Human embryonic stem cell (hES) derived dendritic cells are functionally normal and are susceptible to HIV-1 infection

<p>Abstract</p> <p>Background</p> <p>Human embryonic stem (hES) cells hold considerable promise for cell replacement and gene therapies. Their remarkable properties of pluripotency, self-renewal, and tractability for genetic modification potentially allows for the production of sizeable quantities of therapeutic cells of the hematopoietic lineage. Dendritic cells (DC) arise from CD34+ hematopoietic progenitor cells (HPCs) and are important in many innate and adaptive immune functions. With respect to HIV-1 infection, DCs play an important role in the efficient capture and transfer of the virus to susceptible cells. With an aim of generating DCs from a renewable source for HIV-1 studies, here we evaluated the capacity of hES cell derived CD34+ cells to give rise to DCs which can support HIV-1 infection.</p> <p>Results</p> <p>Undifferentiated hES cells were cultured on S17 mouse bone marrow stromal cell layers to derive CD34+ HPCs which were subsequently grown in specific cytokine differentiation media to promote the development of DCs. The hES derived DCs (hES-DC) were subjected to phenotypic and functional analyses and compared with DCs derived from fetal liver CD34+ HPC (FL-DC). The mature hES-DCs displayed typical DC morphology consisting of veiled stellate cells. The hES-DCs also displayed characteristic phenotypic surface markers CD1a, HLA-DR, B7.1, B7.2, and DC-SIGN. The hES-DCs were found to be capable of antigen uptake and stimulating naïve allogeneic CD4+ T cells in a mixed leukocyte reaction assay. Furthermore, the hES-DCs supported productive HIV-1 viral infection akin to standard DCs.</p> <p>Conclusion</p> <p>Phenotypically normal and functionally competent DCs that support HIV-1 infection can be derived from hES cells. hES-DCs can now be exploited in applied immunology and HIV-1 infection studies. Using gene therapy approaches, it is now possible to generate HIV-1 resistant DCs from anti-HIV gene transduced hES-CD34+ hematopoietic progenitor cells.</p

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy

BACKGROUND: Many novel studies and therapies are possible with the use of human embryonic stem cells (hES cells) and their differentiated cell progeny. The hES cell derived CD34 hematopoietic stem cells can be potentially used for many gene therapy applications. Here we evaluated the capacity of hES cell derived CD34 cells to give rise to normal macrophages as a first step towards using these cells in viral infection studies and in developing novel stem cell based gene therapy strategies for AIDS. RESULTS: Undifferentiated normal and lentiviral vector transduced hES cells were cultured on S17 mouse bone marrow stromal cell layers to derive CD34 hematopoietic progenitor cells. The differentiated CD34 cells isolated from cystic bodies were further cultured in cytokine media to derive macrophages. Phenotypic and functional analyses were carried out to compare these with that of fetal liver CD34 cell derived macrophages. As assessed by FACS analysis, the hES-CD34 cell derived macrophages displayed characteristic cell surface markers CD14, CD4, CCR5, CXCR4, and HLA-DR suggesting a normal phenotype. Tests evaluating phagocytosis, upregulation of the costimulatory molecule B7.1, and cytokine secretion in response to LPS stimulation showed that these macrophages are also functionally normal. When infected with HIV-1, the differentiated macrophages supported productive viral infection. Lentiviral vector transduced hES cells expressing the transgene GFP were evaluated similarly like above. The transgenic hES cells also gave rise to macrophages with normal phenotypic and functional characteristics indicating no vector mediated adverse effects during differentiation. CONCLUSION: Phenotypically normal and functionally competent macrophages could be derived from hES-CD34 cells. Since these cells are susceptible to HIV-1 infection, they provide a uniform source of macrophages for viral infection studies. Based on these results, it is also now feasible to transduce hES-CD34 cells with anti-HIV genes such as inhibitory siRNAs and test their antiviral efficacy in down stream differentiated cells such as macrophages which are among the primary cells that need to be protected against HIV-1 infection. Thus, the potential utility of hES derived CD34 hematopoietic cells for HIV-1 gene therapy can be evaluated

