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J S Miller

Publications and source records attributed to J S Miller.

At least 91 records · Page 5Linked to original sources
PubMed1997

Genome structure of cucumber leaf spot virus: sequence analysis suggests it belongs to a distinct species within the Tombusviridae.

The complete nucleotide sequence of cucumber leaf spot virus (CLSV) has been determined and the sizes and locations of predicted viral proteins deduced. The genome consists of 4432 nucleotides and contains five long ORFs. The 5' proximal ORF encodes a 25 kDa product that terminates in an amber codon which may be readthrough to produce an 84 kDa protein (ORF 2). ORF 3 codes for the 41 kDa coat protein (CP). ORFs 4 and 5 are completely overlapping at the 3' terminus and code for 27 and 17 kDa products, respectively. The CLSV genome structure is similar to that of tombusviruses and nearly identical to pothos latent virus (PoLV), a newly proposed, atypical, member of the Tombusviridae. It is proposed that CLSV and PoLV be considered strains of a new tombusvirus species. Amino acid sequence comparisons of the CLSV CP and the CPs of several small spherical plant viruses suggest that CLSV is most closely related to melon necrotic spot carmovirus (MNSV), red clover necrotic mosaic dianthovirus (RCNMV) and cucumber necrosis tombusvirus (CNV). These viruses, like CLSV, are transmitted by the soil inhabiting fungus, Olpidium bornovanus.

Amino Acid Sequence↗
PubMed1997

Gastric rupture with tension pneumoperitoneum: a complication of difficult endotracheal intubation.

Gastric rupture is a rare complication of difficult airway management. In cases of vigorous mouth-to-mouth ventilation, bag-valve-mask ventilation, or esophageal intubation, gastric rupture and massive intraperitoneal free air may cause tension pneumoperitoneum. Hemodynamic instability necessitates immediate intervention, including needle decompression of the peritoneum followed by surgical exploration. We recently encountered two cases of gastric rupture with tension pneumoperitoneum that occurred after difficult endotracheal intubation. This report describes the presentation, treatment, and prevention of this entity.

Aged↗
PubMed1997

Intra-articular morphine and/or bupivacaine in the management of pain after total knee arthroplasty.

The purpose of this study was to determine if intra-articular injection of morphine or bupivacaine significantly decreased postoperative pain as well as the use of intravenous narcotics for pain relief in patients undergoing total knee arthroplasty (TKA). In a prospective, double-blind, randomized fashion, 105 patients undergoing TKA were divided into the following 4 groups defined by the intra-articular injection they received: group 1 (n = 27) received saline solution, group 2 (n = 26) received morphine sulfate (5 mg), group 3 (n = 24) received bupivacaine (50 mg), and group 4 (n = 28) received a combination of morphine sulfate and bupivacaine. The injections were administered immediately after wound closure by the Hemovac drainage tubing that remained clamped for 45 minutes after surgery to allow for absorption. Before surgery and at 2, 4, 6, 24, and 48 hours after surgery, pain intensity was recorded using a visual analog scale. Postoperative supplemental intravenous morphine and/or meperidine was administered via a patient-controlled analgesia device, and 24-hour drug usage was tabulated. Results were suggestive of a modest short-term reduction in pain scores in the morphine and bupivacaine treatment groups compared with placebo (saline); however, results were statistically significant only at 4 hours because of the great variability in the pain score data. The total amount of postoperative pain medication used in the first 24 hours after surgery was not statistically significant between the 4 treatment groups. Thus, the results put into question the benefit of postoperative intra-articular administration of morphine or bupivacaine in patients undergoing TKA.

Adult↗
PubMed1997

Primary bone marrow progenitors of both granulocytic and monocytic lineages are susceptible to infection with the agent of human granulocytic ehrlichiosis.

