scholarly journals Infection of Human T Lymphocytes with Varicella-Zoster Virus: an Analysis with Viral Mutants and Clinical Isolates

2000 ◽  
Vol 74 (4) ◽  
pp. 1864-1870 ◽  
Author(s):  
Weily Soong ◽  
Julie C. Schultz ◽  
Andriani C. Patera ◽  
Marvin H. Sommer ◽  
Jeffrey I. Cohen
Keyword(s):  
T Cells ◽  
Cord Blood ◽  
Varicella Zoster Virus ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Mononuclear Cells ◽  
Clinical Isolates ◽  
In Vitro System ◽  
Varicella Zoster

ABSTRACT Varicella-zoster virus (VZV) disseminates in the body in peripheral blood mononuclear cells during chickenpox. Up to 1 in 10,000 mononuclear cells are infected during the viremic phase of the disease. We developed an in vitro system to infect human mononuclear cells with VZV by using umbilical cord blood. In this system, 3 to 4% of T cells were infected with VZV. VZV mutants unable to express certain genes, such as open reading frame 47 (ORF47) or ORF66, were impaired for growth in T cells, while other mutants showed little difference from parental virus. VZV unable to express ORF47 was even more impaired for spread from umbilical cord blood cells to melanoma cells in vitro. Early-passage clinical isolates of VZV infected T cells at a similar rate to the Oka vaccine strain; however, the clinical isolates were more efficient in spreading from infected T cells to melanoma cells. This in vitro system for infecting human T cells with VZV should be useful for identifying cellular and viral proteins that are important for virus replication in T cells and for the spread of virus from T cells to other cells.

Proliferation, differentiation, and cytokine secretion of human umbilical cord blood–derived mononuclear cells in vitro

2007 ◽  
Vol 35 (7) ◽  
pp. 1119-1131 ◽  
Author(s):  
Sandra Neuhoff ◽  
Janet Moers ◽  
Maike Rieks ◽  
Thomas Grunwald ◽  
Arne Jensen ◽  
...  
Keyword(s):  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Mononuclear Cells ◽  
Cytokine Secretion ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord

CD45−/ALDHlow/SSEA-4+/Oct-4+/CD133+/CXCR4+/Lin− Very Small Embryonic-Like (VSEL) Stem Cells Isolated from Umbilical Cord Blood as Potential Long Term Repopulating Hematopoietic Stem Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2444-2444
Author(s):  
Ewa K Zuba-Surma ◽  
Magdalena Kucia ◽  
Rui Liu ◽  
Mariusz Z Ratajczak ◽  
Janina Ratajczak
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Mononuclear Cells ◽  
Hematopoietic Stem ◽  
Hematopoietic Activity

Abstract Recently, we identified a population of very small embryonic-like (VSEL) stem cells in umbilical cord blood (CB) (Leukemia2007;21:297–303) These VSELs are: smaller than erythrocytes; SSEA-4+/Oct-4+/CD133+/CXCR4+/Lin−/CD45−; responsive to SDF-1 gradient; and iv) possessing large nuclei that contain unorganized chromatin (euchromatin). Data obtained in a murine model indicate that a similar cell population isolated from bone marrow (BM) does not reveal hematopoietic activity after isolation. However, in appropriate models (i.e., in vitro co-culture over OP-9 cells or in vivo after intra bone injection), these cells contribute to hematopoiesis and thus possesses potential of long term repopulating hematopoietic stem cells (LT-HSCs). To investigate the hematopoietic activity of CB-derived, CD45 negative VSELs, we employed staining with Aldefluor detecting aldehyde dehydrogenase (ALDH), the enzyme expressed in primitive hematopoietic cells. We sorted CD133+/CD45−/ALDHhigh and CD133+/CD45−/ALDHlow sub-fractions of VSELs from CB samples and established that both freshly sorted CB-derived populations did not grow hematopoietic colonies in vitro. However, when activated/expanded over OP-9 stroma cells, they exhibit hematopoietic potential and initiate hematopoietic colonies composed of CD45+ cells when replated into methylcellulose cultures. Furthermore, while CD133+/CD45−/ALDHhigh VSELs gave raise to hematopoietic colonies after the first replating, the formation of colonies by CD133+/CD45−/ALDHlow VSELs was delayed. The data indicate that both populations of CD45− cells may acquire hematopoietic potential; however hematopoietic specification is delayed for CD133+/CD45−/ALDHlow cells (Fig. 1A). In parallel, real time PCR analysis revealed that freshly isolated CD133+/CD45−/ALDHhigh VSELs express more hematopoietic transcripts (e.g., c-myb, 80.2±27.4 fold difference) while CD133+/CD45−/ALDHlow exhibit higher levels of pluripotent stem cell markers (e.g., Oct-4, 119.5±15.5 fold difference) as compared to total CB mononuclear cells (Fig. B). Furthermore and somewhat unexpectedly, we found that because of their unusually small size, these important cells may be partially depleted (in 42.5±12.6%) during standard preparation strategies of CB units for storage that employ volume reduction. In conclusion, our data suggest very small CB mononuclear cells expressing VSEL markers that are CD133+/CD45−/ALDHlow are highly enriched for the most primitive population of LT-HSCs. These cells may be responsible for long term CB engraftment and be a population of cells from which HSCs should be expanded. We are currently testing this in an in vivo model by performing heterotransplants of CD45− ALDHlow VSELs into immunodeficient mice. It is important to stress that currently employed, routine CB processing strategies may lead up to ~50% loss of these small cells that are endowed with such remarkable hematopoietic activity. Figure Figure

