[Extraction of Extracelluar Vesicles Derived from Mycobacterium tuberculosis and Their Effect on the Production of Reactive Oxygen Species and Expression of Inflammatory Factors in Mouse Bone Marrow-Derived Dendritic Cells].

Objective: To isolate extracellular vesicles (EVs) from Mycobacterium tuberculosis ( Mtb), to examine their morphology, particle size, and distribution, to study the effect of EVs derived from Mtb ( Mtb-EVs) on intracellular reactive oxygen species (ROS) production and cytokine secretion in dendritic cells (DCs), and to make preliminary exploration of Mtb-EVs' effect on the immune regulation of DCs. Methods: Mtb-EVs were obtained by ultrafiltration concentration and the protein concentration was determined by BCA assay. The morphology of Mtb-EVs was observed through negative staining electron microscopy (EM). The particle size distribution and concentration of Mtb-EVs were determined by nanoparticle tracking analysis (NTA). Mouse bone marrow was isolated through sterile procedures and mice myeloid DCs were induced and amplified by the combined use of recombinant mouse granulocyte-macrophage colony-stimulating factor (rm GM-CSF) and recombinant mouse interleukin-4 (rm IL-4). Then, morphological and immunophenotypic characterization was performed. After that, the DCs were treated with Mtb-EVs at different concentrations and CCK-8 assay was done to measure their effect on the survival rate of DCs and to identify the appropriate stimulation concentration for subsequent experimental procedures. The intracellular ROS levels of DCs were evaluated with DCFH-DA fluorescence probe and the cytokine secretion of DCs was determined by ELISA. Results: EM observation showed that Mtb-EVs isolated by ultrafiltration concentration were spherical vesicles of varied sizes, all being approximately 100 nm in diameter and displaying typical morphology. NTA results from NanoSight nanoparticle tracker showed that the peak particle size was 98.5 nm, that the average particle size was 110.2 nm, and that the particle size was mainly distributed between 68.4-155.7 nm. Mtb-EVs that were smaller than 250 nm accounted for 98.39% of the total. Mouse myeloid DCs directionally induced and amplified in vitro displayed typical DC phenotype and morphology, and the purity exceeded 85%. EM verified the abundance of microvilli and radial protuberance on the surface of DCs, which had uniform cytoplasm and clear nuclear membrane. Loaded with Mtb-EVs at different concentrations, including 10 2, 10 3, and 10 4 particles/cell, the DCs had significantly upregulated levels of intracellular ROS ( P<0.05). In addition, Mtb-EVs induced the release of IL-1ß and IL-6 in a dose-dependent manner ( P<0.05). Conclusion: We established in the study a technical process for the extraction of Mtb-EVs by ultrafiltration concentration and obtained Mtb-EVs with sound morphology, high purity, and concentrated particle size distribution. Furthermore, Mtb-EVs can upregulate the intracellular ROS level in DCs and induce the release of IL-1ß and IL-6 in a dose-dependent manner.

[Immune Modulatory Effect of Outer Membrane Vesicles Derived from Salmonella on Mouse Bone Marrow-Derived Dendritic Cells].

OBJECTIVE: To study the effect of outer membrane vesicles (OMVs) derived from Salmonella typhimurium (ST) on the ultrastructural features and immune function of dendritic cells (DC). METHODS: Mice bone marrow cells were collected aseptically, and myeloid DC were generated by the combined induction and amplification with recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) and recombinant mouse interleukin-4 (rm IL-4). Cell morphology was observed under inverted phase contrast microscope and the phenotype was identified with flow cytometry. ST-OMVs were isolated through ultracentrifugation. The survival rate of DC was assessed with CCK-8 assay, and the stimulus concentration of OMVs was henceforth determined. The ultrastructural characteristics of DC loaded with OMVs were observed with transmission electron microscopy. The cytokine secretion, surface molecule expression and phagocytic capacity of DC were examined with flow cytometry. RESULTS: The DC induced and amplified in vitro displayed typical DC phenotype in morphological analysis and the purity of DC exceeded 85%. Transmission electron microscopy showed that there were large numbers of protrusions on the cell surface. After stimulation with ST-OMVs, it was observed that the dendritic structures on the surface of DC were reduced and a large number of phagolysosomes were found in the cytoplasm. In addition, increased numbers of mitochondria, swelling and typical apoptosis were observed. After treatment with ST-OMVs at 5 µg/mL and 10 µg/mL, the secretion of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) of DC increased significantly ( P<0.05). Furthermore, the immature DC could differentiate into mature DCs after stimulation with ST-OMVs, which were characterized by a decrease in phagocytic capacity ( P<0.05) and an upregulation of phenotypic markers ( P<0.05). CONCLUSION: ST-OMVs can stimulate DC to produce TNF-α and IL-1ß and promote DC maturation and antigen presentation.

