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Background: In August 2013, a naloxone distribution program was implemented in North Carolina (NC). This study evaluated that program by quantifying the association between the program and county-level opioid overdose death (OOD) rates and conducting a cost-benefit analysis., Methods: One-group pre-post design. Data included annual county-level counts of naloxone kits distributed from 2013 to 2016 and mortality data from 2000-2016. We used generalized estimating equations to estimate the association between cumulative rates of naloxone kits distributed and annual OOD rates. Costs included naloxone kit purchases and distribution costs; benefits were quantified as OODs avoided and monetized using a conservative value of a life., Results: The rate of OOD in counties with 1-100 cumulative naloxone kits distributed per 100,000 population was 0.90 times (95% CI: 0.78, 1.04) that of counties that had not received kits. In counties that received >100 cumulative kits per 100,000 population, the OOD rate was 0.88 times (95% CI: 0.76, 1.02) that of counties that had not received kits. By December 2016, an estimated 352 NC deaths were avoided by naloxone distribution (95% CI: 189, 580). On average, for every dollar spent on the program, there was $2742 of benefit due to OODs avoided (95% CI: $1,237, $4882)., Conclusions: Our estimates suggest that community-based naloxone distribution is associated with lower OOD rates. The program generated substantial societal benefits due to averted OODs. States and communities should continue to support efforts to increase naloxone access, which may include reducing legal, financial, and normative barriers., (Copyright © 2019 Elsevier B.V. All rights reserved.)

GLI1 is one of three transcription factors (GLI1, GLI2 and GLI3) that mediate the Hedgehog signal transduction pathway and play important roles in normal development. GLI1 and GLI2 form a positive-feedback loop and function as human oncogenes. The mouse and human GLI1 genes have untranslated 5' exons and large introns 5' of the translational start. Here we show that Sonic Hedgehog (SHH) stimulates occupancy in the introns by H3K27ac, H3K4me3 and the histone reader protein BRD4. H3K27ac and H3K4me3 occupancy is not significantly changed by removing BRD4 from the human intron and transcription start site (TSS) region. We identified six GLI binding sites (GBS) in the first intron of the human GLI1 gene that are in regions of high sequence conservation among mammals. GLI1 and GLI2 bind all of the GBS in vitro. Elimination of GBS1 and 4 attenuates transcriptional activation by GLI1. Elimination of GBS1, 2, and 4 attenuates transcriptional activation by GLI2. Eliminating all sites essentially eliminates reporter gene activation. Further, GLI1 binds the histone variant H2A.Z. These results suggest that GLI1 and GLI2 can regulate GLI1 expression through protein-protein interactions involving complexes of transcription factors, histone variants, and reader proteins in the regulatory intron of the GLI1 gene. GLI1 acting in trans on the GLI1 intron provides a mechanism for GLI1 positive feedback and auto-regulation. Understanding the combinatorial protein landscape in this locus will be important to interrupting the GLI positive feedback loop and providing new therapeutic approaches to cancers associated with GLI1 overexpression., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The safety and efficacy of ibrutinib (420 mg) in chronic lymphocytic leukemia (CLL) were evaluated in a phase 2 study; 51 patients had TP53 aberration (TP53 cohort) and 35 were enrolled because of age 65 years or older (elderly cohort). Both cohorts included patients with treatment-naive (TN) and relapsed/refractory (RR) CLL. With the median follow-up of 4.8 years, 49 (57.0%) of 86 patients remain on study. Treatment was discontinued for progressive disease in 20 (23.3%) patients and for adverse events in 5 (5.8%). Atrial fibrillation occurred in 18 (20.9%) patients for a rate of 6.4 per 100 patient-years. No serious bleeding occurred. The overall response rate at 6 months, the primary study endpoint, was 95.8% for the TP53 cohort (95% confidence interval, 85.7%-99.5%) and 93.9% for the elderly cohort (95% confidence interval, 79.8%-99.3%). Depth of response improved with time: at best response, 14 (29.2%) of 48 patients in the TP53 cohort and 9 (27.3%) of 33 in the elderly cohort achieved a complete response. Median minimal residual disease (MRD) in peripheral blood was 3.8 × 10 -2 at 4 years, with MRD-negative (<10 -4 ) remissions in 5 (10.2%) patients. In the TP53 cohort, the estimated 5-year progression-free survival (PFS) was 74.4% in TN-CLL compared with 19.4% in RR-CLL ( P = .0002), and overall survival (OS) was 85.3% vs 53.7%, respectively ( P = .023). In the elderly cohort, the estimated 5-year PFS and OS in RR-CLL were 64.8% and 71.6%, respectively, and no event occurred in TN-CLL. Long-term administration of ibrutinib was well tolerated and provided durable disease control for most patients. This trial was registered at www.clinicaltrials.gov as #NCT01500733., (© 2018 by The American Society of Hematology.)