Human granulocytic ehrlichiosis (HGE) is an emerging tickborne infection resulting in an acute febrile illness associated with cytopenias and characteristic intracellular organisms within peripheral blood granulocytes. The etiologic agent of HGE has recently been isolated and cultivated in the HL-60 promyelocytic leukemia cell line, but the spectrum of host cells that it naturally infects remains unknown. To determine if normal hematopoietic progenitors could be targets of infection, CD34+ primary human bone marrow cells, stimulated to differentiate along myelomonocytic lineages, were incubated with the HGE agent. Immature marrow progenitors and, remarkably, not only granulocytic but also CD14+ monocytic cells from these cultures supported replication of the HGE agent, suggesting that all are potential targets of infection in vivo. Infection of bone marrow progenitors may contribute to the hematologic manifestations of HGE. Furthermore, the ability of the agent to interact with monocytes has significant implications regarding disease pathogenesis and host response.

Cells, Cultured↗
PubMed1997

The role of autologous natural killer cells in chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) is a lethal disease of the hematopoietic stem cell. Bone marrow transplantation has highlighted the importance of allogeneic disparity in maintaining remissions in CML. However, it has been unclear whether the immune effect against CML is mediated by T cells, natural killer cells (NK) or a combination of both. We have previously demonstrated that autologous activated NK are capable of selectively lysing malignant CML progenitors while sparing benign progenitors. NK effectors may play an important role in CML since NK lytic function, clonogenic frequency and proliferative capacity decrease as CML progresses from chronic phase to advanced phase and blast crisis. Incubation of CML NK with IL-2 is capable of restoring cytolytic activity to normal levels. We hypothesize that activated NK represent a potential therapy against CML to maintain remissions in a minimal residual disease setting induced by autologous transplantation. Clinical trials are in progress to test whether IL-2 based immunotherapy and activated cell infusions play a therapeutic role in CML.

Disease Progression↗
PubMed1997

Phenotypic and functional characterization of committed and primitive myeloid and lymphoid hematopoietic precursors in human fetal liver.

We studied the phenotypic and functional properties of colony-forming cells (CFCs), primitive long-term culture initiating cells (LTC-ICs) and lymphoid precursors present in human fetal liver (FL) and compared these with their adult bone marrow (BM) counterparts. FL (7-14-week) cells were selected by fluorescence-activated cell sorting based on increasing CD34 antigen expression (34+, 34++, or 34 ), CD38 antigen expression (CD34++/ CD38+, or CD38-), and HLA-DR antigen expression (CD34++/ HLA-DR+ or HLA-DR-). 13 +/- 0.6% of FL CD34-positive cells were 34 . Significantly more FL CD34++/ cells were CD38- (49 +/- 2.4%) and HLA-DR-(72 +/- 6.7%) than BM CD34++ cells (6.8 +/- 0.7% CD38- and 13.3 +/- 3.2% HLA-DR-). FL and BM CFCs were CD34+/++, CD38+, and HLA-DR+. However, significantly more FL CFCs were erythroid (40%) than adult BM CFCs (15%), and FL colonies were larger (8111 +/- 738 cells/CFC) than BM colonies (3466 +/- 272 cells/CFC, p < 0.001). As is seen in adult BM, FL LTC-ICs were CD34++/ CD38-. In contrast to BM LTC-ICs, FL LTC-ICs were almost exclusively CD34++/ HLA-DR+. In addition, a single FL LTC-IC gave rise to >30 CFCs at 5 weeks compared with only 5 +/- 0.9 CFCs per LTC-IC from BM. Finally, we demonstrate that the FL CD34++/ /CD38-/HLA-DR+ population, which contains 3.7% LTC-ICs, also contains primitive lymphoid progenitors capable of differentiating into natural killer (NK) cells. In conclusion, the phenotype of primitive human FL progenitors such as LTC-IC and primitive NK progenitors is CD34++/ /CD38-/HLA-DR+, suggesting that this population may contain FL hematopoietic stem cells. The phenotypic characterization of FL primitive LTC-ICs and NK progenitors will facilitate further studies of the functional properties of these progenitors.

ADP-ribosyl Cyclase↗
PubMed1997

A circus atmosphere.

Explore the source record for details and available documents.

Complementary Therapies↗
PubMed1997

Low dose subcutaneous interleukin-2 after autologous transplantation generates sustained in vivo natural killer cell activity.

