For in vitro studies, both CD34+ selected cell and mononuclear cell (MNC) can be used to expand hematopoietic stem/progenitor cells. To investigate the expansion characteristics of mononuclear cells (MNC) and CD34+ selected cells the two cell fractions were cultured in the medium containing cytokine cocktails of SCF + IL-3 + IL-6 + FL + Tpo. It was found that the CD34+ selected cells had presented a high proliferation potential. The expansion of CD34+ selected cells could be maintained for 8 weeks while that of MNCs declined after 4 weeks. During the culture period, the maximum expansion of total cells in CD34+ selected cell culture achieved 31,270.9 +/- 8640.5 times, while that of MNC reached 50.9 +/- 8.2 times only. In the culture of MNCs, the colony density and the proportion of CD34+ cells increased from day 0 to day 7. However, in the culture of CD34+ selected cells, both the colony density and the proportion of CD34+ cells declined continuously during the whole culture period. During the ex vivo culture of CD34+ selected cells, the maximum expansion of CFU-GM and CD34+ cells achieved 185.7 +/- 14.1 fold and 191.7 +/- 188.8 fold, respectively. They are much higher than that of MNC, which were 12.4 +/- 3.2 fold and 50.6 +/- 33.2 fold only. While the BFU-E of both cell fractions only expanded by few times, which were 7.2 +/- 5.2 and 10.1 +/- 3.4 times, respectively. The results showed that the CD34+ selected cells culture could obtain more CFU-GM cells and CD34+ cells during the whole culture period.
Antigens, CD34 ; analysis ; Cell Count ; Cell Separation ; Cells, Cultured ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; physiology ; Leukocytes, Mononuclear ; cytology

OBJECTIVETo identify predictors of treatment retention problems at the two initial methadone maintenance treatment (MMT) clinics in Guangdong province, and to provide reference in reducing the rate of drop-outs.

METHODSAll of the patients were investigated at baseline and followed during the treatment period. Kaplan-Meier method on Survival Analysis was used to analyze retention related factors and predictors.

RESULTS509 subjects were recruited in this study with median of retention time as 108 days (95%CI: 74 - 142 days). The retention rate at 1-, 3-, 6-, 12-months were 75.9%, 52.7%, 41.6%, 30.1%, respectively. Data from Multivariate Cox Proportional Hazard Model analysis showed predictors of retention would involve factors as HIV infection state at baseline (HR = 1.241, P = 0.047), daily methadone dose (HR = 0.633, P = 0.004) and secretly using drugs during treatment period (HR = 5.345, P = 0.000).

CONCLUSIONThe retention rates at the two initial MMT clinics in Guangdong province were low. Patients who were HIV negative at baseline but still secretly using heroine during treatment or accepted low daily average dosage of methadone, had the tendency to drop out. The results implied that retention time could be prolonged by increasing daily methadone dosage.

Acquired Immunodeficiency Syndrome ; mortality ; Adult ; China ; Female ; Heroin Dependence ; therapy ; Humans ; Male ; Methadone ; administration & dosage ; therapeutic use ; Opiate Substitution Treatment ; Patient Compliance ; statistics & numerical data ; Patient Dropouts ; statistics & numerical data ; Substance Abuse Treatment Centers ; Substance Abuse, Intravenous ; drug therapy ; Survival Rate

Stirred culture offers a number of advantages over static systems as it maintains a stable, homogeneous culture environment and is easy to scale-up. This paper focused on the development and application of stirred tank bioreactor to culture hematopoietic cells. Preliminary study of stirred culture of hematopoietic cells was carried out in cord blood mononuclear cells culture in spinner flask. The results showed that the amplification rates of total cell, CFU-GM and BFU-E, with the exception of CFU-Mk, were greater in spinner flask than T-flask. The number of total cells increased 20 fold after 14 days incubation in spinner flask. The amplification rates of CFU-GM, CFU-Mk and BFU-E reached maximum at 10th day, 10th day and 7th day respectively, and the maximal amplification rates were 9.2-fold, 5.5-fold and 2.4-fold respectively, whereas the rate of CD34+ cells in spinner flask was (6.7 +/- 4.0)-fold at day 10. These results indicated that the stirred culture system is better than the static culture systems for hematopoietic cell proliferation. The biocompatibility of cord blood MNC to different types of materials used in bioreactors was also tested. The results showed that glass, stainless steel 316L and polytetraflouroethylene (PTFE) supported the growth of hematopoietic cells well. A higher cell density was reached in stirred bioreactors with controlled pH and DO than static culture. These findings suggested that the controlled large-scale culture could be used to overcome the clinical shortage of hematopoietic cells.
Antigens, CD34 ; metabolism ; Bioreactors ; Cell Culture Techniques ; instrumentation ; methods ; Erythroid Precursor Cells ; cytology ; Fetal Blood ; cytology ; Granulocyte-Macrophage Progenitor Cells ; cytology ; Humans ; Polytetrafluoroethylene ; Stainless Steel