Intrabone (IB) hematopoietic cell transplantation (HCT) of umbilical cord blood in humans remains experimental and the technique has not been optimized. It is unknown whether hematopoietic progenitor cells (HPCs) injected IB are initially retained in the marrow or rapidly enter into the venous circulation before homing to the marrow. To develop an IB-injection technique that maximizes HPC marrow-retention, we tracked radiolabeled human HPCs following IB-injection into swine. We developed a method to radionuclide-label HPCs using a long-lived positron emitter (89) Zr and protamine sulfate that resulted in cellular-retention of low-dose radioactivity. This approach achieved radioactivity levels sufficient for detection by positron emission tomography with both high sensitivity and spatial resolution when fused with computed tomography. We found that conditions utilized in pilot IB-HCT clinical trials conducted by others led to both rapid drainage into the central venous circulation and cellular extravasation into surrounding muscle and soft tissues. By optimizing the needle design, using continuous real-time intra-marrow pressure monitoring, and by reducing the infusion-volume and infusion-rate, we overcame this limitation and achieved high retention of HPCs in the marrow. This method of IB cellular delivery is readily applicable in the clinic and could be utilized in future investigational IB-HCT trials aimed at maximizing marrow retention of HPCs., (Published 2015. This article is a U.S. Government work and is in the public domain in the USA.)

Background: North Carolina, like much of the U.S. South, is disproportionately affected by HIV and hepatitis. This persistently high disease burden may be driven in part by laws that criminalize the possession and distribution of syringes for illicit drug use. Legal change to decriminalize syringes may reduce infection rates in the state, but is unlikely absent support from law enforcement actors., Methods: We analyzed the responses of 350 North Carolina law enforcement officers to a confidential, anonymous survey. The survey instrument collected data regarding self-reported needle-stick injury (NSI), blood borne disease risk perception and attitudes toward syringe decriminalization., Results: 82% of respondents reported that contracting HIV was a "big concern" for them. 3.8% of respondents reported ever receiving a job-related NSI, a rate of 36 NSI per 10,000 officer-years. Majorities of respondents reported positive views regarding syringe decriminalization, with approximately 63% agreeing that it would be "good for the community" and 60% agreeing that it would be "good for law enforcement." Black and female officers were significantly less likely to agree that on-the-job NSI was a "big concern" and significantly more likely to agree that it would be good for law enforcement., Conclusions: These findings suggest that many North Carolina LEOs understand the public health benefits of syringe access programs and may be inclined to support syringe decriminalization legislation. Further research is indicated to determine the causes of observed differences in perceptions of bloodborne disease risk and attitudes toward syringe decriminalization by race and sex., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Background Aims: Although cytomegalovirus (CMV) infection after allogeneic stem cell transplantation (SCT) is rarely fatal, the management of CMV by pre-emptive medication for viral reactivation has toxicity and carries a financial burden. New strategies to prevent CMV reactivation with vaccines and antiviral T cells may represent an advance over pre-emptive strategies but have yet to be justified in terms of transplantation outcome and cost., Methods: We compared outcomes and post-transplantation treatment cost in 44 patients who never required pre-emptive CMV treatment with 90 treated patients undergoing SCT at our institute between 2006 and 2012. Eighty-one subjects received CD34+ selected myeloablative SCT, 12 umbilical cord blood transplants, and 41 T-replete non-myeloablative SCT. One hundred nineteen patients (89%) were at risk for CMV because either the donor or recipient was seropositive. Of these, 90 patients (75.6%) reactivated CMV at a median of 30 (range 8-105) days after transplantation and received antivirals., Results: There was no difference in standard transplantation risk factors between the two groups. In multivariate modeling, CMV reactivation >250 copies/mL (odds ratio = 3, P < 0.048), total duration of inpatient IV antiviral therapy (odds ratio = 1.04, P < 0.001), type of transplantation (T-deplete vs. T-replete; odds ratio = 4.65, P < 0.017) were found to be significantly associated with increased non-relapse mortality. The treated group incurred an additional cost of antiviral medication and longer hospitalization within the first 6 months after SCT of $58,000 to $74,000 per patient., Conclusions: Our findings suggest that to prevent CMV reactivation, treatment should be given within 1 week of SCT. Preventative treatment may improve outcome and have significant cost savings., (Published by Elsevier Inc.)

