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POINTS CLÉSLa prise en charge de la tuberculose (TB) chez les personnes âgées, enceintes ou aux prises avec des maladies concomitantes ou des troubles liés à la consommation de substances présente des défis; on recommande donc de consulter un expert en tuberculose.Les événements indésirables et les interruptions de traitement sont plus fréquents dans ces populations particulières que dans la population générale; une surveillance étroite et un soutien additionnel sont souvent nécessaires.Étant donné les modifications de la pharmacocinétique des médicaments antituberculeux et le potentiel de graves interactions médicament-médicament chez les patients présentant des maladies concomitantes, nous recommandons de consulter un pharmacien expérimenté.La TB active et ses traitements ont d’importantes répercussions sur la prise en charge des maladies concomitantes; une étroite collaboration avec des médecins spécialistes et des professionnels paramédicaux est souvent requise.La durée prolongée du traitement antituberculeux est une bonne occasion de dépister des maladies concomitantes et de faciliter l’orientation et les liens vers les soins pertinents.

SommaireLa prépondérance des données indique qu’un traitement approprié rend rapidement non contagieuses les personnes atteintes de tuberculose (TB), voire au bout d’environ quelques jours suivant le début du traitement, même pour les cas initialement positifs au frottis.Les études indiquent également que le résultat du frottis d’expectorations et de la culture est moins révélateur de la contagiosité, une fois que les patients reçoivent un traitement efficace.Il subsiste cependant des incertitudes quant au moment précis où les personnes qui reçoivent un traitement antituberculeux ne sont plus du tout contagieuses.L’insistance concernant la négativation du frottis avant de lever les précautions contre la transmission par voie aérienne pourrait prolonger inutilement l’isolement, être préjudiciable au patient et avoir peu d’avantages sur le plan de la santé publique.Pour les personnes atteintes de tuberculose dont l’état de santé est suffisamment bon, un traitement ambulatoire et un isolement à domicile doivent être préférables à un isolement prolongé en milieu hospitalier. Cela permettra d’atténuer au moins certains des inconvénients de l’isolement prolongé.

Canada has accepted more than 25,000 Syrian refugees fearing persecution in their homeland. Canadian guidelines recommend screening recent refugees from high incidence countries for tuberculosis (TB) and latent TB infection (LTBI). The prevalence of TB in this population is unknown. A refugee clinic in Edmonton presented a unique opportunity to estimate prevalence of TB and LTBI in Syrian refugees arriving in Canada. In January 2016, 100 consecutive Syrian refugees were screened for TB with clinical assessment and LTBI using QuantiFERON Gold In-Tube assay (QFT-GIT). Patients with positive QFT-GIT were referred to Edmonton Tuberculosis Clinic (ETBC) for evaluation and, if appropriate, offered prophylaxis. No cases of active TB were found. Valid QFT-GIT were measured in 99 of 100 individuals and of these, nine (9%) were positive using a threshold concentration of blood interferon-γ greater than 0.35 IU/L. Eight of the nine patients attended follow-up appointments; of these, seven began LTBI prophylaxis and all seven (78%) completed same. The 9% (95% confidence interval 3–15%) prevalence of LTBI was higher than expected in this population. Le Canada a accepté plus de 25 000 réfugiés syriens fuyant la persécution dans leur patrie. Les lignes directrices canadiennes recommandent le dépistage de la tuberculose et de la tuberculose-infection latente (TBIL) chez les réfugiés récents des pays à forte incidence. La prévalence de la tuberculose dans cette population est inconnue. Une clinique pour les réfugiés d’Edmonton a représenté une occasion unique d’estimer la prévalence de la tuberculose et de la TBIL chez les réfugiés syriens arrivant au Canada. En janvier 2016, 100 réfugiés syriens de suite ont subi des tests de dépistage de la tuberculose avec évaluation clinique et de dépistage de la TBIL à l’aide du test QuantiFERON Gold In-Tube Assay (QFT-GIT). Les patients séropositifs selon le QFT-GIT ont subi une évaluation à la clinique de tuberculose d’Edmonton (ETBC) et, le cas échéant, se sont fait offrir une prophylaxie. Aucun cas de tuberculose active n’a été décelé. Des épreuves QFT-GIT valides ont été réalisées chez 99 personnes sur 100, et neuf d’entre elles (9 %) ont affiché des résultats positifs, soit un seuil de concentration supérieur à 0,35 UI/l selon le test sanguin interféron gamma. Huit patients sur neuf se sont présentés aux rendez-vous de suivi, sept ont commencé un traitement contre la TBIL en prophylaxie, et tous les sept (78 %) l’ont terminé. La prévalence de 9 % (intervalle de confiance de 95 %: 3–15 %) de la TBIL était plus élevée que prévu dans cette population.

