Dog hepatocytes are key effector cells in the liver innate immune response to Leishmania infantum.

Hepatocytes constitute the majority of hepatic cells, and play a key role in controlling systemic innate immunity, via pattern-recognition receptors (PRRs) and by synthesizing complement and acute phase proteins. Leishmania infantum, a protozoan parasite that causes human and canine leishmaniasis, infects liver by establishing inside the Kupffer cells. The current study proposes the elucidation of the immune response generated by dog hepatocytes when exposed to L. infantum. Additionally, the impact of adding leishmanicidal compound, meglumine antimoniate (MgA), to parasite-exposed hepatocytes was also addressed. L. infantum presents a high tropism to hepatocytes, establishing strong membrane interactions. The possibility of L. infantum internalization by hepatocytes was raised, but not confirmed. Hepatocytes were able to recognize parasite presence, inducing PRRs [nucleotide oligomerization domain (NOD)1, NOD2 and Toll-like receptor (TLR)2] gene expression and generating a mix pro- and anti-inflammatory cytokine response. Reduction of cytochrome P 450s enzyme activity was also observed concomitant with the inflammatory response. Addition of MgA increased NOD2, TLR4 and interleukin 10 gene expression, indicating an immunomodulatory role for MgA. Hepatocytes seem to have a major role in coordinating liver's innate immune response against L. infantum infection, activating inflammatory mechanisms, but always balancing the inflammatory response in order to avoid cell damage.

A functional glycoproteomics approach identifies CD13 as a novel E-selectin ligand in breast cancer.

BACKGROUND: The glycan moieties sialyl-Lewis-X and/or -A (sLeX/A) are the primary ligands for E-selectin, regulating subsequent tumor cell extravasation into distant organs. However, the nature of the glycoprotein scaffolds displaying these glycans in breast cancer remains unclear and constitutes the focus of the present investigation. METHODS: We isolated glycoproteins that bind E-selectin from the CF1_T breast cancer cell line, derived from a patient with ductal carcinoma. Proteins were identified using bottom-up proteomics approach by nanoLC-orbitrap LTQ-MS/MS. Data were curated using bioinformatics tools to highlight clinically relevant glycoproteins, which were validated by flow cytometry, Western blot, immunohistochemistry and in-situ proximity ligation assays in clinical samples. RESULTS: We observed that the CF1_T cell line expressed sLeX, but not sLeA and the E-selectin reactivity was mainly on N-glycans. MS and bioinformatics analysis of the targeted glycoproteins, when narrowed down to the most clinically relevant species in breast cancer, identified CD44 glycoprotein (HCELL) and CD13 as key E-selectin ligands. Additionally, the co-expression of sLeX-CD44 and sLeX-CD13 was confirmed in clinical breast cancer tissue samples. CONCLUSIONS: Both CD44 and CD13 glycoforms display sLeX in breast cancer and bind E-selectin, suggesting a key role in metastasis development. Such observations provide a novel molecular rationale for developing targeted therapeutics. GENERAL SIGNIFICANCE: While HCELL expression in breast cancer has been previously reported, this is the first study indicating that CD13 functions as an E-selectin ligand in breast cancer. This observation supports previous associations of CD13 with metastasis and draws attention to this glycoprotein as an anti-cancer target.

Leishmania infantum exerts immunomodulation in canine Kupffer cells reverted by meglumine antimoniate.

Kupffer cells (KC) are the liver macrophage population that resides in the hepatic sinusoids and efficiently phagocyte pathogens by establishing an intimate contact with circulating blood. KC constitute the liver host cells in Leishmania infection, nevertheless little is described about their role, apart from their notable contribution in granulomatous inflammation. The present study aims to investigate how canine KC sense and react to the presence of Leishmania infantum promastigotes and amastigotes by evaluating the gene expression of specific innate immune cell receptors and cytokines, as well as the induction of nitric oxide and urea production. Complementarily, the impact of a leishmanicidal drug - meglumine antimoniate (MgA) - in infected KC was also explored. KC revealed to be susceptible to both parasite forms and no major differences were found in the immune response generated. L. infantum parasites seem to interact with KC innate immune receptors and induce an anergic state, promoting immune tolerance and parasite survival. The addition of MgA to infected KC breaks the parasite imposed silence and increased gene expression of Toll-like receptors (TLR) 2 and TLR4, possibly activating downstream pathways. Understanding how KC sense and react to parasite presence could bring new insights into the control or even elimination of canine leishmaniasis.