The ethics of diagnosis and management of fetal genetic disorders are particularly controversial because of the contested status of the fetus and perceptions of genetics. An additional complicating factor is the potential conflict between mother and fetus. Ethical issues in diagnosis include the nature and purpose of the diagnosis itself, and management of the information. Management of the disorder includes issues of termination as an option, and the emerging field of fetal gene therapy with associated issues of somatic versus germ-line interventions., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background Aims: Multipotent mesenchymal stromal cells, also known as mesenchymal stem cells (MSC), can be isolated from adult and fetal tissues. Recently, there has been considerable interest in MSC because they have features favorable for transplantation, namely their multipotency and non-immunogenic properties., Methods: We analyzed how human MSC derived from first-trimester fetal liver and adult bone marrow interact with naive and activated innate natural killer (NK) cells. NK cell function was studied by measuring killing of MSC, as well as degranulation (CD107a) induced by MSC. To assess the importance of NK cell killing, expression of surface epitopes was analyzed by flow cytometry on MSC before and after stimulation with interferon (IFN)γ., Results: Fetal and adult MSC express several ligands to activating NK cell receptors as well as low levels of HLA class I, with large inter-individual variation. Naive peripheral blood NK cells did not lyse fetal or adult MSC, whereas interleukin (IL)2 activated allogeneic as well as autologous NK cells did. Pre-incubation of MSC with IFN-γ increased their levels of HLA class I, protecting them from NK cell recognition. Fetal and adult MSC were preferably killed via the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL) pathways, respectively. Blocking NKG2D reduced NK cell degranulation in both fetal and adult MSC., Conclusions: Fetal and adult MSC differ in their interactions with NK cells. Both fetal and adult MSC are susceptible to lysis by activated NK cells, which may have implications for the use of MSC in cell therapy.

Ligation of inhibitory receptors renders natural killer (NK) cells inactive against autologous tumors. Recently, the proteasome inhibitor bortezomib was shown to sensitize tumors to autologous NK-cell cytotoxicity in vitro. Here, we show bortezomib augments the antitumor effects of syngeneic NK-cell infusions in tumor-bearing animals; this effect is further enhanced in regulatory T cell (Treg cell)-depleted hosts. In vitro, bortezomib-treated tumors had higher tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and perforin/granzyme-mediated caspase-8 activity, which enhanced their susceptibility to NK-cell lysis. Bioluminescence imaging of mice with established tumors showed treatment with bortezomib and syngeneic NK cells reduced tumor growth and prolonged survival compared with controls receiving bortezomib or NK cells alone. In contrast, tumor progression was not delayed when animals received bortezomib and perforin-deficient NK cells, showing drug-induced augmentation in NK-cell cytotoxicity was mediated through perforin/granzyme. Furthermore, tumor growth was slower in bortezomib-treated recipients when host Treg cells were eradicated with anti-CD25 antibody before infusing NK cells compared with mice without Treg-cell ablation (tumor doubling time, 16.7 vs 4.9 days, respectively; P = .02). These findings suggest that depletion of Treg cells followed by bortezomib-induced tumor sensitization to autologous NK cells could be used as a novel strategy to treat cancer.