Internal carotid artery pseudoaneurysm caused by parapharyngeal abscess: A case report / 北京大学学报(医学版)

Pseudoaneurysms of the neck are seldom, and those caused by neck infections especially parapharyngeal abscess are even rarer. However, it is life-threatening and may bring sudden death due to the obstruction of airway and the pseudoaneurysms rupture. We analyzed the clinical features, diagnosis and treatment of the disease through a case summary and literature review in order to guide clinical diagnosis and treatment of pseudoaneurysms. The patient, whom we presented was an 87-year-old male and admitted in emergency of our hospital with the chief complaint of neck swelling for 7 days and shortness of breath for 2 days. Cervical ultrasound examination showed that there was an liquid dark area next to the left common carotid artery which was approximately 8.0 cm × 5.0 cm, consideration of formation of left carotid artery pseudoaneurysm, and the liquid dark area which was visible on the right considered of pseudoaneurysm or infection. Angiography of neck showed a clustered high-density shadow around the bifurcation of the left carotid artery, with an overall range of approximately 65 mm × 52 mm × 72 mm, the pseudoaneurysms for sure, while on the right side of the lesion, mixed low density shadows with air could be seen, the parapharyngeal abscess for sure.Then he was diagnosed as the pseudoaneurysm of left internal carotid artery which was caused by parapharyngeal abscess. After tracheal intubation and anti-infection treatment, the patient died due to hemorrhagic shock of the ruptured of the pseudoaneurysm. Morever we performed literature search on PubMed, Wanfang database and CNKI with keywords of "neck pseudoaneurysm, neck infection, parapharyngeal abscess" and enrolled 10 cases. Then we summarized the clinical characteristics and treatment. We analyzed and summarized the 10 case reports, in which the number of male was 7. Among them, there were 4 pediatric, and 6 adults were enrolled overall. Most of the symptoms were neck swelling, and the diseased blood vessel was mainly the right internal carotid artery which accounted for half overall. All the patients underwent surgical intervention, and recovered well. So we draw the conclusion that the clinical incidence of cervical pseudoaneurysms is low and can be caused by a variety of factors, especially caused by infectious factors. When a patient has a progressive pulsating mass in the neck, the preliminary diagnosis should be made by ultrasound as soon as possible, and the aortic enhancement CT should be used to further confirm.For a patient with cervical pseudo-aneurysms caused by parapharyngeal infections, he should take operation timely combined with antibiotic treatment in time.

Polymorphism of Platelet Specific Antigens (HPA-1-5, 15) in Han Donors in the North Area of Henan Province / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the gene polymorphism distribution characteristics of human platelet HPA-1-5 and 15 blood group antigens and construct a certain scale of platelet HPA database in the north area of Henan Province so as to provide platelet apheresis for clinical departments.</p><p><b>METHODS</b>Using polymerase chain reaction with sequence-specific primers (PCR-SSP), the genotyping of HPA-1-5 and 15 system was carried out; the periperal blood of 500 healthy Han donors in north area of Henan Province was collected randomly, the gene and genotype frequencies were detected by direct counting method, and the population distribution frequncy of HPA genes was analyzed by Hardy-Weinberg balance test, and compared with other regions and ethnics by using χ(2) test.</p><p><b>RESULTS</b>There was statistically significant (P < 0.05) of increase HPA-3b and HPA-5a in North area of Henan Province, compared with Chinese Han population; the HPA-3b and 5a increase and HPA-2a decrease were statistically significant (P < 0.05), compared with Ethnic minority of China. There was partly increase of HPA-1a, 2a, 3a and 5a, compared with different regions and ethnic in abroad. HPA allele genes of 500 Han donors in the North area of Henan Province were as follows: 0.985 and 0.015 for 1a and 1b; 0.924 and 0.076 for 2a and 2b; 0.469 and 0.531 for 3a and 3b; 1.000 and 1.000 for 4a and 5a; 0.532 and 0.468 for 15a and 15b, respectively. HPA allele gene frequencies were 1aa0.970, 1ab0.030; 2aa0.848, 2ab0.152; 3aa0.222, 3ab0.494, 3bb0.284; 4aa1.000; 5aa1.000; 15aa0.282, 15ab0.500, 15bb0.218. Compared with other regions and ethnic, HPA gene frequencies partly had statistical significance.</p><p><b>CONCLUSION</b>Distribution of HPA allele frequencies in the North area of Henan province is in accordence with the Hardy-Weinberg law. There are race and regional differences in HPA allele gene frequencies, compared with other regions and countries. And the HPA systems HPA-3 and 15 display the genetic polymorphisms, which provides a theoretical basis for the relevant research of the same type platelet infusion and alloimmune thrombocytopenia.</p>

