Factors affecting the transduction of pluripotent hematopoietic stem cells: long-term expression of a human adenosine deaminase gene in mice

An amphotropic retroviral vector, LgAL(delta Mo + PyF101) containing a human adenosine deaminase (ADA) cDNA was used to optimize procedures for the lasting genetic modification of the hematopoietic system of mice. The highest number of retrovirally infected cells in the hematopoietic tissues of long-term reconstituted mice was observed after transplantation of bone marrow (BM) cells that had been cocultured in the presence of both interleukin-1 alpha (IL-1 alpha) and IL-3. A significantly lower number was detected when IL-1 alpha was omitted from such cocultures. The yield of cells that generate spleen colony-forming cells (CFU-S) in the BM of lethally irradiated recipients (MRA-CFU-S) significantly improved on inclusion of the adherent cell fraction of cocultures in the transplant. Retroviral integration patterns in MRA-CFU-S-derived spleen colonies showed that an MRA-CFU-S can produce many CFU-S during BM regeneration. Expression of hADA was detected in the circulating white blood cells of long-term reconstituted animals, demonstrating that the LgAL(delta Mo + PyF101) vector is capable of directing the sustained expression of hADA, and in approximately 35% of the transduced MRA-CFU-S-derived spleen colonies. These results should facilitate the development of gene therapy protocols for the treatment of severe combined immunodeficiency caused by a lack of functional ADA.     

Outcomes following cardiopulmonary resuscitation in an emergency department of a low- and middle-income country

Background

Cardiopulmonary resuscitation (CPR) is a key component of emergency care following cardiac arrest. A better understanding of factors that influence CPR outcomes and their prognostic implications would help guide care. A retrospective analysis of 800 adult patients that sustained an in- or out-of-hospital cardiac arrest and underwent CPR in the emergency department of a tertiary care facility in Karachi, Pakistan, between 2008 and 15 was conducted.

Methods

Patient demographics, clinical history, and CPR characteristics data were collected. Logistic regression model was applied to assess predictors of return of spontaneous circulation and survival to discharge. Analysis was conducted using SPSS v.21.0.

Results

Four hundred sixty-eight patients met the study’s inclusion criteria, and overall return of spontaneous circulation and survival to discharge were achieved in 128 (27.4%) and 35 (7.5%) patients respectively. Mean age of patients sustaining return of spontaneous circulation was 52 years and that of survival to discharge was 49 years. The independent predictors of return of spontaneous circulation included age ≤?49 years, witnessed arrest, ≤?30 min interval between collapse-to-start, and 1–4 shocks given during CPR (aOR (95% CI) 2.2 (1.3–3.6), 1.9 (1.0–3.7), 14.6 (4.9–43.4), and 3.0 (1.4–6.4) respectively), whereas, age ≤?52 years, bystander resuscitation, and initial rhythm documented (pulseless electrical activity and ventricular fibrillation) were independent predictors of survival to discharge (aOR (95% CI) 2.5 (0.9–6.5), 1.4 (0.5–3.8), 5.3 (1.5–18.4), and 3.1 (1.0–10.2) respectively).

Conclusion

Our study notes that while the majority of arrests occur out of the hospital, only a small proportion of those arrests receive on-site CPR, which is a key contributor to unfavorable outcomes in this group. It is recommended that effective pre-hospital emergency care systems be established in developing countries which could potentially improve post-arrest outcomes. Younger patients, CPR initiation soon after arrest, presenting rhythm of pulseless ventricular tachycardia and ventricular fibrillation, and those requiring up to four shocks to revive are more likely to achieve favorable outcomes.

     

Inhibition of microglial activity alters spinal wide dynamic range neuron discharge and reduces microglial Toll‐like receptor 4 expression in neuropathic rats

It is believed that neuropathic pain results from aberrant neuronal discharges although some evidence suggests that the activation of glia cells contributes to pain after an injury to the nervous system. This study aimed to evaluate the role of microglial activation on the hyper‐responsiveness of wide dynamic range neurons (WDR) and Toll‐like receptor 4 (TLR4) expressions in a chronic constriction injury (CCI) model of neuropathic pain in rats. Adult male Wistar rats (230 ± 30 g) underwent surgery for induction of CCI neuropathy. Six days after surgery, administration of minocycline (10, 20, and 40 mg/kg, i.p.) was initiated and continued until day 14. After administration of the last dose of minocycline or saline, a behavioral test was conducted, then animals were sacrificed and lumbar segments of the spinal cord were collected for Western blot analysis of TLR4 expression. The electrophysiological properties of WDR neurons were investigated by single unit recordings in separate groups. The findings showed that after CCI, in parallel with thermal hyperalgesia, the expression of TLR4 in the spinal cord and the evoked response of the WDR neurons to electrical, mechanical, and thermal stimulation significantly increased. Post‐injury administration of minocycline effectively decreased thermal hyperalgesia, TLR4 expression, and hyper‐responsiveness of WDR neurons in CCI rats. The results of this study indicate that post‐injury, repeated administration of minocycline attenuated neuropathic pain by suppressing microglia activation and reducing WDR neuron hyper‐responsiveness. This study confirms that post‐injury modulation of microglial activity is a new strategy for treating neuropathic pain.     