Specific Regulation of NFAT (Nuclear Factors of Activated T Cells) Expression in CD34+ Cells Differentiating into Diverse Hematopoietic Lineages.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4213-4213
Author(s):  
Alexander Kiani ◽  
Hanna Kuithan ◽  
Friederike Kuithan ◽  
Satu Kyttaelae ◽  
Ivonne Habermann ◽  
...  
Keyword(s):  
T Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Peripheral Blood ◽  
Family Members ◽  
Specific Pattern ◽  
Activated T Cells ◽  
Cd34 Cells

Abstract NFAT (Nuclear Factor of Activated T cells) transcription factors are a family of five proteins that are primarily known for their central role in the regulation of inducible gene expression in activated T cells. It is now clear that NFAT proteins are also expressed in various non-immune cell types, where they regulate the expression of genes involved in such diverse cellular processes as proliferation, apoptosis and differentiation. We have previously shown that NFATc2 is strongly expressed in human CD34+ cells and megakaryocytes, but not in purified peripheral blood neutrophil granulocytes and monocytes. Furthermore, granulocytic differentiation of CD34+ cells in vitro was paralleled by the rapid and profound suppression of NFATc2 mRNA and protein. The function of NFATc2 in CD34+ cells, however, is unknown, and no information exists on the expression or regulation of other NFAT family members in CD34+ cells or during heamtopoietic differentiation. To provide a systematic basis for further functional analysis, we established in the present study a comprehensive expression profile of all five NFAT family members in CD34+ cells and during their in vitro differentiation into neutrophil, eosinophil, erythroid and megakaryocytic lineages. CD34+ cells were purified from umbilical cord blood and cultured in the presence of cytokines or cytokine combinations inducing differentiation of the respective lineages. At several time-points during the culture, the efficacy and specificity of the differentiation was monitored by morphological examination of cytospin preparations as well as by analysis of lineage-specific cell surface markers. By quantitative RT-PCR, NFATc3 and NFAT5 were the NFAT family member found to be most prominently expressed in CD34+ cells of both peripheral blood and umbilical cord blood, as well as in the immature CD34+CD38− subpopulation of cells. NFAT expression during the differentiation of umbilical cord blood CD34+ cells into the diverse hematopoietic lineages followed a family member- and lineage-specific pattern. Neutrophil differentiation was accompanied by a rapid suppression of transcript level for all NFAT family members. In contrast, eosinophil, erythrocyte and megakaryocyte differentiation was paralleled by an upregulation of NFATc3, NFATc1/NFATc3 and NFATc1 mRNA, respectively. The most obvious lineage-specific pattern was observed for NFATc4, where transcript levels were low in CD34+ cells and either not or only transiently increased in neutrophil, eosinophil and erythrocyte differentiation; in contrast, they were specifically upregulated about 10-fold in the megakaryocytic lineage. The expression profile of NFAT family members in developing hematopoietic cells of diverse lineages presented here will allow predicting and directly assessing the role of individual NFAT family members in hematopoietic differentiation.

scholarly journals Toxicological profile of umbilical cord blood-derived small extracellular vesicles

