admitted to intensive care unit (ICU) of the First Hospital of Hebei Medical University from March 2015 to April 2017,with length of ICU stay＞24 h,were enrolled.The patients were divided into 3 groups according to the Sequential Organ Failure Assessment (SOFA) score within 24 h after admission to ICU:2＜SOFA score ≤ 6 mild sepsis group (n =50),6＜SOFA score ≤ 12 moderate sepsis group (n =50) and SOFA score＞ 12 severe sepsis group (n =45).Peripheral venous blood samples were collected immediately after admission to hospital for determination of serum pro-ADM concentrations by enzyme-linked immunosorbent assay.The receiver operating characteristic (ROC) curve of pro-ADM in predicting sepsis at different degrees of severity was plotted,and the area under the curve and 95％ confidence interval,cut-off value,sensitivity and specificity were calculated.Results The serum pro-ADM concentrations were significantly increased with the severity of sepsis,and the length of ICU stay was prolonged with the severity of sepsis (P＜0.05).In mild sepsis group,the area under the ROC curve was 0.770,95％ confidence interval 0.591-0.949,sensitivity 0.725,specificity 0.700 and cut-off value 6.45 nmol/L.In moderate sepsis group,the area under the ROC curve was 0.776,95％ confidence interval 0.645-0.907,sensitivity 0.813,specificity 0.760,cut-off value 8.30 nmol/L.In severe sepsis group,the area under the ROC curve was 0.83,95％ confidence interval 0.715-0.963,sensitivity 0.73,specificity 0.800,cut-off value 5.70 nmol/L.Conclusion Serum pro-ADM concentration can be used as a reliable index in predicting sepsis at different degrees of severity.