Clinical evidence of hematopoietic restoration with umbilical cord blood (UCB) grafts indicates the UCB can be a useful source of hematopoietic stem cells for routine bone marrow reconstitution. Considering (10 +/- 5) x 10(8) nucleared cells per cord blood unit, there is a potential limitation for the use of cord blood in adults, which, however, can be overcome by ex vivo expansion of cells. A prerequisite for expansion is the significantly higher recovery of MNC, CD34+ cells and colony-forming cells (CFC) by thawing cryopreserved MNC. Cooling rate always acts as a critical factor that can affect the recovery of cells. Although the rate of - 1 degrees C/min is adopted in most of the cryopreservations, no data has been reported about the detailed effects of different cooling rates. The aim of the study was to reveal the different effects of cooling rates on cryopreservation of hematopoietic stem cells from cord blood. UCB samples were collected, and cryopreserved as mononuclear cells (MNC) with different cooling rates of - 0.5 degrees C/min, - 1 degrees C/min, - 5 degrees C/min, and the recovery and viability of MNC and CD34+ cells, the clonogenic capacity and the ex vivo expansion potential of UCB progenitor cells were evaluated after thawing. With - 1 degrees C/min cooling rate, the recovery of MNC reached 93.3% +/- 1.8% , viability 95.0% +/- 3.9% , recovery of CD34+ cells 80.0% +/- 17.9% , and clonogenic recovery were 87.1% +/- 5.5%, 88.5% +/- 8.9%, 86.2% +/- 7.4% for BFU-E CFU-GM CFU-MK, respectively. After 14 days of liquid culture, no significant difference was detected in CFC expansion between fresh and cryopreserved MNC cells with - 1 degrees C/min cooling rate, but this was not the case with - 0.5 degreesC/min and - 5 degrees C/min. In conclusion, it was demonstrated that controlling the rate at - 1 degrees C/min is more suitable for cryopreservation of hematopoietic stem cells than - 0.5 degrees C/min and - 5 degrees C/min.
Cell Survival ; physiology ; Cells, Cultured ; Cryopreservation ; methods ; Erythroid Precursor Cells ; cytology ; Fetal Blood ; cytology ; Flow Cytometry ; Granulocyte-Macrophage Progenitor Cells ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans

OBJECTIVETo explore the ex vivo expansion characteristics of selected CD(34)(+) cells and mononuclear cells (MNC).

METHODSCD(34)(+) cells were isolated from umbilical cord blood MNC by MiniMACS system, expanded under the same conditions as that for MNC. The effects of re-isolation and the MNC supernatant (MNC-SN) on the selected CD(34)(+) cells were investigated. And the CD(34)(-) cells of MNC were cultured ex vivo.

RESULTSIn the culture of selected CD(34)(+) cells, both the colony density and the proportion of the CD(34)(+) cells declined continuously with the culturing, although they presented a high proliferation potential. However, in the culture of the MNC, from day 0 to day 7, the colony density and the proportion of CD(34)(+) cells were increased from 412 +/- 167/10(5) cells and (1.12 +/- 0.42)% to 1 162 +/- 566/10(5) cells and (4.17 +/- 1.44)%, respectively. It was found that both the total cells and the CD(34)(+) cells restored expansion potential by re-isolating. CD(34)(-) cells of MNC had the ability to form colony and could transform to CD(34)(+) cells. MNC-SN can promote colony forming ability and lead to CD(34)(+) cells differentiation at the same time.

CONCLUSIONSIn ex vivo culture, selected CD(34)(+) cells presented a high proliferation and differentiation potentials, and the CD(34)(-) cells produced during the cultivation had inhibition effect on CD(34)(+) cells expansion. CD(34)(-) cell population from cord blood MNC contained hematopoietic stem/progenitor cells and the cytokines secreted by CD(34)(-) cells could induce CD(34)(+) cells to more mature colony-forming cells.

Antigens, CD34 ; analysis ; Cell Count ; Cell Differentiation ; immunology ; Cell Division ; immunology ; Cells, Cultured ; Colony-Forming Units Assay ; Fetal Blood ; cytology ; immunology ; Hematopoietic Stem Cells ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; Time Factors

OBJECTIVETo investigate the characteristics of inhibitory and activating receptor expressions on natural killer (NK) cells in HIV/HCV co-infected patients.

METHODSNumbers, frequencies and expressions of activating and inhibitory receptors of NK cells were measured with flow cytometry (FCS) from HIV/HCV co-infected group (n = 24), HCV mono-infected group (n = 34), HIV mono-infected group (n = 21) and healthy control group (HC, n = 20), then analysis and compare were performed among those groups.

RESULTSThe NK cell absolute counts in HIV/HCV group were significantly lower than those in other three groups. The NKP30 and NKP46 frequencies on NK cells in HIV/HCV, HIV and HCV groups were all significantly lower than those in HC group, but there were no significant differences of NKP30 among former three groups; and NKP46 frequencies in HIV/HCV and HIV groups were lower than those in HCV group, but there were no significant differences between former two groups. The NKG2A frequencies in HIV/HCV and HCV groups were all higher than those in HIV and HC groups significantly, but the NKG2A frequencies in HIV group were lower than those in HC group; There were no significant differences of NKG2D, CD158a and CD158b among those four groups.