Thermal barrier coating (TBC) resistance to varied CMAS deposition rates was experimentally assessed using a temperature gradient rig using a CMAS precursor spray. TBCs were composed of EB-PVD YSZ. Five different deposition rates were tested quantifying life versus deposition rate. A high deposition rate and low deposition rate were selected from those tests for sequence testing. For sequence effect testing CMAS deposition occurred at the initial rate until the expected half-life and then switched to the other rate until failure, in the order of high deposition rate first followed by low deposition rate and vice versa. The results suggest that the order of deposition rates can affect ultimate failure behavior. Specifically applying the CMAS at the low deposition rate extended the life of the sample by a nearly a factor of 2 compared to a virgin sample. At the high deposition rate, the CMAS infiltrated all the way to the bond coat past the estimated freeze line. Data showing the coating life versus CMAS deposition rate and the existence of sequence effects in CMAS dosing are fundamentally important to coating life prediction.

Next-generation sequencing (NGS) is essential to the care of patients with NSCLC. Nevertheless, NGS is dependent on adequate material from biopsy. We evaluated the impact of biopsy method and needle gauge necessary for optimizing success in tissue NGS.

We investigated the effects of circle hook size and angling technique on hooking location, capture success, and postrelease mortality in salmonids by using hatchery-reared Rainbow Trout Oncorhynchus mykiss. Fish were caught using a range of inline circle hook sizes (4, 6, 8, and 10) baited and fished both actively and passively. While hook size had no influence on capture rates, active fishing resulted in significantly higher capture rates. Very few mortalities (2%) were observed across angling methods or hook sizes during 72 h of postrelease monitoring. However, significantly more fish were deeply hooked with passive fishing. Contrary to claims by manufacturers and sportfishing entities, mounting evidence indicates that passively fished circle hooks have lower capture rates and might actually increase deep hooking in salmonids. Received May 19, 2015; accepted November 12, 2015 Published online March 21, 2016

We compared the effectiveness of baited circle hooks and J hooks in catch-and-release angling for wild, stream-dwelling Brook Trout Salvelinus fontinalis. We caught 201 Brook Trout using active and passive techniques to assess the influence of hook type and angling method on capture efficiency, hooking location, handling time, deep-hooking rates, and postrelease mortality (up to 48 h). Anglers recorded an average catch rate of 7.6 trout/h over 26.25 total hours of fishing. Actively fishing with J hooks was the most effective at landing Brook Trout (82.3%). Only 10% (n= 20) of all fish were deeply hooked, and half of those were caught when anglers were passively fishing with J hooks. Larger fish were significantly more likely to be deeply hooked. However, no fish were deeply hooked when anglers passively fished with circle hooks. The most common hooking location among treatments was the corner of the mouth (40%, n= 80). Eye injuries due to hooking were relatively uncommon (6.5%) and occurred less frequently than deep hooking. Overall mortality rates were very low (2%, n= 4) and were probably influenced by low deep-hooking rates in concert with cool water temperatures and experienced anglers equipped with the necessary tools to efficiently release the trout. Received May 19, 2015; accepted November 12, 2015 Published online March 16, 2016

AbstractWe compared the effectiveness of baited circle hooks and J hooks in catch-and-release angling for wild, stream-dwelling Brook Trout Salvelinus fontinalis. We caught 201 Brook Trout using active and passive techniques to assess the influence of hook type and angling method on capture efficiency, hooking location, handling time, deep-hooking rates, and postrelease mortality (up to 48 h). Anglers recorded an average catch rate of 7.6 trout/h over 26.25 total hours of fishing. Actively fishing with J hooks was the most effective at landing Brook Trout (82.3%). Only 10% (n= 20) of all fish were deeply hooked, and half of those were caught when anglers were passively fishing with J hooks. Larger fish were significantly more likely to be deeply hooked. However, no fish were deeply hooked when anglers passively fished with circle hooks. The most common hooking location among treatments was the corner of the mouth (40%, n= 80). Eye injuries due to hooking were relatively uncommon (6.5%) and occurred less frequently than deep hooking. Overall mortality rates were very low (2%, n= 4) and were probably influenced by low deep-hooking rates in concert with cool water temperatures and experienced anglers equipped with the necessary tools to efficiently release the trout.Received May 19, 2015; accepted November 12, 2015 Published online March 16, 2016