Autologous transplantation can induce extended remission in some patients with advanced breast cancer and lymphoma yet nearly 80% and 50%, respectively, will ultimately relapse. In vitro studies suggest that activated natural killer cells (NK) mediate lytic activity against breast cancer and lymphoma cell lines. Therefore, immunotherapy with interleukin-2 (IL-2, Amgen) to activate NK may improve long-term disease-free survival when administered in a post-transplant minimal residual disease setting. To determine the feasibility of administering IL-2 and activation of NK post-transplant, twelve patients (6 breast cancer, 6 lymphoma) were enrolled on a phase I dose escalation study after autologous transplantation (median day + 94, range 50-166). IL-2 was self administered at 0.25 x 10(6) (n = 6) or 0.5 x 10(6) (n = 6) U/m2/day subcutaneously for 84 consecutive days. The best tolerated dose was 0.25 x 10(6) U/m2/day (75% of planned doses given vs. 48% at the higher dose). Dose limiting toxicity occurred in 6 patients (n = 2 at 0.25 x 10(6) U/m2/day, n = 4 at 0.5 x 10(6) U/m2/day) consisting of decreased performance status (n = 2), thrombocytopenia (n = 3, 1 at the lower dose), and mild neutropenia (n = 1 at the lower dose). However, all symptoms resolved within a week following discontinuation of IL-2 and no patient required hospitalization. Circulating soluble IL-2 receptor levels were significantly increased in all patients receiving IL-2. Patients receiving at least 28 days of IL-2 exhibited a greater than 10-fold increment in circulating CD56+bright/CD3- NK. Furthermore, lytic function was increased against NK resistant targets, MCF-7 (breast cancer), and Raji (lymphoma). In vivo IL-2 primed NK cells obtained by lymphapheresis were activated in large-scale ex vivo incubation in high dose IL-2 (1,000 U/mL) at high cell density (10 x 10(6)/mL), in gas permeable bags, and using serum-free media. NK lytic function against MCF-7 and Raji targets was further enhanced. We conclude that low dose subcutaneous IL-2 based immunotherapy is feasible, relatively safe, can be administered in an outpatient setting and hypothesize that additional ex vivo incubation in IL-2 may be used to generate NK cells with potent antitumor effects in vivo.

Adjuvants, Immunologic↗
PubMed1997

A clinically suitable ex vivo expansion culture system for LTC-IC and CFC using stroma-conditioned medium.

FACS-selected CD34+ HLA-DR- cells (DR- cells) may provide a source of benign stem cells suitable for autografting in chronic myelogenous leukemia (CML) and other hematological malignancies. However, DR- cell selection depletes the majority of committed hematopoietic progenitors, which may be important for early engraftment. Furthermore, only a small number of DR- cells may be selectable in certain patients. These impediments to the use of DR- cells for autografting may be overcome through the development of ex vivo culture systems that support expansion and initial differentiation of primitive progenitors. Because 2-week culture of DR- cells in a stroma "noncontact" system supplemented with interleukin-3 (IL-3) and macrophage inflammatory protein 1-alpha (MIP-1alpha) expands both long-term culture-initiating cells (LTC-ICs) and colony-forming cells (CFCs), we adapted this system to a clinically applicable method for expanding LTC-ICs and CFCs ex vivo. In initial small-scale studies, DR cells were grown in stroma conditioned medium (SCM) supplemented with IL-3 with or without additional growth-promoting cytokines and the chemokines PF-4 and BB10010, all approved for clinical use. An IL-3 dose-dependent expansion of committed progenitors and LTC-ICs was observed when DR- cells were cultured in tissue culture plates in SCM+IL-3 for 2 weeks. Similar CFC expansion along with increased (5-fold) LTC-IC expansion was observed following addition of PF-4 to SCM+IL-3 cultures. The addition of stem cell factor (SCF), but not of IL-6, IL-11, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage (GM)-CSF, IL-1, and IL-7, increased CFC and LTC-IC expansion beyond the levels observed with SCM+IL-3 alone. We next evaluated the suitability of this culture system for scale-up. Culture of 2-6 x 10(5) DR- cells in gas-permeable bags with SCM+IL-3 resulted in similar CFC and LTC-IC expansion as seen in small-scale cultures. In addition, we observed that progenitors capable of differentiating to natural killer (NK)-cells were maintained under these conditions. Finally, we found that BCR/ABL mRNA-negative CFCs and LTC-ICs present in DR- cells selected from steady-state CML marrow could be expanded in large-scale SCM+IL-3 cultures. We conclude that culture of DR- cells for 2 weeks in SCM+IL-3 culture, with or without PF-4 or SCF, results in significant CFC and LTC-IC expansion and lymphoid NK progenitor maintenance. This culture system is readily adaptable to the expansion of primitive progenitors for autotransplantation.

