Summary Like mesenchymal stem cells from bone marrow (BM‐MSCs), adipose tissue‐derived adult stem cells (ADAS cells) can differentiate into several lineages and present therapeutical potential for repairing damaged tissues. The use of allogenic stem cells can enlarge their therapeutical interest, provided that the grafted cells could be tolerated. We investigate here, for the first time, the immunosuppressive properties of ADAS cells compared with the well‐characterized immunosuppressive properties of BM‐MSCs. ADAS cells did not provoke in vitro alloreactivity of incompatible lymphocytes and, moreover, suppressed mixed lymphocyte reaction (MLR) and lymphocyte proliferative response to mitogens. The impairment of inhibition when ADAS cells and BM‐MSCs were separated from lymphocytes by a permeable membrane suggests that cell contact is required for a full inhibitory effect. Hepatocyte growth factor is secreted by both stem cells but, similar to interleukin‐10 and transforming growth factor‐β (TGF‐β), the levels of which were undetectable in supernatants of MLR inhibited by ADAS cells or BM‐MSCs, it did not seem implicated in the stem cell suppressive effect. These findings support that ADAS cells share immunosuppressive properties with BM‐MSCs. Therefore, ADAS cell‐based reconstructive therapy could employ allogenic cells and because of their immunosuppressive properties, ADAS cells could be an alternative source to BM‐MSCs to treat allogenic conflicts.
Background In the high-prevalence setting of Pakistan, screening, diagnosis and treatment services for chronic hepatitis C (CHC) patients are commonly offered in specialized facilities. We aimed to describe the cascade of care in a Médecins Sans Frontières primary health care clinic offering CHC care in an informal settlement in Karachi, Pakistan. Methods This was a retrospective cohort analysis using routinely collected data. Three different screening algorithms were assessed among patients with one or more CHC risk factors. Results Among the 87 348 patients attending the outpatient clinic, 5003 (6%) presented with one or more risk factors. Rapid diagnostic test (RDT) positivity was 38% overall. Approximately 60% of the CHC patients across all risk categories were in the early stage of the disease, with an aspartate aminotransferase:platelet ratio index score <1. The sequential delays in the cascade differed between the three groups, with the interval between screening and treatment initiation being the shortest in the cohort tested with GeneXpert onsite. Conclusions Delays between screening and treatment can be reduced by putting in place more patient-centric testing algorithms. New strategies, to better identify and treat the hidden at-risk populations, should be developed and implemented.
We have used laser-assisted confocal microscopy to evaluate the intracellular distribution of daunorubicin (DNR) in acute myeloid leukemia (AML) cell lines and fresh AML cells according to their differentiation phenotype. In KG1a, KG1, TF-1 and HEL cells, which express the early differentiation marker CD34, DNR was distributed in perinuclear vesicles which could be associated with the Golgi apparatus, as suggested by the distribution of fluorescent probes specific for intracellular organelles. In contrast, U937 and HL-60 cells, which display a more mature phenotype, exhibited nuclear and diffuse cytoplasmic DNR fluorescence. DNR sequestration was not correlated with P-glycoprotein (P-gp) or multidrug resistance protein expression. Furthermore, PSC833, a potent P-gp blocker, had little effect on drug sequestration in CD34 1 AML cells. We also tested the effect of metabolic inhibitors, cytoskeleton inhibitors and carboxy-ionophores on DNR distribution in both CD34 2 and CD34 1 AML cells. However, only non-specific metabolic inhibitors restored nucleic/cytoplasmic distribution in CD34 1 cells. In these cells, the intracellular distribution of doxorubicin and idarubicin was very similar to that of DNR, while the distribution of methoxymorpholinyl-doxorubicin was nuclear and diffusely cytoplasmic. In fresh AML cells, DNR was also concentrated in the perinuclear region in CD34 1 but not in CD34 2 cells. However, DNR sequestration was not observed in normal CD34 1 cells. Finally, our results show that DNR is sequestered in organelles in CD34 1 AML cells via an active mechanism which appears to be different from P-gp-mediated transport. Abnormal DNR distribution may account for the natural resistance of immature AML cells to anthracyclines.
We have used laser-assisted confocal microscopy to evaluate the intracellular distribution of daunorubicin (DNR) in acute myeloid leukemia (AML) cell lines and fresh AML cells according to their differentiation phenotype. In KG1a, KG1, TF-1 and HEL cells, which express the early differentiation marker CD34, DNR was distributed in perinuclear vesicles which could be associated with the Golgi apparatus, as suggested by the distribution of fluorescent probes specific for intracellular organelles. In contrast, U937 and HL-60 cells, which display a more mature phenotype, exhibited nuclear and diffuse cytoplasmic DNR fluorescence. DNR sequestration was not correlated with P-glycoprotein (P-gp) or multidrug resistance protein expression. Furthermore, PSC833, a potent P-gp blocker, had little effect on drug sequestration in CD34 1 AML cells. We also tested the effect of metabolic inhibitors, cytoskeleton inhibitors and carboxy-ionophores on DNR distribution in both CD34 2 and CD34 1 AML cells. However, only non-specific metabolic inhibitors restored nucleic/cytoplasmic distribution in CD34 1 cells. In these cells, the intracellular distribution of doxorubicin and idarubicin was very similar to that of DNR, while the distribution of methoxymorpholinyl-doxorubicin was nuclear and diffusely cytoplasmic. In fresh AML cells, DNR was also concentrated in the perinuclear region in CD34 1 but not in CD34 2 cells. However, DNR sequestration was not observed in normal CD34 1 cells. Finally, our results show that DNR is sequestered in organelles in CD34 1 AML cells via an active mechanism which appears to be different from P-gp-mediated transport. Abnormal DNR distribution may account for the natural resistance of immature AML cells to anthracyclines.