Acute exercise amplifies inflammation in obese patients with COPD.

Systemic inflammation has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD) systemic effects. However, most COPD patients do not suffer from persistent systemic inflammation even after exacerbations and exercise and scientific evidence has provided conflicting results. Our aim is to evaluate inflammatory gene expression at rest and at 1 and 24h after strenuous exercise in COPD patients and study the patient variables associated with inflammatory expression. A cross-sectional study was conducted in COPD patients who were recruited on entry to a pulmonary rehabilitation (PR) program. Demographic, clinical and functional data were collected. Blood samples were collected and gene expression was analyzed by reverse transcriptase polymerase chain reaction for IFNg, IL1b, IL6, IL8, TNFa, TGFb1 and iNOS. The study included 21 patients (15 men, 71.4%), mean age 66.1 years old (SD=8.27), mean FEV1 46.76% (SD 20.90%), 67% belonging to GOLD grade D, mean BODE index of 3.9, 90.5% with smoking history, mean BMI 25.81 (SD=4.87), median of 1.29 exacerbations in the previous year. There was no statistical significant difference between inflammatory expression at rest and at 1h and 24h after the maximal exercise test for all tested genes. We found an association between BMI and inflammatory expression at all the points of time checked, a slight inverse association occurs with low BMI for mRNA IL1b, IL6, TNFa, TGFb1 and iNOS, and there was a more pronounced positive association for obese patients for all tested genes. This preliminary study did not show an enhanced inflammatory gene expression from rest to 1h and 24h after short-term exercise, but did show an increased inflammatory gene expression in both BMI extremes, both at rest and after exercise, suggesting not only malnourishment, but also obesity as potential links between COPD and systemic inflammation. Studies with larger samples and designed to definitely exclude OSA or OHS as confounding factors in obese patients are required.

KIR genotypic diversity in Portuguese and analysis of KIR gene allocation after allogeneic hematopoietic stem cell transplantation.

The diversity of killer-cell immunoglobulin-like receptors (KIR) genes was evaluated in Portuguese and the observed genotypic profiles were found related to the ones reported in European populations. The KIR repertoire after hematopoietic stem cell transplantation is determined by these gene frequencies and the KIR group B motifs are the less common. We estimated donor-KIR/recipient-ligand interactions in transplants with related donors and unrelated donors found in a local registry or from abroad. A large fraction of transplants had all three ligands of inhibitory receptors, and therefore, in theory were not prone to natural killer cell (NK) mediated alloreactivity. Furthermore, the distribution of KIR alloreactive interactions was found independent of the donor-recipient genetic proximity, probably because of different gene segregation and comparable KIR frequencies in the donor pools.

Clinical outcomes and genetic expression profile in human liver graft dysfunction during ischemia/reperfusion injury.

INTRODUCTION: This study aims to compare the molecular gene expression during ischemia reperfusion injury. Several surgical times were considered: in the beginning of the harvesting (T0), at the end of the cold ischemia period (T1), and after reperfusion (T2) and compared with graft dysfunction after liver transplant (OLT). METHODS: We studied 54 patients undergoing OLT. Clinical, laboratory data, and histologic data (Suzuki classification) as well as the Survival Outcomes Following Liver Transplantation (SOFT) score were used and compared with the molecular gene expression of the following genes: Interleukin (IL)-1b, IL-6, tumor necrosis factor-α, perforin, E-selectin (SELE), Fas-ligand, granzyme B, heme oxygenase-1, and nitric oxide synthetase. RESULTS: Fifteen patients presented with graft dysfunction according to SOFT criteria. No relevant data were obtained by comparing the variables graft dysfunction and histologic variables. We observed a statistically significant relation between SELE at T0 (P = .013) and IL-1ß at T0 (P = .028) and early graft dysfunction. CONCLUSIONS: We conclude that several genetically determined proinflammatory expressions may play a critical role in the development of graft dysfunction after OLT.

Resolution of HLA-B*44:02:01G, -DRB1*14:01:01G and -DQB1*03:01:01G reveals a high allelic variability among 12 European populations.