Primitive quiescent CD34(+) chronic myeloid leukemia (CML) cells are more biologically resistant to tyrosine kinase inhibitors than their cycling counterparts; however, graft-versus-leukemia (GVL) effects after allogeneic stem cell transplantation (SCT) probably eliminate even these quiescent cells in long-term surviving CML transplant recipients. We studied the progeny of CD34(+) cells from CML patients before SCT, which were cultured 4 days in serum-free media with hematopoietic growth factors. BCR-ABL expression was similar in both cycling and quiescent noncycling CD34(+) populations. Quiescent CD34(+) cells from CML patients were less susceptible than their cycling CD34(+) and CD34(-) counterparts to lysis by natural killer (NK) cells from their HLA-identical sibling donors. Compared with cycling populations, quiescent CD34(+) CML cells had higher surface expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5. Bortezomib up-regulated TRAIL receptor expression on quiescent CD34(+) CML cells, and further enhanced their susceptibility to cytotoxicity by in vitro expanded donor NK cells. These results suggest that donor-derived NK cell-mediated GVL effects may be improved by sensitizing residual quiescent CML cells to NK-cell cytotoxicity after SCT. Such treatment, as an adjunct to donor lymphocyte infusions and pharmacologic therapy, may reduce the risk of relapse in CML patients who require treatment by SCT.

Background Aims: Cancer immunotherapy involving natural killer (NK) cell infusions and administration of therapeutic agents modulating the susceptibility of tumors to NK-cell lysis has been proposed recently. We provide a method for expanding highly cytotoxic clinical-grade NK cells in vitro for adoptive transfer following bortezomib treatment in patients with advanced malignancies., Methods: NK cells were expanded with irradiated Epstein-Barr virus-transformed lymphoblastoid cells. Expanded cells were evaluated for their phenotype, cytotoxicity, cytokine secretion, dependence on interleukin (IL)-2 and ability to retain function after cryopreservation., Results: A pure population of clinical-grade NK cells expanded 490+/-260-fold over 21 days. Expanded NK cells had increased TRAIL, FasL and NKG2D expression and significantly higher cytotoxicity against bortezomib-treated tumors compared with resting NK cells. Expanded NK cells, co-cultured with K562 and renal cell carcinoma tumor targets, secreted significantly higher levels of soluble Fas ligand 6; fgjhd IFN-gamma, GM-CSF, TNF-alpha, MIP-1alpha and MIP-1beta compared with resting NK cells. Secretion of the above cytokines and NK-cell cytolytic function were IL-2 dose dependent. Cryopreservation of expanded NK cells reduced expression of NKG2D and TRAIL and NK-cell cytotoxicity, although this effect could be reversed by exposure of NK cells to IL-2., Conclusions: We describe a method for large-scale expansion of NK cells with increased expression of activating receptors and death receptor ligands resulting in superior cytotoxicity against tumor cells. This ex vivo NK-cell expansion technique is currently being utilized in a clinical trial evaluating the anti-tumor activity of adoptively infused NK cells in combination with bortezomib.

Recently there has been a substantial gain in our understanding of the role NK-cells play in mediating innate host immune responses. Although NK cells have long been known to mediate antigen independent tumor cytotoxicity, the therapeutic potential of NK cell-based immunotherapy has yet to be realized. Manipulating the balance between inhibitory and activating NK receptor signals, sensitization of tumor target cells to NK cell-mediated apoptosis, and recent discoveries in NK-cell receptor biology have fueled translational research that has led to clinical trials investigating a number of novel methods to potentiate NK cytotoxicity against human malignancies.

Perforin is a cytolytic protein expressed mainly in activated cytotoxic lymphocytes and natural killer cells. Inherited perforin mutations account for 20% to 40% of familial hemophagocytic lymphohistiocytosis, a fatal disease of early childhood characterized by the absence of functional perforin. Aplastic anemia, the paradigm of immune-mediated bone marrow failure syndromes, is characterized by hematopoietic stem cell destruction by activated T cells and Th1 cytokines. We examined whether mutations in the perforin gene occurred in acquired aplastic anemia. Three nonsynonymous PRF1 mutations among 5 unrelated patients were observed. Four of 5 patients with the mutations showed some hemophagocytosis in the bone marrow at diagnosis. Perforin protein levels in these patients were very low or absent, and perforin granules were completely absent. Natural killer (NK) cell cytotoxicity from these patients was significantly decreased. Our data suggest that PRF1 genetic alterations help explain the aberrant proliferation and activation of cytotoxic T cells and may represent genetic risk factors for bone marrow failure.