CONCLUSIONNK cell numbers and expressions of activiting receptors on NK cells obviously decreased in HIV/HCV co-infected patients, but some inhibitory receptors expressions increased, even higher than those of HIV mono-infected patients. NK cells impairments in HIV/HCV co-infection is more severe than HIV or HCV mono-infection.

Adult ; Female ; Flow Cytometry ; HIV Infections ; genetics ; metabolism ; Hepatitis C ; genetics ; metabolism ; Humans ; Killer Cells, Natural ; metabolism ; Male ; Middle Aged ; NK Cell Lectin-Like Receptor Subfamily C ; genetics ; metabolism ; NK Cell Lectin-Like Receptor Subfamily K ; genetics ; metabolism ; Natural Cytotoxicity Triggering Receptor 1 ; genetics ; metabolism ; Natural Cytotoxicity Triggering Receptor 3 ; genetics ; metabolism ; Receptors, KIR2DL1 ; genetics ; metabolism ; Receptors, KIR2DL3 ; genetics ; metabolism ; Young Adult

Purpura fulminans is a serious condition that can result in severe morbidity in the pediatric population. Although autologous skin grafts remain the gold standard for the coverage of partial- to full-thickness wounds, they have several limitations in pediatric patients, including the lack of planar donor sites, the risk of hemodynamic instability, and the limited graft thickness. In Singapore, an in-house skin culture laboratory has been available since 2005 for the use of cultured epithelial autografts (CEAs), especially in burn wounds. However, due to the fragility of CEAs, negative-pressure wound therapy (NPWT) dressings have been rarely used with CEAs. With several modifications, we report a successful case of NPWT applied over a CEA in an infant who sustained 30% total body surface area full-thickness wounds over the anterior abdomen, flank, and upper thigh secondary to purpura fulminans. We also describe the advantages of using NPWT dressing over a CEA, particularly in pediatric patients.

Since the outbreak of Novel Coronavirus Pneumonia(NCP), hospitals have taken the fight against the virus as its own responsibility, and keep standing in the front line of epidemic prevention and control. The continuous input of anti-epidemic forces in hospitals also brings challenges to the medical supplies support, including the management of protective supplies and the maintenance of medical equipment. In the face of increasing security pressure, the medical materials support team broke the game on multiple fronts. Firstly, the team implements active material procurement strategy, sets material distribution priority according to risk level, releases materials uniformly based on stock and use, and implements traceability management of donated materials to ensure material supply. Secondly, centralized allocation management of equipment, emergency installation, advanced maintenance and emergency maintenance work is effectively completed. Thirdly, disinfection strategies for items and equipment are developed safely and effectively with the aid of disinfection equipment functions. At last, personnel management and training have been strengthened. These measures have provided strong support for the orderly prevention and control of the epidemic.

Purpura fulminans is a serious condition that can result in severe morbidity in the pediatric population. Although autologous skin grafts remain the gold standard for the coverage of partial- to full-thickness wounds, they have several limitations in pediatric patients, including the lack of planar donor sites, the risk of hemodynamic instability, and the limited graft thickness. In Singapore, an in-house skin culture laboratory has been available since 2005 for the use of cultured epithelial autografts (CEAs), especially in burn wounds. However, due to the fragility of CEAs, negative-pressure wound therapy (NPWT) dressings have been rarely used with CEAs. With several modifications, we report a successful case of NPWT applied over a CEA in an infant who sustained 30% total body surface area full-thickness wounds over the anterior abdomen, flank, and upper thigh secondary to purpura fulminans. We also describe the advantages of using NPWT dressing over a CEA, particularly in pediatric patients.

Vpr, an auxiliary protein of HIV-1(Human immunodeficiency virus type 1), exerts important functions to promote viral replication and AIDS progression. In this study, we performed a yeast two-hybrid screening assay using human cDNA library to further investigate the molecular mechanism of various functions of Vpr RelB, a key protein in NF-kappaB signaling pathway, was identified as a Vpr interaction protein by co-immunoprecipitation. Further investigations indicated that RelB not only promoted the Vpr-mediated activation of NF-kappaB reporter gene, but also enhanced the transactivation of HIV LTR. Moreover, the results showed that RelB promoted Vpr-induced cell cycle G2/M arrest. Collectively, these results indicated that RelB might interact with Vpr and regulate its transcriptional activation and cell cycle arrest.
Cell Cycle Checkpoints ; Cell Division ; G2 Phase ; HIV Long Terminal Repeat ; HeLa Cells ; Humans ; NF-kappa B ; genetics ; Transcription Factor RelB ; physiology ; Transcriptional Activation ; vpr Gene Products, Human Immunodeficiency Virus ; physiology