AbstractWe investigated the effects of circle hook size and angling technique on hooking location, capture success, and postrelease mortality in salmonids by using hatchery-reared Rainbow Trout Oncorhynchus mykiss. Fish were caught using a range of inline circle hook sizes (4, 6, 8, and 10) baited and fished both actively and passively. While hook size had no influence on capture rates, active fishing resulted in significantly higher capture rates. Very few mortalities (2%) were observed across angling methods or hook sizes during 72 h of postrelease monitoring. However, significantly more fish were deeply hooked with passive fishing. Contrary to claims by manufacturers and sportfishing entities, mounting evidence indicates that passively fished circle hooks have lower capture rates and might actually increase deep hooking in salmonids.Received May 19, 2015; accepted November 12, 2015 Published online March 21, 2016

Graphene is an ideal candidate for lightweight, high-strength composite materials given its superior mechanical properties (specific strength of 130 GPa and stiffness of 1 TPa). To date, easily scalable graphene-like materials in a form of separated flakes (exfoliated graphene, graphene oxide, and reduced graphene oxide) have been investigated as candidates for large-scale applications such as material reinforcement. These graphene-like materials do not fully exhibit all the capabilities of graphene in composite materials. In the current study, we show that macro (2 inch × 2 inch) graphene laminates and fibers can be produced using large continuous sheets of single-layer graphene grown by chemical vapor deposition. The resulting composite structures have potential to outperform the current state-of-the-art composite materials in both mechanical properties and electrical conductivities (>8 S/cm with only 0.13% volumetric graphene loading and 5 × 103S/cm for pure graphene fibers) with estimated graphene contributions of >10 GPa in strength and 1 TPa in stiffness.

We surveyed large wood volumes in relation to the distribution and density of rainbow trout Oncorhynchus mykissand steelhead (anadromous rainbow trout) in 15 stream reaches in the upper Salinas River watershed, California, which represents the southern end of the species’ range. The main tree species contributing to large wood were hardwoods: coast live oak Quercus agrifolia, California sycamore Platanus racemosa, red willow Salix laevigata, and valley oak Q. lobata. Large wood jams were important in pool formation and typically had red willow as their key pieces. Temperatures were exceptionally warm during the study period. No steelhead were observed at sites where the mean water temperature exceeded 21.5°C or the maximum water temperature exceeded 26°C. The combined importance of high temperatures and large wood on the distribution and abundance of southern steelhead indicates that suitable habitat may be reduced if climate change continues on its present course of warming and the frequency of fires increases.

AbstractThe introduction of potent combination antiretroviral therapy (ART) in the treatment of HIV infection has permitted reliable control of disease progression and has markedly improved survival among people with HIV. As a result, health care providers and patients have shifted clinical priorities; whereas once delaying opportunistic illness was a primary focus, increasing emphasis is now placed on preventative health, management of comorbid chronic disease and avoiding long-term toxicities of ART. Although renal disease is common in people with HIV, renal disease specifically due to ART remains relatively rare. Still, as the use of ART continues to increase, health care providers are likely to encounter this potentially serious complication with increasing frequency. Distinguishing ART-related nephrotoxicity from the myriad of other potential causes of renal disease in people with HIV is important in order to avoid unnecessary discontinuation of an appropriate ART regimen. This review focuses on the early recognition of renal disease associated with ART and suggests strategies for management and prevention.Copyright © 2011 S. Karger AG, Basel

To improve the purity of lentiviral vector supernatants for clinical studies we have evaluated plasmid DNA removal from lentiviral vectors and also the extent of plasmid DNA associated with transduced CD34 cells in an ex vivo transduction protocol. Optimal conditions of plasmid DNA removal by benzonase treatment were established by varying the temperature, time, and benzonase concentrations in the reaction mix and were determined to be 50 units of benzonase per milliliter of vector supernatant at 37°C, for 15 min. No plasmid DNA was detected, suggesting efficient plasmid degradation was achieved under these experimental conditions. The infectious titer of benzonase-treated lentiviral vector (RRL-CMV-GFP) was nearly identical to the titer of untreated vector (2.3 ± 0.3 × 106 transduction units per milliliter (TU/ml) and 2.7 ± 0.3 × 106 TU/ml, respectively). Analysis of plasmid DNA in concentrated lentiviral vectors shows that concentration substantially decreases the amount of DNA per TU. Analysis of the extent of plasmid DNA associated with transduced CD34 cells in an ex vivo transduction protocol suggests that a minimal amount of plasmid is transferred to transduced cells if the vector supernatant was not previously treated with benzonase. In conclusion, benzonase treatment is effective in eliminating plasmid DNA from vector supernatants and treatment does not affect infectious titers. However, because there is minimal transfer of plasmid DNA to transduced cells under ex vivo transduction conditions, DNA removal from lentiviral vectors may not be essential for all ex vivo clinical applications.