2021 ◽  
Author(s):  
Silvia C Rodrigues ◽  
Renato M S Cardoso ◽  
Claudia F Gomes ◽  
Filipe V Duarte ◽  
Patricia C Freire ◽  
...  
Keyword(s):  
Cord Blood ◽  
Umbilical Cord ◽  
Extracellular Vesicles ◽  
Umbilical Cord Blood ◽  
Mononuclear Cells ◽  
High Dose ◽  
Cell Therapies ◽  
Cell Counts ◽  
Blood Mononuclear Cells

The development and adoption of cell therapies has been largely limited by difficulties associated with their safety, handling and storage. Extracellular vesicles (EV) have recently emerged as a likely mediator for the therapeutic effect of cells, offering several advantages over cell therapies. Due to their small size and inability to expand and metastasize, EV are generally considered safer than cell transplantation. Nevertheless, few studies have scrutinized the toxicity profile of EV, particularly after repeated high dose administration. The present study aimed to evaluate a preparation of small EV obtained from umbilical cord blood mononuclear cells (UCB-MNC-sEV) for its cytotoxicity in different cell lines, as well as its differential accumulation, distribution and toxicity following repeated intravenous (IV) administrations in a rodent model. In vitro, repeated sEV exposure in concentrations up to 1x10^11 particles/ml had no deleterious impact on the viability or metabolic activity of peripheral blood mononuclear cells, THP-1 monocytes, THP-1-derived macrophages, normal dermal human fibroblasts or human umbilical vein endothelial cells. DiR-labeled sEV, injected IV for four weeks in healthy rats, were detected in clearance organs, particularly kidneys, spleen and liver, similarly to control dye. Moreover, repeated administrations during six and twelve weeks of up to 1x10^10 total particles of sEV-dye were well tolerated, with no changes in general hematological cell counts, or kidney and liver toxicity markers. Importantly, unlabeled sEV likewise did not induce significant alterations in cellular and biochemical blood parameters, nor any morphological changes in heart, kidney, lung, spleen, or liver tissue. In sum, our data shows that UCB-MNC-sEV have no significant toxicity in vitro or in vivo, even when administered repeatedly at high concentrations, therefore confirming their safety profile and potential suitability for future clinical use.

scholarly journals Toxicological Profile of Umbilical Cord Blood-Derived Small Extracellular Vesicles

Membranes ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 647 ◽  
Author(s):  
Silvia C. Rodrigues ◽  
Renato M. S. Cardoso ◽  
Claudia F. Gomes ◽  
Filipe V. Duarte ◽  
Patricia C. Freire ◽  
...  
Keyword(s):  
Cord Blood ◽  
Umbilical Cord ◽  
Extracellular Vesicles ◽  
Umbilical Cord Blood ◽  
Mononuclear Cells ◽  
High Dose ◽  
Cell Therapies ◽  
Cell Counts ◽  
Blood Mononuclear Cells

The development and adoption of cell therapies has been largely limited by difficulties associated with their safety, handling, and storage. Extracellular vesicles (EV) have recently emerged as a likely mediator for the therapeutic effect of cells, offering several advantages over cell therapies. Due to their small size and inability to expand and metastasize, EV are generally considered safer than cell transplantation. Nevertheless, few studies have scrutinized the toxicity profile of EV, particularly after repeated high-dose administration. The present study aimed to evaluate a preparation of small EV obtained from umbilical cord blood mononuclear cells (UCB-MNC-sEV) for its cytotoxicity in different cell lines, as well as its differential accumulation, distribution, and toxicity following repeated intravenous (IV) administrations in a rodent model. In vitro, repeated sEV exposure in concentrations up to 1 × 1011 particles/mL had no deleterious impact on the viability or metabolic activity of peripheral blood mononuclear cells, THP-1 monocytes, THP-1-derived macrophages, normal dermal human fibroblasts, or human umbilical vein endothelial cells. DiR-labelled sEV, injected intravenously for four weeks in healthy rats, were detected in clearance organs, particularly the kidneys, spleen, and liver, similarly to control dye. Moreover, repeated administrations for six and twelve weeks of up to 1 × 1010 total particles of sEV dye were well-tolerated, with no changes in general haematological cell counts, or kidney and liver toxicity markers. More importantly, unlabelled sEV likewise did not induce significant alterations in cellular and biochemical blood parameters, nor any morphological changes in the heart, kidney, lung, spleen, or liver tissue. In sum, our data show that UCB-MNC-sEV have no significant toxicity in vitro or in vivo, even when administered repeatedly at high concentrations, therefore confirming their safety profile and potential suitability for future clinical use.