We investigated if an infusion of alloreactive natural killer (NK) cells would reduce GVHD and mediate antitumor effects in mice undergoing MHC-matched allogeneic stem cell transplantation (SCT). Balb/c mice bearing RENCA tumors underwent an allogeneic SCT from MHC-matched B10.d2 donors and were given a single infusion of either Ly49 ligand-matched, ligand-mismatched, or no donor NK cells. Recipients of Ly49 ligand-mismatched NK cells had a reduced incidence of graft-versus-host disease (GVHD; 39% vs 100%; P < .01), and prolonged survival (median 84 days vs 39 days; P < .01) compared with SCT recipients not receiving NK cells. Recipients of Ly49 ligand-matched NK cells had the same incidence of GVHD and similar survival compared with controls not receiving NK cells. Pulmonary tumor burden was significantly (P < .01) lower in recipients that received Ly49-mismatched or Ly49-matched NK cells compared with recipients not receiving NK cells. These data provide in vivo evidence that a single infusion of alloreactive donor NK cells reduces GVHD and mediates antitumor effects following MHC-matched allogeneic transplantation.

Eighty patients with chronic myeloid leukemia (CML) underwent T cell-depleted stem cell transplantation from an HLA-identical sibling, with add-back of donor T cells on days 30 to 45 and days 60 to 100 in patients in whom grade 2 or greater acute graft-versus-host disease (GVHD) developed. The outcomes for 54 patients with chronic-phase (CP) and 26 with advanced-phase (AP) disease were as follows: overall survival, 85% +/- 5% versus 36% +/- 10%; transplantation-related mortality (TRM), 13% +/- 5% versus 43% +/- 11%; and current leukemia-free survival, 76% +/- 6% versus 34% +/- 9%. The day-30 lymphocyte count (LC30) was strongly associated with outcome. For patients in CP with counts greater than the median of 0.30 x 10(9)/L, survival was 100% versus 70% +/- 9% (P = .003); current LFS 100% versus 56% +/- 9% (P = .002); and TRM 0% versus 26% +/- 8% (P = .006). Higher-than-median LC30 correlated significantly with molecular remission (MR) at 3, 6, and 12 months and with higher CD34 doses. Lymphocyte subset analysis performed in 20 patients available for phenotyping showed that LC30 was highly correlated with absolute CD56+CD3- natural killer cell numbers (NK30), which also predicted for survival and MR. CD34 cell dose, LC30, and NK30, but not day-30 CD3+ cell count, were highly correlated and were significant predictors of transplantation outcome. These results suggest that transplanted CD34 cell doses greater than 5 x 10(6)/kg may improve outcomes by increasing the early recovery of NK cells.

We have selectively depleted host-reactive donor T cells from peripheral blood stem cell (PBSC) transplant allografts ex vivo using an anti-CD25 immunotoxin. We report a clinical trial to decrease graft-versus-host disease (GVHD) in elderly patients receiving selectively depleted PBSC transplants from HLA-identical sibling donors. Sixteen patients (median age, 65 years [range, 51-73 years]), with advanced hematologic malignancies underwent transplantation following reduced-intensity conditioning with fludarabine and either cyclophosphamide (n = 5), melphalan (n = 5), or busulfan (n = 6). Cyclosporine was used as sole GVHD prophylaxis. The allograft contained a median of 4.5 x 10(6) CD34 cells/kg (range, 3.4-7.3 x 10(6) CD34 cells/kg) and 1.0 x 10(8)/kg (range, 0.2-1.5 x 10(8)/kg) selectively depleted T cells. Fifteen patients achieved sustained engraftment. The helper T-lymphocyte precursor (HTLp) frequency assay demonstrated successful (mean, 5-fold) depletion of host-reactive donor T cells, with conservation of third-party response in 9 of 11 cases tested. Actuarial rates of acute GVHD were 46% +/- 13% for grades II to IV and 12% +/- 8% for grades III to IV. These results suggest that allodepletion of donor cells ex vivo is clinically feasible in older patients and may reduce the rate of severe acute GVHD. Further studies with selectively depleted transplants to evaluate graft-versus-leukemia (GVL) and survival are warranted.