孟根乌森乌日乐对小鼠的急性毒性研究

目的：了解孟根乌森乌日乐的急性毒性作用剂量及给药后的急性毒性反应和死亡分布情况,确定孟根乌森乌日乐的半数致死量（ LD50）。方法用孔氏综合法（改进寇氏法）分为14．30,9．28,6．04,3．92,2．55,1．66 g? kg－16个剂量组,以0．4 mL／10 g的量灌胃给药1次。实验后观察14 d,记录体重变化及不良反应情况。结果孟根乌森乌日乐小鼠半数致死量为5．1597 g? kg－1（95％CI：3．6652～7．2637 g? kg－1）。14 d内未出现明显不良反应症状且体重有增长趋势。结论孟根乌森乌日乐的急性毒性实验的半数致死量为临床用药量的100倍,提示单次口服较为安全。     

Reserpine-induced altered neuro-behavioral,biochemical and histopathological assessments prevent by enhanced antioxidant defence system of thymoquinone in mice

Metabolic Brain Disease - Thymoquinone (Tq), an active compound of Nigella sativa, has been known for its anti-inflammatory, antioxidant, and neuroprotective characteristics. The present study is...     

五种布鲁氏菌核酸检测试剂盒检测性能的评价北大核心CSCD

目的比较5种布鲁氏菌核酸实时荧光PCR检测试剂盒的一致性和检出能力,为临床实验室选择检测方法和布鲁氏菌的诊断提供参考依据。方法选用经病原学检测确定为布鲁氏菌阳性的血液样本38份,健康人的血液样本24份,潘氏变形杆菌、溶藻弧菌、河弧菌、铜绿假单胞菌、肺炎克雷伯菌DNA各1份,使用5种试剂盒(编号A-E)分别进行核酸检测,比较5种试剂盒临床样本检测的一致性;选择1份阳性样本核酸用无RNA酶水梯度稀释得到5个浓度(浓度1:4453.13 fg/μL,浓度2:1113.28 fg/μL,浓度3:278.32 fg/μL,浓度4:69.58 fg/μL,浓度5:17.40 fg/μL),每个浓度使用5种试剂盒(编号A-E)分别进行3次检测,比较5种试剂盒的阳性检出率及批内重复性。结果5种试剂盒检测67份DNA样品的符合率稍有不同,试剂盒ABDE的符合率均为100%,试剂盒C的符合率为98.51%。批内重复性显示5种试剂盒在浓度1、浓度2、浓度3水平重复检测DNA的Ct值变异系数均<5%;在浓度1与浓度4梯度区间,试剂盒的阳性检出能力比较显示试剂盒A、B、D较高,为11/12,试剂盒C和E较低,为8/12。结论5种试剂盒的真实性和可靠性较好,灵敏度和符合率稍有差别,特异度均为100%;重复性较好,检测性能良好。部分试剂盒对弱阳性样本的检出能力不强,该类样本可使用多种试剂盒复核,以保障结果的准确性。     

Epstein-Barr virus (EBV)-associated lymphoproliferations in severe combined immunodeficient mice transplanted with Hodgkin's disease lymph nodes: implications of EBV-positive bystander B lymphocytes rather than EBV-infected Reed-Sternberg cells