Bone Marrow Cells↗
PubMed1997

Endogenous IL-2 production by natural killer cells maintains cytotoxic and proliferative capacity following retroviral-mediated gene transfer.

Interleukin (IL)-2 therapy given at tolerable doses is insufficient to induce maximum activation of natural killer (NK) cells. We recently demonstrated that NK cells expanded in vivo can be maximally activated by short-term ex vivo incubation with 1000 U/mL IL-2. However, IL-2 withdrawal, which would occur with reinfusion, may lead to a rapid loss of cell viability and function. We hypothesized that retroviral transduction could provide an endogenous source of IL-2 to maintain NK function as measured by proliferation and cytotoxicity. Enriched NK cells were transduced with supernatants containing an MFG-based retrovirus designed to express murine IL-2 cDNA. Several supernatant transduction strategies were evaluated. NK cells were initially cultured in 1000 U/mL of huIL2 for 7-8 days, harvested, and replated prior to transduction (4 hours at 37degrees C); this proved insufficient to sustain NK proliferation or maintain cytotoxicity after exogenous human IL-2 (huIL-2) withdrawal. An alternative transduction procedure using phosphate-depleted medium, centrifugation, and transduction for 16 hours at 32degrees C was then evaluated. NK cells transduced under these conditions maintained significant NK proliferation in the absence of exogenous IL-2 compared with sham-transduced controls. Two consecutive daily transductions resulted in less proliferation, suggesting that several exposures to retroviral supernatant may inhibit subsequent NK proliferation. Cytotoxicity of the transduced NK cells against K562 and Raji was maintained under these conditions without exogenous IL-2. Sham-transduced NK cells produced 8.3+/-2.6 U/mL of murine IL-2 (muIL-2) by ELISA (background) after 7 days without exogenous IL-2. In contrast, 109+/-23 U/mL muIL-2 was produced by NK cells transduced with supernatant from the MFG/muIL-2 producer line. These experiments demonstrate that NK cells can be successfully transduced with retroviruses and induced to express sufficient IL-2 to maintain their proliferative and cytotoxic functions. Transduction of IL-2 genes into NK cells may offer advantages over exogenous IL-2 administration in maintaining maximum function for use in antitumor immunotherapy.

Adult↗
PubMed1997

Port-Access coronary artery bypass grafting.

New techniques for minimally invasive cardiac surgery have recently emerged. This report describes the Port-Access technique for coronary artery bypass grafting, which involves a small left anterior thoracotomy, femoral cannulation for endovascular cardiopulmonary bypass, and cardioplegic arrest using an endoaortic occlusion catheter and cardioplegia delivery system. This technique allows for minimally invasive single or multivessel revascularization in an arrested, protected heart, while maintaining a high level of anastomotic precision. The Port-Access surgical techniques are described, along with the indications and contraindications for this procedure. The initial New York University clinical results with Port-Access coronary bypass grafting are presented.

Adult↗
PubMed1996

CD56+bright and CD56+dim natural killer cells in patients with chronic myelogenous leukemia progressively decrease in number, respond less to stimuli that recruit clonogenic natural killer cells, and exhibit decreased proliferation on a per cell basis.