Strategies that increase the ability of human hematopoietic stem and progenitor cells to repair alkylator-induced DNA damage may prevent the severe hematopoietic toxicity in patients with cancer undergoing high-dose alkylator therapy. In the context of genetic diseases, this approach may allow for selection of small numbers of cells that would not otherwise have a favorable growth advantage. No studies have tested this approach in vivo using human hematopoietic stem and progenitor cells. Human CD34+ cells were transduced with a bicistronic oncoretrovirus vector that coexpresses a mutant form of O6-methylguanine DNA methyltransferase (MGMTP140K) and the enhanced green fluorescent protein (EGFP) and transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Mice were either not treated or treated with O6-benzylguanine (6BG) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). At 8-weeks postinjection, a 2- to 8-fold increase in the percentage of human CD45+EGFP+ cells in 6BG/BCNU-treated versus nontreated mice was observed in the bone marrow and was associated with increased MGMTP140K-repair activity. Functionally, 6BG/BCNU-treated mice demonstrated multilineage differentiation in vivo, although some skewing in the maturation of myeloid and B cells was observed in mice transplanted with granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood compared to umbilical cord blood. Expansion of human cells in 6BG/BCNU-treated mice was observed in the majority of mice previously transplanted with transduced umbilical cord blood cells. In addition, a significant increase in the number of EGFP+ progenitor colonies in treated versus nontreated mice were observed in highly engrafted mice indicating that selection and maintenance of human progenitor cells can be accomplished by expression of MGMTP140K and treatment with 6BG/BCNU.

Two alternatively spliced stem cell factor (SCF) transcripts encode protein products, which differ in the duration of membrane presentation. One form, soluble SCF (S-SCF) gets rapidly processed to yield predominantly secreted protein. The other form, membrane-associated SCF (MA-SCF) lacks the primary proteolytic cleavage site but is cleaved slowly from an alternate site, and thus represents a more stable membrane form of SCF. Mutants of SCF that lack the expression of MA-SCF (Steel-dickie) or possess a defect in its presentation (Steel17H) manifest deficiencies in erythroid cell development. In this study, we have compared the consequence(s) of activating Kit, the receptor for SCF by MA-SCF with S-SCF, and an obligate membrane-restricted (MR) form of SCF (MR-SCF) on erythroid cell survival, proliferation, cell cycle progression, and the activation of p38 and ERK MAP kinase pathways. Activation of Kit by MR-SCF was associated with a significantly lower incidence of apoptosis and cell death in erythroid cells compared to either other isoform. MR- or MA-SCF–induced stimulation of erythroid cells resulted in similar and significantly greater proliferation and cell cycle progression compared to soluble SCF. The increase in proliferation and cell cycle progression via MA- or MR-SCF stimulation correlated with sustained and enhanced activation of p38 and ERK MAP kinase pathways. In addition, MR- or MA-SCF–induced proliferation was more sensitive to the inhibitory effects of ERK inhibitor compared to S-SCF–induced proliferation. In contrast, soluble SCF-induced proliferation was more sensitive to the inhibitory effects of p38 inhibitor compared with MR- or MA-SCF. These results suggest that different isoforms of SCF may use different biochemical pathways in stimulation of survival and/or proliferation of erythroid cells.