HMGB1-a as potential target for therapy of hematological malignancies / 中国实验血液学杂志

HMGB1 is a widely existing DNA-binding nuclear protein, participating in gene transcription, damage repair, recombination and stabilizing nucleosome construction. Under injury, infection and chemotherapy, HMGB1 can be released by nature immunocyte and necrosis cells as a DAMP, exerting pleiotropic biological effects by binding to RAGE, TLR and CXCL12, which lead to activation of CDC42, mitogen-activated protein kinases (MAPKs), c-IAP and NF-κB, thereby promoting angiogenesis, unlimited replicative potential, tissue invasion and metastasis. And it also involves in immune response by regulating immunocyte function as a immunocyte warning signal. Scholars have detected that HMGB1 is over expressed and released following chemotherapy and radiation therapy in cells of hematological malignancies, promoting malignant cell replication, decreasing therapeutic effect. Recently, endogenous HMGB1 has been implied to be an intrinsic modulator of autophagy and referenced to resistant to apoptosis in malignant hematosis cells. In contrast, through suppression of HMGB1 expression, tumor cell apoptosis and chemotherapeutic drug sensitivity were increased, which will be a new strategy for the treatment of hematological malignancies. In this article, the basic characteristics of HMGB1, including structure and biological features, and HMGB1 and tumors such as lymphoma, myeloproliferative neoplasms and acute myeloid leukemia are reviewed.

Advance of clinical study on immune thrombocytopenia caused by irregular antibodies / 中国实验血液学杂志

The platelet antibodies mainly include platelet-specific and related antibodies, which belong to irregular antibodies. They are produced by autoimmune, drug-induced or isoimmunization (such as pregnancy, blood transfusion and so on), the irregular IgG and/or IgM antibodies produce and lead to platelet transfusion refractoriness (PTR), post-transfusion purpura (PTP) and isoimmune neonatal thrombocytopenic purpura (INTP), idiopathic thrombocytopenic purpura and so on. It is very necessary to screen and identify the irregular antibodies before blood transfusion or parturition. Except some serological detections should be done first, flow cytometry and molecular biological techniques such as PCR and PCR-SSP are applied to detect the difficult-matching patients' genotypes and fetal genotypes in order to further predict fetal INTP and to provide the right blood for difficult-matching patients, therefore, some measures must be taken early for prevention and treatment of immune thrombocytopenic purpura. In this review, the production, typing and laboratory tests of irregular antibodies, as well as the pathogenesis and clinical symptoms of diseases caused by irregular antibodies, and the current progress are summarized.

Immune hemolytic diseases caused by irregular antibodies and its research advance in clinic / 中国实验血液学杂志

The irregular antibodies are other than antibodies from ABO blood group system because of pregnancies and blood transfusions, clinical autoimmune, drug-induced etc. The irregular IgG and/or IgM antibodies emerge and lead to the difficult identification of clinical blood type, difficult matching of blood, hemolytic disease of newborn, hemolytic transfusion reaction, and so on. It is very necessary to screen and identify the irregular antibodies before blood transfusion or antepartum. For some difficult identifying samples, some detections on serological level should be done firstly, combining with flow cytometry analysis, the difficult-matching patients' genotypes and fetal genotypes were detected by molecular biology techniques such as PCR and PCR-SSP in order to further predict fetal hemolytic disease of newborn and to provide the right blood to difficult-matching patients, and free fetal DNA extracted from maternal plasma. So that some measures must early be taken for clinical prevention and treatment to reduce immune hemolytic reactions. In this paper, the emergence of irregular antibodies, species, laboratory testing, pathogenesis, clinical symptoms and the current research are reviewed.

Re-discrimination on "reinforcement can be achieved by slow-yet-rapid needling, and rapid-yet-slow needling weakens evil qi" / 中国针灸

<p><b>OBJECTIVE</b>To study on the original meanings of "reinforcement can be achieved by slow-yet-rapid needling, and rapid-yet-slow needling weakens evil-qi", one of important bases of reinforcing-reducing in acupuncture.</p><p><b>METHODS</b>Using the principle of knowledge archaeology and through structure an d through structure analysis on the subjects described by "slow" and "rapid" and the papers appearing the words, the meanings of this sentence are re-discriminated.</p><p><b>CONCLUSION</b>"Slow-rapid" prescribed in the reinforcing-reducing needling method in Huangdi's Internal Classic includes 3 subjects: pulse condition, needle-retained time, and velocity of withdrawing the needle. The "slow-rapid" in "reinforcement can be achieved by slow-yet-rapid needling, and rapid-yet-slow needling weakens evil-qi" should be pulse condition, but was not needling method, which means that the pulse changes from slow to rapid, marked by transformation of channels from deficiency to excess; the transform of the pulse from rapid to slow means that the channel transforms from excess to deficiency.</p>