Human natural killer cells (NK) require accessory cell-derived contact and soluble factors for maximal expansion. However, it is unclear whether increased recruitment of clonogenic NK, increased proliferation on a per cell basis, or a combination of both is responsible for the increased expansion. We show that expansion of both CD56+dim and CD56+bright NK from normal donors is increased in the presence of M2-10B4 accessory cell-soluble factors. In contrast, the addition of M2-10B4 stromal ligands further augments only the expansion of CD56+bright NK. Using single-cell sorting of CD56+bright NK, M2-10B4-soluble and contact factors independently increase both recruitment of clonogenic NK and proliferation on a per cell basis. This well-defined M2-10B4 accessory cell system was used to investigate potential defects in NK from patients with CML. Although we have previously shown diminished interleukin-2 (IL-2)-activated NK outgrowth and function from patients with chronic myelogenous leukemia (CML) as their disease progresses, it has been unclear if this is due to a defect in an accessory cell function or an inherent abnormality of CML NK themselves. CD56+/CD3- NK purified by fluorescence-activated cell sorting from 21 patients (7 early chronic phase [ECP] patients, 10 late chronic and accelerated phase [LCP/AP], and 4 blast crisis [BC] patients) were studied. The proliferative capacity, clonogenic frequency, and cytotoxic capacity of CML NK were compared with NK from normal donors. The absolute number of circulating NK per milliliter of peripheral blood is significantly decreased in patients with CML compared with normal donors (normal, 63,700 +/- 6,400; ECP, 40,700 +/- 6,700; LCP/AP, 31,900 +/- 6,000; BC, 10,700 +/- 5,200). Additionally, the unique CD56+bright NK subset, analyzed as a percentage of the total circulating NK pool, is significantly reduced in all patients with CML (normal, 5.7% +/- 0.8% v CML [all stages combined], 2.5% +/- 0.5%, P = .001]. After purification of NK to correct for differences in circulating NK number, resting NK cytotoxicity against K562 tumor targets is significantly reduced in patients with CML on or recently on hydroxyurea therapy. However, this reduced cytotoxicity can be corrected by 18 hours of incubation with 1,000 U/mL recombinant IL-2. When plated in limiting dilution on viable M2-10B4, which maximally stimulates NK from normal donors, we show that both NK clonogenic frequency and proliferative capacity are significantly reduced as CML progresses, demonstrating an inherent defect in their ability to respond to normal NK stimuli. Although NK cloning efficiency between normal donors and ECP CML patients was the same, significant differences were observed in (1) the absolute number of circulating CD56+/CD3- NK, (2) the absolute number of circulating CD56+bright NK, and (3) proliferation on a per cell basis. Unlike resting NK function, prior cytotoxic therapy alone did not account for these observed abnormalities. These data suggest that, although NK are not derived from the malignant clone, they are inherently affected by their malignant microenvironment.

Adolescent↗
PubMed1996

BCR/ABL-negative primitive progenitors suitable for transplantation can be selected from the marrow of most early-chronic phase but not accelerated-phase chronic myelogenous leukemia patients.

We have previously reported that selection of marrow cells on the basis of the CD34+HLA-DR- phenotype (34+DR-) may result in the recovery of Philadelphia chromosome (Ph)- and BCR/ABL-negative long-term culture-initiating cells (LTC-IC) in selected patients with chronic myelogenous leukemia (CML). We now present data on 27 early chronic-phase ([ECP] studied within 1 year after diagnosis) and 23 advanced-phase ([AP] late chronic phase, ie, studied >1 year from diagnosis, or accelerated phase) CML patients. Fluorescence-activated call-sorting (FACS)-selected 34+DR- and 34+DR+ cells were subjected to reverse transcriptase-polymerase chain reaction and fluorescence in situ hybridization. These cells were also cultured in long-term bone marrow culture for 1 to 5 weeks to examine the number of LTC-IC and the presence or absence of the BCR/ABL gene rearrangement in progeny of primitive LTC-IC. The number of 34+DR- cells and LTC-IC present in ECP CML marrow was similar to that in normal (NL) marrow, whereas the numbers were reduced in AP CML. Furthermore, 34+DR- cells from more than 80% of ECP CML patients were BCR/ABL mRNA- and Ph-negative and contained only BCR/ABL mRNA- and Ph-negative LTC-IC, whereas 34+DR- cells and LTC-IC from less than 40% of AP CML patients were BCR/ABL mRNA- and Ph-negative. In contrast to NL marrow, 34+DR+ cells from CML marrow, irrespective of clinical stage, contained large numbers of LTC-IC. CML 34+DR+ cells and LTC-IC were BCR/ABL mRNA- and Ph-positive. Since these studies suggested that a population of primitive progenitors that are Ph-negative can be selected from steady-state marrow in some ECP CML patients, we determined if similar results could be obtained when large quantities of marrow sufficient for transplantation are processed. We demonstrate that 1 to 3 x 10(5) BCR/ABL mRNA-negative 34+DR- cells/kg recipient body weight, containing only BCR/ABL mRNA-negative LTC-IC, can be obtained from a 2- to 2.5-L marrow collection by sequential COBE Spectra apheresis (COBE BCT, Lakewood, CO), CD34+ enrichment using the CEPRATE SC Cell-Concentrator (CellPro, Bothell, WA), and high-speed FACS. Thus, large-scale selection of a BCR/ABL mRNA- and Ph-negative 34+DR- cell population is possible in a fraction of chronic-phase CML patients, in whom these cells could be used to reconstitute the hematopoietic compartment following autologous transplantation.