Within the framework of the EU-funded HLA-NET action, an analysis of three G-group alleles, HLA-B*44:02:01G, DRB1*14:01:01G and DQB1*03:01:01G, was undertaken in 12 European populations. Ambiguities were resolved by polymerase chain reaction-sequence-specific amplification (PCR-SSP) or PCR-sequence-based typing (PCR-SBT) in a total of 5095 individuals. The results of the DRB1*14:01/14:54 ambiguity showed high relative ratios (24-53%) of DRB1*14:01 in Bulgarians, Croatians, Greeks, Italians and Slovenians, contrasting with low ratios (6-13%) in Austrians, Finnish, French, Hungarians, Norwegians and Swiss. Resolution of the B*44:02/44:27 ambiguity showed that B*44:27 had a high relative ratio in Slovenians (25.5%) and Bulgarians (37%) and low in French and Swiss (0.02-1%), and was not observed in Greeks and Italians. The highest relative ratio of DQB1*03:19 was found in Portuguese (11%), by contrast with low ratios (0-3%) in the other five populations. Analysis of the A, B, DRB1 phenotypes and family-derived haplotypes in 1719 and 403 individuals positive for either HLA-B*44:02G or DRB1*14:01G ambiguities, respectively, showed some preferential associations, such as A*26∼DRB1*14:01, B*35∼DRB1*14:01, B*38∼DRB1*14:01 and B*44:27∼DRB1*16. Because these ambiguities are located outside the peptide-binding site, they may not be recognized by alloreactive T-cells. However, because of strong linkage disequilibrium (LD), the DRB1*14:01 vs DRB1*14:54 and the B*44:02 vs B*44:27 mismatches are associated to DRB3-, and C-mismatches, respectively. These results are informative for algorithms searching unrelated hematopoietic stem cell donors. For B*44:27-positive patients, searches are expected to be more successful when requesting donors from Southeastern-European ancestry. Furthermore, the introduction of human leukocyte antigen (HLA)-typing strategies that allow resolving exon 4 (for class I) and exon 3 (for class II) polymorphisms can be expected to contribute significantly to population genetics studies.

Genetic expression profile of human liver grafts in ischemia-reperfusion injury: comparison of familial amyloidotic polyneuropathy and deceased-donor liver grafts.

This study aimed to compare the histologic and molecular gene expression at several surgical times (beginning of harvesting, T0; end of cold ischemia period, T1; and after reperfusion, T2) to characterize the ischemia-reperfusion injury (IRI) in deceased-donor liver grafts harvested from patients with familial amyloidotic polyneuropathy (FAP). For this purpose, 54 patients undergoing liver transplantation were studied and divided into 3 groups: deceased donor to cirrhotic recipient (group 1; n = 27), deceased donor to FAP recipient (group 2; n = 15), and FAP donor to cirrhotic recipient (group 3; n = 12). The main comparison was performed between a histologic score (Suzuki score, adding steatosis and neutrophil infiltration), and molecular gene expression of the following genes: interleukin (IL) 1ß, IL-6, E-selectin, Fas-ligand, granzyme B, heme oxygenase 1 (HO1), and nitric oxide synthetase (iNOS2A). We observed less neutrophil infiltration levels in group 3 in sample T0 (P = .0082), which was associated with gene expression of HO1 in the biopsies at T2 (P = .022). In group 3, the molecular expression of genes related to attenuated proinflammatory reaction during IRI, iNOS2A at T0 and HO1 at T2, was detected. We conclude that FAP liver grafts express differently the genes associated with an attenuated proinflammatory reaction, presenting less neutrophil infiltration at harvesting. These findings add more knowledge about the better short-term outcomes in patients receiving this type of liver graft.

HLA alleles and HLA-B27 haplotypes associated with susceptibility and severity of ankylosing spondylitis in a Portuguese population.