Two alternatively spliced stem cell factor (SCF) transcripts encode protein products, which differ in the duration of membrane presentation. One form, soluble SCF (S-SCF) gets rapidly processed to yield predominantly secreted protein. The other form, membrane-associated SCF (MA-SCF) lacks the primary proteolytic cleavage site but is cleaved slowly from an alternate site, and thus represents a more stable membrane form of SCF. Mutants of SCF that lack the expression of MA-SCF (Steel-dickie) or possess a defect in its presentation (Steel17H) manifest deficiencies in erythroid cell development. In this study, we have compared the consequence(s) of activating Kit, the receptor for SCF by MA-SCF with S-SCF, and an obligate membrane-restricted (MR) form of SCF (MR-SCF) on erythroid cell survival, proliferation, cell cycle progression, and the activation of p38 and ERK MAP kinase pathways. Activation of Kit by MR-SCF was associated with a significantly lower incidence of apoptosis and cell death in erythroid cells compared to either other isoform. MR- or MA-SCF–induced stimulation of erythroid cells resulted in similar and significantly greater proliferation and cell cycle progression compared to soluble SCF. The increase in proliferation and cell cycle progression via MA- or MR-SCF stimulation correlated with sustained and enhanced activation of p38 and ERK MAP kinase pathways. In addition, MR- or MA-SCF–induced proliferation was more sensitive to the inhibitory effects of ERK inhibitor compared to S-SCF–induced proliferation. In contrast, soluble SCF-induced proliferation was more sensitive to the inhibitory effects of p38 inhibitor compared with MR- or MA-SCF. These results suggest that different isoforms of SCF may use different biochemical pathways in stimulation of survival and/or proliferation of erythroid cells.

The gene transfer efficiency into nonobese diabetic/severe combined immunodeficient (NOD/SCID)-repopulating cells (SRCs) derived from umbilical cord blood (UCB) (n = 11 NOD/SCID mice) and granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood (MPB) (n = 64 NOD/SCID mice) was compared using a clinically relevant protocol and a retrovirus vector expressing the enhanced green fluorescent protein (EGFP). At 6–9 weeks after transplantation, the frequency of transduced human cells in the bone marrow (BM) (40.5% ± 2.4% [mean ± SE]) and spleen (SPL) (36.4% ± 3.2%) in recipients of UCB cells was significantly higher (p < 0.001) than that observed in the BM (2.2% ± 1.8%) and SPL (2.0% ± 2.6%) in recipients of MPB. In subsequent studies, MPB was cultured for 2–8 days in cytokines prior to transduction to determine if longer prestimulation was required for optimal gene transfer. A significant increase in gene transfer into CD45+ human cells and clonogenic cells derived from MPB SRCs was observed when cells were prestimulated for 6 days compared to 2 days prior to transduction (p = 0.019). However, even after 6 days of prestimulation, transduction was still significantly less than UCB. A substantial discrepancy exists in the ability to introduce genes effectively via retrovirus vectors into SRCs derived from MPB as compared to UCB.

A potential therapeutic option for patients with Fanconi anemia is collection of peripheral blood stem cells prior to the development of severe pancytopenia. These hematopoietic cells potentially could be infused when symptomatic bone marrow failure develops, as autologous rescue after chemotherapy in the event of leukemic transformation, or as targets for gene therapy. Eight patients with Fanconi anemia were mobilized with 10 μg/kg per day of granulocyte colony-stimulating factor (median, 10 ± 4 days) to determine the feasibility of collecting peripheral blood stem cells for future use. Six patients achieved a peripheral blood CD34+ count of ≥ 6/μL and underwent apheresis. The collection goal was 2 × 106 CD34+ cells/kg based on a predicted weight 5 years from the date of collection. A mean of 2.6 ± 0.9 × 106 CD34+ cells/kg of the weight at the time of collection were collected, which corresponded to 1.9 ± 0.4 × 106 CD34+cells/kg of the target weight. The collections required a mean of 4 ± 3 days (range, 2-8 days) of apheresis. Six of the 8 subjects had ≥ 1 × 106 CD34+ cells/kg cryopreserved based on both actual and target weights, and 4 subjects had ≥ 2 × 106 CD34+ cells/kg cryopreserved based on the target weight. These results suggest that some patients with Fanconi anemia can have adequate numbers of CD34+ cells mobilized and collected from the peripheral blood prior to the onset of severe bone marrow failure, but they may require an extended mobilization and multiple days of collection.

A potential therapeutic option for patients with Fanconi anemia is collection of peripheral blood stem cells prior to the development of severe pancytopenia. These hematopoietic cells potentially could be infused when symptomatic bone marrow failure develops, as autologous rescue after chemotherapy in the event of leukemic transformation, or as targets for gene therapy. Eight patients with Fanconi anemia were mobilized with 10 μg/kg per day of granulocyte colony-stimulating factor (median, 10 ± 4 days) to determine the feasibility of collecting peripheral blood stem cells for future use. Six patients achieved a peripheral blood CD34+count of ≥ 6/μL and underwent apheresis. The collection goal was 2 × 106CD34+cells/kg based on a predicted weight 5 years from the date of collection. A mean of 2.6 ± 0.9 × 106CD34+cells/kg of the weight at the time of collection were collected, which corresponded to 1.9 ± 0.4 × 106CD34+cells/kg of the target weight. The collections required a mean of 4 ± 3 days (range, 2-8 days) of apheresis. Six of the 8 subjects had ≥ 1 × 106CD34+cells/kg cryopreserved based on both actual and target weights, and 4 subjects had ≥ 2 × 106CD34+cells/kg cryopreserved based on the target weight. These results suggest that some patients with Fanconi anemia can have adequate numbers of CD34+cells mobilized and collected from the peripheral blood prior to the onset of severe bone marrow failure, but they may require an extended mobilization and multiple days of collection.