Antigens implicated in the graft-versus-leukemia (GVL) effect in chronic myeloid leukemia (CML) include WT1, PR1, and BCR-ABL. To detect very low frequencies of these antigen-specific CD8+ T cells, we used quantitative polymerase chain reaction (qPCR) to measure interferon-gamma (IFN-gamma) mRNA production by peptide-pulsed CD8+ T cells from HLA-A*0201+ healthy volunteers and from patients with CML before and after allogeneic stem cell transplantation (SCT). Parallel assays using cytomegalovirus (CMV) pp65 tetramers demonstrated the IFN-gamma copy number to be linearly related to the frequency of tetramer-binding T cells, sensitive to frequencies of 1 responding CD8+ T cell/100 000 CD8+ T cells. Responses to WT1 and PR1 but not BCR-ABL were detected in 10 of 18 healthy donors. Responses to WT1, PR1, or BCR-ABL were observed in 9 of 14 patients with CML before SCT and 5 of 6 after SCT, often to multiple epitopes. Responses were higher in patients with CML compared with healthy donors and highest after SCT. These antigen-specific CD8+ T cells comprised central memory (CD45RO+CD27+CD57-) and effector memory (CD45RO-CD27-CD57+) T cells. In conclusion, leukemia-reactive CD8+ T cells derive from memory T cells and occur at low frequencies in healthy individuals and at higher frequencies in patients with CML. The increased response in patients after SCT suggests a quantitative explanation for the greater effect of allogeneic SCT.

To assess the safety and efficacy of nonmyeloablative allogeneic transplantation in patients with HIV infection, a clinical protocol was initiated in patients with refractory hematologic malignancies and concomitant HIV infection. The results from the first 2 patients are reported. The indications for transplantation were treatment-related acute myelogenous leukemia and primary refractory Hodgkin disease in patients 1 and 2, respectively. Only patient 1 received genetically modified cells. Both patients tolerated the procedure well with minimal toxicity, and complete remissions were achieved in both patients, but patient 2 died of relapsed Hodgkin disease 12 months after transplantation. Patient 1 continues in complete remission with undetectable HIV levels and rising CD4 counts, and with both the therapeutic and control gene transfer vectors remaining detectable at low levels more than 2 years after transplantation. These results suggest that nonmyeloablative allogeneic transplantation in the context of highly active antiretroviral therapy is feasible in patients with treatment-sensitive HIV infection.

Background: Container integrity is critical for maintaining sterility of cryopreserved cellular therapy products. We investigated a series of catastrophic bag failures, first noticed in early 2001., Methods: Process records were reviewed for all PBPC and lymphocyte products cryopreserved in bags from January 2000 through April 2002. Patient charts were also reviewed., Results: One thousand two hundred and four bags were removed from storage for infusion to 261 patients. All products had been cryopreserved in Cryocyte poly(ethylene co-vinyl acetate) (EVA) bags in either 10% DMSO or 5% DMSO and 6% pentastarch. Product volumes were 25-75 mL, and bags were stored with overwrap bags in a liquid nitrogen tank. From January 2000 to April 2001, failure occurred in 10 of 599 (1.7%) bags. From May 2001 to April 2002, 58 of 605 (9.6%) bags failed, typically with extensive fractures that were visible before thaw. Of the 58 that failed, 24 were salvaged by aseptic methods and infused to patients under antibiotic coverage; 10 of those 24 (42%) had positive bacterial cultures. Bag failures were not related to product type, cryoprotectant solution, liquid versus vapor storage, or freezer location. Failures were linked to use of four Cryocyte bag lots manufactured in 2000 and 2001. After replacing these lots with a 1999 Cryocyte lot and with KryoSafe polyfluoroethylene polyfluoropropylene (FEP) bags, no more failures occurred in 75 and 102 bags, respectively, thawed through April 2002., Discussion: High rates of bag failure were associated with four Cryocyte bag lots. No serious adverse patient effects occurred, but bag failures led to microbial contamination, increased product preparation time, increased antibiotic use, and increased resource expenditure to replace products.

Delayed donor red cell engraftment and pure red cell aplasia (PRCA) are well-recognized complications of major ABO-incompatible hematopoietic stem cell transplantation (SCT) performed by means of myeloablative conditioning. To evaluate these events following reduced-intensity nonmyeloablative SCT (NST), consecutive series of patients with major ABO incompatibility undergoing either NST (fludarabine/cyclophosphamide conditioning) or myeloablative SCT (cyclophosphamide/high-dose total body irradiation) were compared. Donor red blood cell (RBC) chimerism (initial detection of donor RBCs in peripheral blood) was markedly delayed following NST versus myeloablative SCT (median, 114 versus 40 days; P <.0001) and strongly correlated with decreasing host antidonor isohemagglutinin levels. Antidonor isohemagglutinins declined to clinically insignificant levels more slowly following NST than myeloablative SCT (median, 83 versus 44 days; P =.03). Donor RBC chimerism was delayed more than 100 days in 9 of 14 (64%) and PRCA occurred in 4 of 14 (29%) patients following NST, while neither event occurred in 12 patients following myeloablative SCT. Conversion to full donor myeloid chimerism following NST occurred significantly sooner in cases with, compared with cases without, PRCA (30 versus 98 days; P =.008). Cyclosporine withdrawal appeared to induce graft-mediated immune effects against recipient isohemagglutinin-producing cells, resulting in decreased antidonor isohemagglutinin levels and resolution of PRCA following NST. These data indicate that significantly delayed donor erythropoiesis is (1) common following major ABO-incompatible NST and (2) associated with prolonged persistence of host antidonor isohemagglutinins. The clinical manifestations of these events are affected by the degree and duration of residual host hematopoiesis.