Molida therapy in the out-patient department for patients with III B prostatitis / 中华男科学杂志

<p><b>OBJECTIVE</b>To study the therapeutic effect of Molida therapy in the out-patient department for patients with III B prostatitis.</p><p><b>METHODS</b>One hundred and thirty-six patients with II B prostatis accompanied with psychological disorder of different degrees were divided into two groups: Group A (68 cases), treated with routine drug and Molida therapy, and Group B (68 cases), treated with routine drug only. The therapeutic effects of the two groups were analyzed.</p><p><b>RESULTS</b>After an 8-week treatment, the scores of self-feeling symptoms of SCL-90, SAS and SDS in Group A were 70.5 +/- 22.3, 18.7 +/- 8.5 and 21.4 +/- 9.0, respectively, significantly lower than those in Group B (P < 0.05) and the rates of efficacy and obvious efficacy in alleviating prostatodynia in Group A were 100% and 72.1%, respectively, both higher than in Group B (P < 0.05).</p><p><b>CONCLUSION</b>Molida therapy in the out-patient department is useful in improving the psychological disorder and enhancing the therapeutic effect for patients with III B prostatitis.</p>

Effect of Progesterone on Progesterone Receptor in Brain Tissue with Hypoxia-Ischemia Brain Damage in Neonatal Rats / 实用儿科临床杂志

ObjectiveTo observe the effect of progesterone on progesterone receptor in brain tissue with hypoxia-ischemia brain da-mage(HIBD) in neonatal rats,and discuss the protective mechanism of progesterone on HIBD of neonatal rats.MethodsTwenty-four 7-day-old neonatal rats were randomly divided into 3 groups:sham-operated group,hypoxia-ischemia group and pretreatment group.Rats in hypoxia-ischemia group and pretreatment group were subjected to left common carotid artery ligation,then were exposed to 80 mL/L oxygen and 920 mL/L nitrogen gas in 37 ℃ closed container for up to 2.5 h to establish hypoxia-ischemia encephalopathy(HIE) model.Progestero-ne was injected intraperitoneally into pretreatment group rats respectively at 30 minutes before hypoxia,solution was injected into the first 2 groups.All rats were killed at the 24 hour after operation,immunohistochemistry staining was used to examine the expression of progesterone receptor in brain.ResultsTotally 24 neonatal rats entered the result analysis without loss.Progesterone receptor was expressed in both endochylema and nucleus in every group rat.The amount of the positive cell of progesterone receptor in hypoxia-ischemia group significantly decreased compared with that in sham-operated group(P

Distribution features of Gleason score and its relationship with clinical stages in 346 prostate cancer patients / 中华男科学杂志

<p><b>OBJECTIVE</b>To analyze the distribution features of Gleason score and evaluate the relationship between Gleason score and clinical stages in patients with prostate cancer.</p><p><b>METHODS</b>Surveys were made of the inpatients with prostate cancer diagnosed by pathology from January 1992 to June 2005 in our hospital. Gleason score and clinical stages were determined on the basis of pathological examination and clinical data of the prostate cancer patients. The patients were divided into three groups (1992-1999, 2000-2002 and 2003-2005). The Chi-square test was used to evaluate the distribution and differences of Gleason score among the three groups. Spearman rank correlation was applied to the evaluation of the relationship between Gleason score and clinical stages.</p><p><b>RESULTS</b>We found a statistically significant shift in the distribution of Gleason score (chi2 = 17.703, P < 0.01), and a slight increase in the mean Gleason score. The proportion of moderately differentiated tumor increased (chi2 = 10.736, P < 0.01). There was little change in the proportion of Gleason score 7, 8, 9 and 10 (chi2 = 4.038, P > 0.05). Gleason score had a significant positive correlation with clinical stages in the 346 cases of prostate cancer (r = 0.452, P < 0.01). Significant difference was observed between Gleason score 2-6 and 7 or 8-10 (chi2 = 8.786, P < 0.01, chi2 = 22.956, P < 0.01), but not between the latter 2 groups (chi2 = 0.787, P > 0.05) in prediction of organ-confined disease.</p><p><b>CONCLUSIONS</b>Gleason score 7 shows the similar value to Gleason score 8-10 in predicting the progression of the disease. Gleason score was significantly correlated with clinical stages, which suggests that Gleason score is also an important indicator for the prognosis of prostate cancer.</p>