Erythroid progenitor cells (EPCs) are deficient in mice lacking either the ligand stem cell factor (SCF), its receptor c-Kit, or β1-integrins. In nonhematopoietic cells, integrins and receptor tyrosine kinases can collaborate to modulate cellular functions, providing evidence for cross-talk between signals emerging from these cell surface molecules. Using specific recombinant fibronectin peptides that contain the binding site for the integrin α4β1(FN-H296) or α5β1(FN-CH271) or both α4β1and α5β1(FN-CH296), this study investigated the effect of adhesion alone, or in combination with activation of c-Kit, on functional and biochemical outcomes in an EPC line, G1E-ER2, and primary EPCs. G1E-ER2 cells and primary EPCs cultured on FN-CH271 in the presence of c-Kit activation led to a significant increase in proliferation in comparison with cells grown on FN-H296 or FN-CH296. G1E-ER2 cells cultured on FN-H296 or FN-CH296 resulted in significant cell death in comparison to cells grown on FN-CH271. Activation of c-Kit enhanced the survival of G1E-ER2 cells grown on FN-H296 or FN-CH296; however, the rescue was only partial. The reduced survival of G1E-ER2 cells on FN-H296 correlated with reduced activation of Akt and expression of Bcl-2 and Bcl-xL, whereas increase in proliferation on FN-CH271 correlated with significantly enhanced and sustained activation of focal adhesion kinase (FAK) and extracellular-regulated kinase (ERK) pathways. These data demonstrate that adhesion-induced signals emanating from ligation of α4β1and α5β1result in distinct biologic outcomes, including death via α4β1and survival/proliferation via α5β1.

Erythroid progenitor cells (EPCs) are deficient in mice lacking either the ligand stem cell factor (SCF), its receptor c-Kit, or β1-integrins. In nonhematopoietic cells, integrins and receptor tyrosine kinases can collaborate to modulate cellular functions, providing evidence for cross-talk between signals emerging from these cell surface molecules. Using specific recombinant fibronectin peptides that contain the binding site for the integrin α4β1 (FN-H296) or α5β1 (FN-CH271) or both α4β1 and α5β1(FN-CH296), this study investigated the effect of adhesion alone, or in combination with activation of c-Kit, on functional and biochemical outcomes in an EPC line, G1E-ER2, and primary EPCs. G1E-ER2 cells and primary EPCs cultured on FN-CH271 in the presence of c-Kit activation led to a significant increase in proliferation in comparison with cells grown on FN-H296 or FN-CH296. G1E-ER2 cells cultured on FN-H296 or FN-CH296 resulted in significant cell death in comparison to cells grown on FN-CH271. Activation of c-Kit enhanced the survival of G1E-ER2 cells grown on FN-H296 or FN-CH296; however, the rescue was only partial. The reduced survival of G1E-ER2 cells on FN-H296 correlated with reduced activation of Akt and expression of Bcl-2 and Bcl-xL, whereas increase in proliferation on FN-CH271 correlated with significantly enhanced and sustained activation of focal adhesion kinase (FAK) and extracellular-regulated kinase (ERK) pathways. These data demonstrate that adhesion-induced signals emanating from ligation of α4β1 and α5β1 result in distinct biologic outcomes, including death via α4β1 and survival/proliferation via α5β1.