We reevaluate here an earlier report of monosaccharide binding by the C-type lectin-like, leukocyte surface protein CD69 in the form of a recombinant soluble dimer, and we examine polysaccharide binding by the protein. We have expressed in Escherichia coli a new construct of the extracellular part (Q(65)-K(199)) of human CD69. We describe the folding in vitro to produce, in good yield, the protein in a soluble, disulphide-linked, dimeric form, and the results of binding experiments with monosaccharides: glucose, galactose, mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine, linked to bovine serum albumin. Monosaccharide-binding signals are not detectable. Among the polysaccharides, heparin, chondroitin sulphates A, B, and C, fucoidan, and dextran sulphate, CD69 dimer gives a weak binding signal with fucoidan., (Copyright 1999 Academic Press.)

Nonmyeloablative allogeneic stem cell transplantation has recently been explored as a safer alternative to conventional high-dose transplant regimens. Although a high incidence of mixed chimerism after nonmyeloablative procedures has been reported, the exact kinetics of engrafting donor cells in specific cellular lineages has yet to be defined. We investigated lineage-specific chimerism in 15 patients receiving an allogeneic peripheral blood stem cell (PBSC) transplant from an HLA-identical (n = 14) or a 5/6 antigen-matched sibling donor after a preparative regimen of cyclophosphamide and fludarabine. Donor chimerism was assessed weekly in T lymphocytes and myeloid cells by polymerase chain reaction (PCR) of minisatellite regions. Eight patients survived between 121 to 409 days after transplant. Ten of 14 patients surviving more than 30 days (71.4%) had delayed disease regression consistent with a graft-versus-malignancy (GVM) effect. One patient rejected the transplant with subsequent recovery of autologous hematopoiesis. Hematological recovery was rapid (median, 11 days to >/=500 neutrophils/microL) and was initially predominantly recipient in origin. Donor myeloid chimerism gradually supplanted recipient hematopoiesis and became fully donor in all survivors by 200 days after transplantation. In contrast, T-cell engraftment was more rapid, with full chimerism in 7 patients by day 30 and in 6 further patients by day 200 after cyclosporine withdrawal and donor lymphocyte infusion. Full donor T-cell engraftment preceded donor myeloid engraftment, acute graft-versus-host disease, and disease regression, consistent with a requirement for 100% donor T-cell chimerism for full expression of the alloresponse. These results emphasize the importance of lineage-specific chimerism analysis to successfully manipulate engraftment after nonmyeloablative allogeneic PBSC transplantation.

This study was aimed at investigating the efficacy of presentation of biotinylated oligosaccharides on streptavidin-coated microwells for interactions with (a) three monoclonal antibodies directed at sialyl-Lewisa (Le(a)) or sulfo-Le(a)-related sequences, and (b) the endothelium-leukocyte adhesion molecules, the E-, L- and P-selectins which recognize both the sulfo- and sialyl-Le(a) series. With the antibodies it was observed that if the biotinylated oligosaccharide incorporated the entire antigenic determinant, and additional saccharide length was not included, the biotinyl tag spacer length was a critical factor in the strength of the binding signal. If oligosaccharide chain beyond the determinant was included, the biotinyl tag spacer length was less important. The E-selectin binding data with the biotinylated sialyl- and sulfo-oligosaccharides were in overall accord with previous knowledge. With the L- and P-selectins, however, unexpectedly low binding signals were elicited by biotinyl sulfo-Le(a) sequences relative to those with the sialyl-analogs. This suppression was more pronounced with the rodent than the human L-selectin. Such differential availabilities of oligosaccharides displayed on streptavidin may relate to biological situations, such as the differential reactivities of the three selectins with a given oligosaccharide ligand presented on different carrier proteins, or on different O-glycan cores on mucin-type glycoproteins.