Antigens, CD34↗
PubMed1996

Malignant neoplasms following bone marrow transplantation.

We undertook an analysis of 2,150 recipients of bone marrow transplant (BMT) at the University of Minnesota to determine the incidence of post-BMT malignant neoplasms (MNs). Fifty-one patients developed 53 MNs, compared with 4.3 expected from general population rates (standardized incidence ratio [SIR], 11.6, 95% confidence interval [CI], 8.2-14.5). These included 22 occurrences of B-cell lymphoproliferative disorder (BLPD), 17 solid nonhematopoietic tumors, 10 myelodysplastic syndromes (MDS), 1 acute myelogenous leukemia (AML), 2 non-Hodgkin's lymphoma (NHL), and 1 Hodgkin's disease (HD). The estimated actuarial incidence of any post-BMT malignancy was 9.9% +/- 2.3% at 13 years posttransplant. The cumulative probability of BLPD plateaued at 1.6% +/- 0.3% by 4 years from transplant and factors independently associated with increased risk included in vitro T-cell depletion of marrow (relative risk (RR) = 11.9, P < .001), HLA mismatch (RR = 8.9, P < .001), use of antithymocyte globulin (ATG) for graft versus host disease (GVHD) prophylaxis (RR = 5.9, P < .001) or in the preparative regimen (RR = 3.1, P = .03) and primary immunodeficiency (RR = 2.5, P = .06). The cumulative probability of developing solid malignancy was 5.6% +/- 2.2% at 13 years from BMT. Malignant melanomas were the most common (SIR, 10.3, 95% CI 1.9 to 25.4). The actuarial incidence of MDS/AML plateaued at 2.1% +/- 0.8% at 9 years and was seen most often in older patients receiving autologous peripheral blood stem cells for HD or NHL. These data document that BMT recipients are at an increased risk of later malignancy, which may add significant morbidity and mortality to the transplant process. Methods for screening and identification of individuals at increased risk need to be addressed in future studies.

Adolescent↗
PubMed1996

Autologous activated natural killer cells suppress primitive chronic myelogenous leukemia progenitors in long-term culture.

A graft-versus-leukemia effect has been well documented to prevent relapse in chronic myelogenous leukemia (CML) after allogeneic marrow transplantation. One type of lymphocytes that may contribute to this effect are natural killer cells (NK), which after activation with interleukin (IL)-2, exhibit a broad range of cytolytic activity against allogeneic and autologous cells. We have previously demonstrated that IL-2-activated NK (ANK) can be generated from blood of patients with CML and are benign in origin. Their proliferation and function, however, diminish with disease progression in CML, suggesting a role in tumor surveillance. We studied the effect of IL-2-activated NK (ANK) on normal and malignant primitive and committed progenitors in a novel long-term bone marrow culture (LTBMC) assay. Because ANK destroy marrow stromal layers, the use of classic stroma-dependent long-term cultures is not possible. Therefore, we used the stroma noncontact LTBMC system developed in our laboratory to analyze the effect of autologous ANK cells on primitive hematopoietic progenitors. Autologous ANK (CD56+/CD3-) were generated from the peripheral blood of 10 patients with chronic phase CML and from six normal individuals by culturing CD5/CD8-depleted mononuclear cells for 14 days in 1,000 U/mL IL-2. At the same time ANK cultures were initiated, sorted normal (CD34+/DR+) marrow populations were plated in Transwell inserts of the stroma noncontact culture. On day 15, hydrocortisone, which rapidly inhibits ANK function, was removed, and autologous ANK were added to the Transwell inserts with fresh LTBMC medium without hydrocortisone but supplemented with 1,000 U/mL IL-2. After 48 hours, the number of colony-forming cells (CFC) was enumerated in methylcellulose culture. To determine the effect of ANK on more primitive long-term culture-initiating cells (LTCIC), the IL-2-supplemented LTBMC medium was replaced with fresh hydrocortisone containing LTBMC medium, and cultures were maintained for an additional 5 weeks. We demonstrate that autologous ANK did not suppress normal CFC or LTCIC. In contrast, ANK from eight patients with CML with potent cytotoxicity against NK-sensitive (K562) NK-resistant (Raji) tumor targets exhibited an ANK dose-dependent suppression of both CFC and LTCIC. Interestingly, ANK from two patients with CML who exhibited diminished cytotoxicity also did not suppress autologous CFC and LTCIC. These studies indicate that ANK with potent major histocompatibility complex unrestricted cytotoxic activity suppress malignant hematopoiesis. This effect was not mediated by soluble factors and was absolutely dependent on direct cell-to-cell contact. We further demonstrate that the beta2 integrin receptor is involved in ANK recognition of CML targets. These observations support the use of autologous ANK therapy to prevent relapse of CML after autologous marrow transplantation or use of ANK to purge CML marrow for autologous transplantation.