CD34+ cells from human umbilical cord blood (CB) were isolated and investigated for megakaryocytic (MK) colony formation in response to recombinant human (rh) stimulatory and suppressive cytokines and compared with their counterparts in normal BM and G-CSF-mobilized peripheral blood (mPBL). First, we observed that IL-11 by itself at any dosage had no stimulator activity on MK colony formation derived from CD34+ cells in CB, mPBL, and BM. IL-3, steel factor (SLF), or thrombopoietin (Tpo) alone stimulated numbers of colony-forming unit-megakaryocyte (CFUMK) in a dose-dependent fashion. Maximum growth of MK progenitor cells was noted in the presence of a combination of cytokines: IL-11, IL-3, SLF, and Tpo. The frequency of CFU-MK in CB and mPBL was significantly greater than that in BM, and the size of colonies in CB and mPBL was significantly greater than that in BM, and the size of colonies was larger as well. In addition, an increased number of big mixed colonies containing MK were observed in CB and mPBL. In the presence of IL-11, IL-3, SLF, and Tpo, CFU-MK derived from CB, mPBL, and BM was suppressed by tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor- beta 1 (TGF-beta 1). CFU-MK derived from normal BM was inhibited by some chemokines evaluated, whereas CFU-MK derived from CB was suppressed only by platelet factor-4 (PF-4), IFN-inducible protein-10 (IP-10), Exodus-1, Exodus-2, and Exodus-3, but to a lesser degree. In CB, unlike granulocyte-macrophage (CFU-GM), erythroid (BFU-E), high-proliferative potential (HPP-CFC), or multipotential (CFU-GEMM) progenitors, at least a subpopulation of MK progenitors are in S-phase. Therefore, CB MK progenitors respond to the suppressive effects of some members of the chemokine family. Similar results were noted for burst-forming unit-MK (BFU-MK). Our results indicate that CB and mPBL are rich sources of MK progenitors and that MK progenitors in CB are responsive to the suppressive effects of TNF-alpha and TGF-beta 1 and some members of the chemokine family.

Stem cell factor (SCF) is expressed as an integral membrane growth factor that may be differentially processed to produce predominantly soluble (S) (SCF248) or membrane-associated (MA) (SCF220) protein. A critical role for membrane presentation of SCF in the hematopoietic microenvironment (HM) has been suggested from the phenotype of the Steel-dickie(Sld) mice, which lack MA SCF, and by studies performed in our laboratory (and by others) using long-term bone marrow cultures and transgenic mice expressing different SCF isoforms.Steel17H (Sl17H) is an SCF mutant that demonstrates melanocyte defects and sterility in males but not in females. The Sl17H allele contains a intronic mutation resulting in the substitution of 36 amino acids (aa’s) in the SCF cytoplasmic domain with 28 novel aa’s. This mutation, which affects virtually the entire cytoplasmic domain of SCF, could be expected to alter membrane SCF presentation. To investigate this possibility, we examined the biochemical and biologic properties of the Sl17H-encoded protein and its impact in vivo and in vitro on hematopoiesis and on c-Kit signaling. We demonstrate that compound heterozygous Sl/Sl17H mice manifest multiple hematopoietic abnormalities in vivo, including red blood cell deficiency, bone marrow hypoplasia, and defective thymopoiesis. In vitro, both S and MA Sl17H isoforms of SCF exhibit reduced cell surface expression on stromal cells and diminished biological activity in comparison to wild-type (wt) SCF isoforms. These alterations in presentation and biological activity are associated with a significant reduction in the proliferation of an SCF-responsive erythroid progenitor cell line and in the activation of phosphatidylinositol 3-Kinase/Akt and mitogen-activated protein-Kinase signaling pathways. In vivo, transgene expression of the membrane-restricted (MR) (SCFX9/D3) SCF in Sl/Sl17H mutants results in a significant improvement in peripheral red blood cell counts in comparison toSl/Sl17H mice.

Primary human T lymphocytes were transduced at high efficiency with the Moloney murine leukemia virus (Mo-MuLV) vector, LNC-mB7-1, in which an internal cytomegalovirus (CMV) promoter drives expression of the murine B7-1 cDNA. Compared with transduced T cells expanded in IL-2 or reactivated with soluble antibodies to CD3 or CD28, transgene expression was significantly increased after activation on immobilized anti-CD3 antibodies (CD3i) or by simultaneous activation on immobilized anti-CD3 and anti-CD28 antibodies (CD3i/CD28i). A similar pattern of transgene expression was observed in T cells transduced with MoMuLV LNC-EGFP. Proviral copy number was maintained in LNC-mB7-1-transduced T cells expanded in IL-2 or reactivated on CD3i/CD28i. Substantial increases in LNC-mB7-1 steady state mRNA in reactivated T lymphocytes, compared with those maintained in IL-2, correlated with increased transcription of the LNC-mB7-1 proviral DNA. Furthermore, T cells transduced with the Mo-MuLV ZIPPGK-mADA, in which the mADA cDNA is driven by an internal human phosphoglycerate kinase (PGK) promoter, showed increases in steady state ZIPPGK-mADA RNA on reactivation. High levels of transgene expression were evident irrespective of cell cycle position in both CD4+ and CD8+ lymphocytes. After reactivation, increases in LNC-mB7-1 mRNA were observed in the presence of the protein synthesis inhibitor cycloheximide, indicating that proteins involved in upregulating transgene expression preexisted in transduced lymphocytes. Induction of transgene expression on CD3i/CD28i showed a dose-dependent decrease in transgene expression when incubated with selective protein kinase inhibitors. These data provide new insights into the mechanisms governing transgene expression driven by Mo-MuLV constructs containing internal promoters in transduced primary T lymphocytes.