The aim of the present study has been to investigate the binding specificity of the soluble 14,000-dalton lectin of bovine heart muscle towards immobilised oligosaccharides in clustered form. To this end, chromatogram overlay assays and quantitative plastic-microwell-binding assays have been performed using several natural glycolipids and neoglycolipids containing one or more of the disaccharide units, beta-D-Galp-(1----4 or 3)-D-GlcNAc or beta-D-Galp-(1----4)-D-Glc and related structures. The microwell assay gave the most consistent results. It was observed that for binding by the soluble lectin the optimal sequence, which is beta-D-Galp-(1----4 or 3)-D-GlcNAc, must occur at the nonreducing end of longer oligosaccharides when linked to lipid. These oligosaccharides may be of poly(N-acetyllactosamine) type or they may be mono- or multi-antennary, complex-type chains in which the disaccharide is joined directly to a trimannosyl core. The lectin bound to such immobilised lipid-linked oligosaccharides on which the terminal D-galactosyl groups are substituted with alpha-L-Fucp-(1----2), alpha-D-Galp-(1----3), or alpha-NeuAc-(2----3) groups. However, no binding was detected if the terminal D-galactosyl groups were substituted with an alpha-NeuAc-(2----6) group or the subterminal N-acetylglucosamine units with an alpha-L-Fucp-(1----3 or -4) group. Internally located N-acetyllactosamine units where the D-galactose units are disubstituted by beta-D-GlcNacp-(1----3) and -(1----6) units, as in branched poly(N-acetyllactosamine) backbones were not bound by the bovine lectin. These results are in accord with previous observations on the bovine lectin and the corresponding human and rat lectins, using structurally defined oligosaccharides as inhibitors of binding. The results of comparative binding experiments using paragloboside and ceramide hexasaccharide which contain one and two N-acetyllactosamine units, respectively, joined in linear sequence to the lactosylceramide core, were equivocal with respect to the availability of the internal N-acetyllactosamine units for binding by the bovine lectin.

High molecular weight glycoproteins of human B and T lymphocytes known as leucocyte common antigen or T200 have been shown to carry O- and N-glycosidically linked, sialylated, carbohydrate chains. The O-linked chains are polydisperse and those of B rather than T cell type are highly susceptible to degradation by endo-beta-galactosidase. These differences among lymphocytes that are functionally distinct raise the possibility that the oligosaccharides may contribute to the functions of these differentiation molecules as well as to their electrophoretic diversity.

Isotopes of iodine are often employed as radiolabels for antibodies used in radioimmunodetection studies in which tumor localization is determined by external imaging. Because of drawbacks associated with the use of these isotopes, alternative labeling methods have been considered; such as covalently attaching strong chelators so that the coupled protein may be radiolabeled with metallic radionuclides by chelation. We have developed a method of coupling the strong chelator diethylenetriaminepentaacetic acid (DTPA) which is simple, efficient, and superior to reported methods. Using the cyclic anhydride, coupling to IgG antibody is about 75% efficient and is completed in less than 1 min at neutral pH. Because the concentration of hydrolytic products is small, the coupled protein is rapidly purified for use or storage. Labeling of the protein is also accomplished rapidly and the labeled product has been shown to be stable both in vitro and in vivo.

A series of oligosaccharides derived from glycoproteins or from human milk were coupled to lipid and used as probes of the binding specificities of Escherichia coli isolated from patients with urinary tract infections. Selective binding to the glycoprotein oligosaccharide probes rich in mannose residues (high-mannose type) was demonstrated with fimbriated E coli that give mannose-inhibitable haemagglutination. This observation is in accordance with predictions from inhibition studies. Binding studies with the human milk oligosaccharide probes, which resemble structures found on host-cell membranes, revealed adhesive specificity unrelated to the presence of fimbriae. This new type of host oligosaccharide receptor is affected by the presence of the blood group genetic markers. It involves the disaccharide sequence linked to the membrane-associated lipid moiety of host-cell glycolipids, and may have a role in initiation of infection on damaged epithelial cell membranes.