scholarly journals In Vitro Induction Of Adenovirus Specific T-cells In Response To 15-mer Peptides In Umbilical Cord Blood

2010 ◽  
Vol 16 (2) ◽  
pp. S159-S160
Author(s):  
L.M. Haveman ◽  
M. Bulatovic ◽  
J.M. Meerding ◽  
B. Prakken ◽  
M. Bierings
Keyword(s):  
T Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
In Vitro Induction

Cytotoxic T Lymphocytes (CTL) Specific for Adenovirus and CMV Can Be Generated from Umbilical Cord Blood for Adoptive Immunotherapy

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3505-3505
Author(s):  
Catherine M. Bollard ◽  
Patrick Hanley ◽  
Conrad Russell Cruz ◽  
Ann M. Leen ◽  
Jeffrey J. Molldrem ◽  
...  
Keyword(s):  
T Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Adoptive Immunotherapy ◽  
Immune Reconstitution ◽  
Mononuclear Cells ◽  
Cell Transplant ◽  
Hematopoietic Stem ◽  
Promising Alternative

Abstract Umbilical cord blood (UCB) transplantation is a promising alternative source of hematopoietic stem cells for patients lacking HLA-matched donors. Nearly 60% of UCB transplants to date have been performed on minority individuals for whom an unrelated donor was not available; moreover, the naïve phenotype of UCB cells may be responsible for the lower incidence and reduced severity of GvHD in these patients. The relatively low cell numbers and naïvety of T lymphocyte populations in UCB grafts has, however, lead to delayed immune reconstitution and higher mortality due to infection. Reactivations of latent viruses such as cytomegalovirus (CMV) are particularly problematic, as is overt infection from adenovirus (Adv). Previous studies have shown that prophylactic adoptive immunotherapy with peripheral blood-derived CTL directed against CMV and Adv can effectively prevent the clinical manifestations of these viruses after hematopoietic stem cell transplant raising the possibility that a similar approach could be developed after UCB transplant. We hypothesized that virus-specific CTL could be generated from UCB for clinical use to restore anti-viral immunity and reduce viral infection post UCB transplant. Bi-virus specific CTL were generated from frozen UCB mononuclear cells using a clinical-grade recombinant adenovirus type5 vector pseudotyped with a type35 fiber carrying a transgene for CMVpp65 as a source of Adv and CMV antigens. UCB-derived dendritic cells were transduced with this Ad5f35pp65 vector as the initial source of antigen presenting cells to stimulate virus-specific CTL in the presence of IL-7, IL-12 and IL-15. This was followed by 2 rounds of weekly stimulation with autologous UCB-derived EBV-lymphoblastoid cell lines (LCL) transduced with the same vector in the presence of IL-15 and IL-2. UCB from donors of varied HLA types were selected. 40×106 UCB mononuclear cells (available in the 20% fraction of frozen UCB units) were thawed and used in the manufacturing process. After 3 rounds of stimulation, 9 CTL cultures contained a mean of 83% (range 64–94%) CD8+ve T-cells and 27% (range 12–40%) CD4+ve T-cells. Flow cytometric analysis of memory markers after 3 weeks expansion revealed a predominance of CD45RA− CD62L− T-cells (69±18%; range 25–93%) with a smaller population of CD45RA− CD62L+ T-cells (10±5%; range 1–23%). Evaluable UCB CTL lines showed specific cytolytic activity in 51Cr release assays against targets loaded with CMV and Adv antigens. The observed cytotoxicity was specific because unloaded targets and MHC-mismatched targets were not killed. IFNγ ELISPOT assays on CTL lines demonstrated a mean of 209 (range 45–694) and 74 (range 0–188) spot forming cells/1×105 T-cells following incubation with CMV-pp65 and Adv-hexon/penton peptides respectively. No significant response to CMV-IE1 peptides was demonstrated. The expanded UCB CTL had a broad Vβ repertoire and were specific for multiple viral epitopes. In addition, the virus-specific T cells were shown to be expanded only from T-cells with a naïve phenotype (CD45RA+/CCR7+). These results demonstrate that, despite the generally naïve nature of UCB lymphocytes, bi-virus-specific responses can be expanded in vitro and could potentially be used clinically in UCBT patients who develop infectious complications prior to immune reconstitution.

Sign in / Sign up

Export Citation Format

Share Document