Mobilized CD34+ cells from human peripheral blood (PB) are increasingly used for hematopoietic stem-cell transplantation. However, the mechanisms involved in the mobilization of human hematopoietic stem and progenitor cells are largely unknown. To study the mobilization of human progenitor cells in an experimental animal model in response to different treatment regimens, we injected intravenously a total of 92 immunodeficient nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with various numbers of granulocyte colony-stimulating factor (G-CSF) –mobilized CD34+ PB cells (ranging from 2 to 50 × 106cells per animal). Engraftment of human cells was detectable for up to 6.5 months after transplantation and, depending on the number of cells injected, reached as high as 96% in the bone marrow (BM), displaying an organ-specific maturation pattern of T- and B-lymphoid and myeloid cells. Among the different mobilization regimens tested, human clonogenic cells could be mobilized from the BM into the PB (P= .019) with a high or low dose of human G-CSF, alone or in combination with human stem-cell factor (SCF), with an average increase of 4.6-fold over control. Therefore, xenotransplantation of human cells in NOD/SCID mice will provide a basis to further study the mechanisms of mobilization and the biology of the mobilized primitive human hematopoietic cell.

Parvovirus B19 infection leads to transient aplastic crises in individuals with chronic hemolytic anemias or immunodeficiency states. An additional unexplained sequela of B19 infection is thrombocytopenia. Because B19 is known to have a remarkable tropism for human erythropoietic elements, and is not known to replicate in nonerythroid cells, the etiology of this thrombocytopenia is uncertain. We sought to define the pathobiology of B19-associated thrombocytopenia by examining the role of B19 on in vitro megakaryocytopoiesis. B19 infection of normal human bone marrow cells significantly suppressed megakaryocyte (MK) colony formation compared with mock-infected cells. No such inhibition was observed with a nonpathogenic human parvovirus, the adeno-associated virus 2 (AAV). The B19-MK cell interaction was also studied at the molecular level. Whereas low-density bone marrow cells containing erythroid precursor cells supported B19 DNA replication, no viral DNA replication was observed in B19-infected MK-enriched fractions as determined by the presence of viral DNA replicative intermediates on Southern blots. However, analysis of total cytoplasmic RNA isolated from B19-infected MK fractions showed a low-level expression of the B19 genome as detected by quantitative RNA dot blots as well as by Northern analysis. Furthermore, a frame-shift mutation in a recombinant AAV-B19 hybrid genome segment that encodes the viral nonstructural (NS1) protein significantly reduced the observed inhibition of MK colony formation. These studies indicate tissue-tropism of B19 beyond the erythroid progenitor cell, and lend support to the hypothesis that B19 genome expression may be toxic to cell populations that are nonpermissive for viral DNA replication.

Mutations of c-kit, which encodes a transmembrane receptor tyrosine kinase, have been identified in mice by abnormal coat color, anemia, and germ cell defects. Mice heterozygous for mutations of c-kit have a white forehead blaze and a white ventral spot, leading these mutants to be termed dominant White spotting(W).We have previously demonstrated that the membrane-associated isoform of human stem cell factor (hSCF220, the ligand for c-kit) is inefficiently processed in murine stromal cell transfectants. Thus, in murine cell lines analyzed in vitro, hSCF220transfectants present SCF as a membrane restricted protein in contrast to the murine SCF220cDNA protein product, which is slowly cleaved and secreted. We show here that transgenic mice expressing the human SCF220isoform in vivo display a phenotype indistinguishable from some alleles of W.Specifically, hSCF220-ex-pressing transgenic mice display a prominent forehead blaze and a white ventral spot. Generations of doubly heterozygous animals that carry both a mutated c-kitallele and the hSCF220transgene display a more severe coat color abnormality. This phenotype appears to be due to occupancy of murine c-kit by human SCF and diminished cell surface expression of endogenous murine SCF. Normal signaling events that lead to cell survival or proliferation appear to be disrupted in vivo in these transgenic mice.