To establish an in vivo model for the study of Hodgkin's disease and Reed-Sternberg (RS) cells, 25 lymph node tissue samples involved by Hodgkin's disease were grafted into severe combined immunodeficiency (SCID) mice. Ten Epstein-Barr virus (EBV)-associated tumors were obtained in SCID mice. EBV-positive tumors growing in SCID mice were correlated with the presence of EBV-positive nonneoplastic B cells in patient tumors (90% v 26.6%; P<.01) and was independent of the EBV status of RS cells. Our results suggested that EBV-positive tumors growing in SCID mice originated from normal EBV-positive small lymphocytes (bystander B lymphocytes). We also compared the characteristics of these tumors with those obtained after transplantation of 15 non-Hodgkin's lymphoma and four reactive lymph nodes. The latent period to observe a growing tumor in SCID mice was similar between the two groups (12.86 +/- 5.59 weeks for Hodgkin's disease v 13.6 +/- 5.36 weeks for non-Hodgkin's lymphoma and reactive lymph nodes). The relatively high number of EBV-positive small lymphocytes detected in Hodgkin's disease and T-cell lymphoma compared with B-cell lymphoma may account for the greater percentage of EBV- positive tumors obtained in SCID mice. Our results show that SCID mice do not provide the growth conditions that are required for in vivo growth of RS cells. We noted in some SCID tumors, the presence of binucleated and/or multinucleated giant cells resembling RS cells. However, the presence of such cells was not restricted to mice grafted with lymph nodes involved by Hodgkin's disease. We postulate that in previous reports, cells resembling RS cells were just binucleated EBV- positive lymphoma blastoid cells rather than actual RS cells.     

Diagnosis and management of asthma in preschoolers: A Canadian Thoracic Society and Canadian Paediatric Society position paper

Asthma often starts before six years of age. However, there remains uncertainty as to when and how a preschool-age child with symptoms suggestive of asthma can be diagnosed with this condition. This delays treatment and contributes to both short- and long-term morbidity. Members of the Canadian Thoracic Society Asthma Clinical Assembly partnered with the Canadian Paediatric Society to develop a joint working group with the mandate to develop a position paper on the diagnosis and management of asthma in preschoolers.In the absence of lung function tests, the diagnosis of asthma should be considered in children one to five years of age with frequent (≥8 days/month) asthma-like symptoms or recurrent (≥2) exacerbations (episodes with asthma-like signs). The diagnosis requires the objective document of signs or convincing parent-reported symptoms of airflow obstruction (improvement in these signs or symptoms with asthma therapy), and no clinical suspicion of an alternative diagnosis. The characteristic feature of airflow obstruction is wheezing, commonly accompanied by difficulty breathing and cough. Reversibility with asthma medications is defined as direct observation of improvement with short-acting ß₂-agonists (SABA) (with or without oral corticosteroids) by a trained health care practitioner during an acute exacerbation (preferred method). However, in children with no wheezing (or other signs of airflow obstruction) on presentation, reversibility may be determined by convincing parental report of a symptomatic response to a three-month therapeutic trial of a medium dose of inhaled corticosteroids with as-needed SABA (alternative method), or as-needed SABA alone (weaker alternative method). The authors provide key messages regarding in whom to consider the diagnosis, terms to be abandoned, when to refer to an asthma specialist and the initial management strategy. Finally, dissemination plans and priority areas for research are identified.     

The effect of lithium on growth factor production in long-term bone marrow cultures

We have previously reported that lithium chloride (LiCl) stimulates the production of granulocyte-macrophage colony-forming cells (GM-CFC), pluripotent stem cells (CFU-S), and differentiated granulocytes, macrophages and megakaryocytes in murine Dexter marrow cultures and that this effect appears to be mediated indirectly by a radioresistant adherent marrow cell. In this study we have established that exposure of murine Dexter cultures to LiCl (4 mEq/L) causes an increase of colony-forming cell megakaryocytes (CFU-meg) over 1 to 6 weeks of culture in both supernatant (188% to 611%) and stromal phases (123% to 246%). Moreover, we have shown that lithium treatment of either irradiated (1,100 rad) or unirradiated stromal cells increased production of activities stimulating formation of megakaryocyte, granulocyte, macrophage, and mixed lineage colonies and proliferation of the factor-dependent cell line, FDC-P1. This FDC-P1 stimulatory activity was completely blocked by an antibody to purified recombinant granulocyte-macrophage colony stimulating factor (rGM-CSF). The baseline or lithium-induced--stromal-derived bone marrow colony stimulating activity was partially blocked by the antibody to rGM-CSF and by an antibody to purified colony stimulating factor I (CSF-1); the two antibodies combined resulted in greater than 90% inhibition of the lithium-induced marrow stimulatory activity. In addition, radioimmunoassay (RIA) showed that although CSF-1 was detectable in supernatants of these cultures, exposure to lithium did not increase CSF-1 levels. These data indicate that Dexter stromal cells produce CSF- 1 and GM-CSF and that lithium appears to exert its stimulatory effects on in vitro myelopoiesis by inducing production of GM-CSF.