This study was designed to correlate the clonogenic capacity of acute myeloid leukemia (AML) cells with P-glycoprotein (Pgp) expression level and P-gp-mediated efflux capacity. Fifty AML cell samples were tested for P-gp expression using MRK16 monoclonal antibody and flow cytometry. Among them, 12 samples were selected for sorting experiments according to the following two criteria: their clonogenic capacity in methylcellulose in the presence of 5637 conditioned medium, and the heterogeneity of P-gp distribution in leukemic cells. For each of these 12 samples, leukemic cells which displayed the highest P-gp expression level (P-gp ؉؉ ) and P-gp ؊ leukemic cells were sorted after MRK16 staining and seeded into methylcellulose for primary clonogenic assay. In each case, the number of CFU-L in the P-gp − fraction was significantly higher than that of the P-gp dull and Rh 123 bright populations, respectively. Altogether this study suggests that, for an individual AML cell population, the clonogenic fraction is preferentially recruited in AML cells which display low P-gp expression and high P-gp-mediated efflux capacity.
Background National healthcare financing strategy recommends tax-based equity funds and insurance schemes for the poor and extreme poor living in urban slums and pavements as the majority of these population utilise informal providers resulting in adverse health effects and financial hardship. We assessed the effect of a health voucher scheme (HVS) and micro-health insurance (MHI) scheme on healthcare utilisation and out-of-pocket (OOP) payments and the cost of implementing such schemes. Methods HVS and MHI schemes were implemented by Concern Worldwide through selected NGO health centres, referral hospitals, and private healthcare facilities in three City Corporations of Bangladesh from December 2016 to March 2020. A household survey with 1,294 enrolees, key-informant interviews, focus group discussions, consultative meetings, and document reviews were conducted for extracting data on healthcare utilisation, OOP payments, views of enrolees, and suggestions of implementers, and costs of services at the point of care. Results Healthcare utilisation including maternal, neonatal and child health (MNCH) services, particularly from medically trained providers, was higher and OOP payments were lower among the scheme enrolees compared to corresponding population groups in general. The beneficiaries were happy with their access to healthcare, especially for MNCH services, and their perceived quality of care was fair enough. They, however, suggested expanding the benefits package, supported by an additional workforce. The cost per beneficiary household for providing services per year was €32 in HVS and €15 in MHI scheme. Conclusion HVS and MHI schemes enabled higher healthcare utilisation at lower OOP payments among the enrolees, who were happy with their access to healthcare, particularly for MNCH services. However, they suggested a larger benefits package in future. The provider’s costs of the schemes were reasonable; however, there are potentials of cost containment by purchasing the health services for their beneficiaries in a competitive basis from the market. Scaling up such schemes addressing the drawback would contribute to achieving universal health coverage.
In this study, we evaluated the individual in vitro sensitivity of fresh acute myeloid leukemia (AML) cells to VP-16, and attempted to correlate VP-16 cytotoxicity with AML cell growth characteristics and drug-induced DNA single-strand breaks (SSB). Primary (PE1) colony inhibition assays allowed us to characterize two distinct groups of AML: group I (patients 1 through 6), which displayed sensitivity to VP- 16 similar to that of normal CFU-GM (IC90 of 20.52 +/- 2.44 micrograms/mL v 20.48 +/- 2.23 micrograms/mL after 1 hour drug exposure, respectively); and group II (patients 7 through 11), which was more sensitive to VP-16 (IC90 of 7.26 +/- 2.93 micrograms/mL, P = .004). Subsequently, groups I and II were termed normosensitive and hypersensitive, respectively. This objective VP-16 sensitivity classification, as determined by PE1, remained unaltered when assessed by secondary (PE2) colony inhibition assay (evaluating the self-renewal fraction of AML progenitors), or by cytofluorometric viability assay (evaluating the ultimately differentiated blast cell population). These findings would suggest that individual sensitivity to VP-16 of a particular cell population is maintained throughout CFU-AML differentiation. Finally, we report that sensitivity of AML cells to VP- 16 did not correlate either with cell growth characteristics or with SSB generation. Indeed, AML cell sensitivity to VP-16 appeared more closely related to DNA repair kinetics after drug removal, ie, hypersensitivity being essentially characterized by a prolonged retention of SSB during the posttreatment period. Interestingly, the established HL-60 cell line, which presented greater sensitivity to VP- 16 cytotoxicity than KG1, HEL, and K562, was also found to exhibit delayed DNA SSB repair kinetics, as compared with the other AML cell lines. These results suggest that hypersensitivity to VP-16 of some AML cells may be related to a deficient DNA-repair mechanism.
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