Pooled analysis of WHO Surgical Safety Checklist use and mortality after emergency laparotomy

Background The World Health Organization (WHO) Surgical Safety Checklist has fostered safe practice for 10 years, yet its place in emergency surgery has not been assessed on a global scale. The aim of this study was to evaluate reported checklist use in emergency settings and examine the relationship with perioperative mortality in patients who had emergency laparotomy. Methods In two multinational cohort studies, adults undergoing emergency laparotomy were compared with those having elective gastrointestinal surgery. Relationships between reported checklist use and mortality were determined using multivariable logistic regression and bootstrapped simulation. Results Of 12 296 patients included from 76 countries, 4843 underwent emergency laparotomy. After adjusting for patient and disease factors, checklist use before emergency laparotomy was more common in countries with a high Human Development Index (HDI) (2455 of 2741, 89.6 per cent) compared with that in countries with a middle (753 of 1242, 60.6 per cent; odds ratio (OR) 0.17, 95 per cent c.i. 0.14 to 0.21, P <0001) or low (363 of 860, 422 per cent; OR 008, 007 to 010, P <0.001) HDI. Checklist use was less common in elective surgery than for emergency laparotomy in high-HDI countries (risk difference -94 (95 per cent c.i. -11.9 to -6.9) per cent; P <0001), but the relationship was reversed in low-HDI countries (+121 (+7.0 to +173) per cent; P <0001). In multivariable models, checklist use was associated with a lower 30-day perioperative mortality (OR 0.60, 0.50 to 073; P <0.001). The greatest absolute benefit was seen for emergency surgery in low- and middle-HDI countries. Conclusion Checklist use in emergency laparotomy was associated with a significantly lower perioperative mortality rate. Checklist use in low-HDI countries was half that in high-HDI countries.Peer reviewe

Global variation in anastomosis and end colostomy formation following left-sided colorectal resection

Background End colostomy rates following colorectal resection vary across institutions in high-income settings, being influenced by patient, disease, surgeon and system factors. This study aimed to assess global variation in end colostomy rates after left-sided colorectal resection. Methods This study comprised an analysis of GlobalSurg-1 and -2 international, prospective, observational cohort studies (2014, 2016), including consecutive adult patients undergoing elective or emergency left-sided colorectal resection within discrete 2-week windows. Countries were grouped into high-, middle- and low-income tertiles according to the United Nations Human Development Index (HDI). Factors associated with colostomy formation versus primary anastomosis were explored using a multilevel, multivariable logistic regression model. Results In total, 1635 patients from 242 hospitals in 57 countries undergoing left-sided colorectal resection were included: 113 (6·9 per cent) from low-HDI, 254 (15·5 per cent) from middle-HDI and 1268 (77·6 per cent) from high-HDI countries. There was a higher proportion of patients with perforated disease (57·5, 40·9 and 35·4 per cent; P < 0·001) and subsequent use of end colostomy (52·2, 24·8 and 18·9 per cent; P < 0·001) in low- compared with middle- and high-HDI settings. The association with colostomy use in low-HDI settings persisted (odds ratio (OR) 3·20, 95 per cent c.i. 1·35 to 7·57; P = 0·008) after risk adjustment for malignant disease (OR 2·34, 1·65 to 3·32; P < 0·001), emergency surgery (OR 4·08, 2·73 to 6·10; P < 0·001), time to operation at least 48 h (OR 1·99, 1·28 to 3·09; P = 0·002) and disease perforation (OR 4·00, 2·81 to 5·69; P < 0·001). Conclusion Global differences existed in the proportion of patients receiving end stomas after left-sided colorectal resection based on income, which went beyond case mix alone