Bone Marrow↗
PubMed1996

Human natural killer cell expansion is regulated by thrombospondin-mediated activation of transforming growth factor-beta 1 and independent accessory cell-derived contact and soluble factors.

Natural killer cells (NK) were studied to determine factors important in their expansion. Flourescence-activated cell sorter (FACS) purified CD56+/CD3- NK cells cultured alone for 18 days in rIL-2 containing medium (1,000 U/mL) showed enhanced cytotoxicity but only minimal expansion. NK expansion was increased (12.5 +/- 1.6-fold) by coculturing NK with soluble factors produced by irradiated peripheral blood mononuclear cells (PBMNC) in which the two populations were separated by a microporous membrane. However, maximal NK expansion was always observed when NK were cocultured in direct contact with irradiated PBMNC (49.4 +/- 5.9-fold). To determine if marrow stroma, which supports differentiation of primitive NK progenitors, was a better accessory cell population than irradiated PBMNC, NK were cocultured in direct contact with primary marrow stromal layers. NK expansion with marrow stroma was similar to PBMNC. Fibroblast cell lines (M2-10B4, NRK-49F, NIH-3T3) and human umbilical vein endothelial cells (HUVEC), all homogeneous populations and devoid of monocytes, also exhibited a similar contact-dependent increase in NK expansion. Experiments were designed using fixed M2-10B4 stromal cells to separate the contact-induced proliferative stimuli from soluble factors. NK plated directly on ethanol/acetic acid-fixed M2-10B4, which leaves stromal ligands (cell membrane components and ECM) intact, resulted in increased NK expansion compared with medium alone. We further show that the combination of independent contact and soluble factors is responsible for maximal late NK expansion (days 28 through 40) but paradoxically inhibits early NK expansion (day 7). The proliferation inhibitory effects were verified by 3H-thymidine uptake and could be detected at days 2 through 6 but no longer 14 days after the initiation of the culture. We show that both laminin and thrombospondin inhibit early NK proliferation, whereas only thrombospondin was capable of also stimulating late NK expansion. The effect of thrombospondin on early NK proliferation is related to activation of transforming growth factor-beta 1 (TGF-beta) because anti-TGF-beta neutralizing antibody completely abrogated thrombospondin-mediated inhibition of early NK proliferation. Although inhibitory early in culture, active TGF-beta added only at culture initiation increases late NK expansion similar to thrombospondin. TGF-beta was not present in the thrombospondin preparation but latent TGF-beta in serum, or TGF-beta transcripts identified in IL-2-activated NK could explain paracrine or autocrine mechanisms for the regulation of NK proliferation. Finally, anti-TGF-beta neutralizing antibody only minimally affects stroma-mediated inhibition of early NK proliferation suggesting that aside from thrombospondin/TGF-beta, additional contact factors are important for the regulation of NK proliferation.

3T3 Cells↗