CD34+ cells were cultured in cytokine media and analyzed by FACS for CD14 and CD1a markers at different days by staining with CD1a-PECY5 and CD14-PE conjugated antibodies

<p><b>Copyright information:</b></p><p>Taken from "Human embryonic stem cell (hES) derived dendritic cells are functionally normal and are susceptible to HIV-1 infection"</p><p>http://www.aidsrestherapy.com/content/5/1/1</p><p>AIDS Research and Therapy 2008;5():1-1.</p><p>Published online 23 Jan 2008</p><p>PMCID:PMC2248203.</p><p></p> Dot plots are representative of triplicate experiments

The allogeneic stimulatory properties of DCs were assessed in a mixed leukocyte reaction assay using allogeneic T-cells

<p><b>Copyright information:</b></p><p>Taken from "Human embryonic stem cell (hES) derived dendritic cells are functionally normal and are susceptible to HIV-1 infection"</p><p>http://www.aidsrestherapy.com/content/5/1/1</p><p>AIDS Research and Therapy 2008;5():1-1.</p><p>Published online 23 Jan 2008</p><p>PMCID:PMC2248203.</p><p></p> Graded numbers of sorted and irradiated DCs were co-cultured with 5 × 10allogeneic T cells. BrdU incorporation was determined by FACS using a PE-conjugated antibody against BrdU. Histograms depict relative percent of BrdU uptake when compared to positive control cells stimulated with IL-2 and PHA. The X-axis is expressed as ratio of stimulator DCs cells to allogeneic responder T cells

Undifferentiated hES cells were cocultured with S17 mouse stromal cells to derive cystic bodies

<p><b>Copyright information:</b></p><p>Taken from "Human embryonic stem cell (hES) derived dendritic cells are functionally normal and are susceptible to HIV-1 infection"</p><p>http://www.aidsrestherapy.com/content/5/1/1</p><p>AIDS Research and Therapy 2008;5():1-1.</p><p>Published online 23 Jan 2008</p><p>PMCID:PMC2248203.</p><p></p> Later, purified CD34+ cells derived from cystic bodies and fetal liver were cultured in cytokine media to derive DCs as described in Methods. A and B, representative hES colony and an cystic body respectively. C and D, morphology of DCs differentiated from hES and FL derived CD34+ cells

To determine virus susceptibility, FL and hES DCs were infected with a replication competent X4-tropic HIV-GFP reporter virus strain at an m

<p><b>Copyright information:</b></p><p>Taken from "Human embryonic stem cell (hES) derived dendritic cells are functionally normal and are susceptible to HIV-1 infection"</p><p>http://www.aidsrestherapy.com/content/5/1/1</p><p>AIDS Research and Therapy 2008;5():1-1.</p><p>Published online 23 Jan 2008</p><p>PMCID:PMC2248203.</p><p></p>o.i. of 0.2. At 6 days post infection, cells were visualized by fluorescence microscopy to determine GFP expression in productively infected cells at the single cell level. Phase contrast and fluorescence images are shown for the respective cell types (A). Infected culture supernatants were assayed for viral p24 antigen by ELISA at different days post-infection (B). Data is representative of duplicate experiments

HES-DCs and FL-DCs were stained with antibodies CD1a-PECY5, HLA-DR-PE, B7

<p><b>Copyright information:</b></p><p>Taken from "Human embryonic stem cell (hES) derived dendritic cells are functionally normal and are susceptible to HIV-1 infection"</p><p>http://www.aidsrestherapy.com/content/5/1/1</p><p>AIDS Research and Therapy 2008;5():1-1.</p><p>Published online 23 Jan 2008</p><p>PMCID:PMC2248203.</p><p></p>1-PE, B7.2-PE, and DC-SIGN-PE. Expression of these respective markers was analyzed by FACS. Percent positive cells are indicated in respective plots for each of the cell surface markers. The isotype controls are shown in the left panel. Data is representative of triplicate experiments