Human leukocyte antigen (HLA)-B27 is the mostly known major histocompatibility complex (MHC) gene associated with ankylosing spondylitis (AS). Nonetheless, there is substantial evidence that other MHC genes appear to be associated with the disease, although it has not yet been established whether these associations are driven by direct associations or by linkage disequilibrium (LD) mechanisms. We aimed to investigate the contributions of HLA class I and II alleles and B27-haplotypes for AS in a case-control study. A total of 188 HLA-B27 AS cases and 189 HLA-B27 healthy controls were selected and typed for HLA class I and II by the Luminex polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSOP) method. Allelic and haplotypic distributions were estimated by maximum likelihood method using Arlequin v3.11 and statistical analysis were performed by Stata10.1. No associations were found between non-HLA-B27 loci and AS susceptibility, but several associations were observed for phenotypic features of the disease. DRB1*08 was identified as a risk factor for uveitis and DQB1*04 seems to provide protection for AS severity (functional, metrological and radiological indexes). A*02/B27/C*02/DRB1*01/DQB1*05 [P<0.0001; odds ratio (OR) = 39.06; 95% confidence interval (CI) (2.34-651)] is the only haplotype that seems to confer susceptibility to AS. Moreover, the haplotype A*02/B27/C*01/DRB1*08/DQB1*04 seems to provide protection for disease functional and radiological repercussions. Our findings are compatible with the hypothesis that other genes within the HLA region besides HLA-B27 might play some role in AS susceptibility and severity.

Postprandial insulin resistance in Zucker diabetic fatty rats is associated with parasympathetic-nitric oxide axis deficiencies.

The Zucker diabetic fatty (ZDF) rat is an obesity and type 2 diabetes model. Progression to diabetes is well characterised in ZDF rats, but only in the fasted state. We evaluated the mechanisms underlying postprandial insulin resistance in young ZDF rats. We tested the hypothesis that the overall postprandial action of insulin is affected in ZDF rats as a result of impairment of the hepatic parasympathetic-nitric oxide (PSN-NO) axis and/or glutathione (GSH), resulting in decreased indirect (PSN-NO axis) and direct actions of insulin. Nine-week-old male ZDF rats and lean Zucker rats (LZR, controls) were used. The action of insulin was assessed in the fed state before and after parasympathetic antagonism atropine. Basal hepatic NO and GSH were measured, as well as NO synthase (NOS) and γ-glutamyl-cysteine synthethase (GCS) activity and expression. ZDF rats presented postprandial hyperglycaemia (ZDF, 201.4 ± 12.9 mg/dl; LZR, 107.7 ± 4.3 mg/dl), but not insulinopaenia (ZDF, 5.9 ± 0.8 ng/ml; LZR, 1.5 ± 0.3 ng/ml). Total postprandial insulin resistance was observed (ZDF, 78.6 ± 7.5 mg glucose/kg; LZR, 289.2 ± 24.7 mg glucose/kg), with a decrease in both the direct action of insulin (ZDF, 54.8 ± 7.0 mg glucose/kg; LZR, 173.3 ± 20.5 mg glucose/kg) and the PSN-NO axis (ZDF, 24.5 ± 3.9 mg glucose/kg; LZR, 115.9 ± 19.4 mg glucose/kg). Hepatic NO (ZDF, 117.2 ± 11.4 µmol/g tissue; LZR, 164.6 ± 4.9 µmol/g tissue) and GSH (ZDF, 4.9 ± 0.3 µmol/g; LZR, 5.9 ± 0.2 µmol/g) were also compromised as a result of decreased NOS and GCS activity, respectively. These results suggest a compromise of the mechanism responsible for potentiating insulin action after a meal in ZDF rats. We show that defective PSN-NO axis and GSH synthesis, together with an impaired direct action of insulin, appears to contribute to postprandial insulin resistance in this model.

Strategies to work with HLA data in human populations for histocompatibility, clinical transplantation, epidemiology and population genetics: HLA-NET methodological recommendations.

HLA-NET (a European COST Action) aims at networking researchers working in bone marrow transplantation, epidemiology and population genetics to improve the molecular characterization of the HLA genetic diversity of human populations, with an expected strong impact on both public health and fundamental research. Such improvements involve finding consensual strategies to characterize human populations and samples and report HLA molecular typings and ambiguities; proposing user-friendly access to databases and computer tools and defining minimal requirements related to ethical aspects. The overall outcome is the provision of population genetic characterizations and comparisons in a standard way by all interested laboratories. This article reports the recommendations of four working groups (WG1-4) of the HLA-NET network at the mid-term of its activities. WG1 (Population definitions and sampling strategies for population genetics' analyses) recommends avoiding outdated racial classifications and population names (e.g. 'Caucasian') and using instead geographic and/or cultural (e.g. linguistic) criteria to describe human populations (e.g. 'pan-European'). A standard 'HLA-NET POPULATION DATA QUESTIONNAIRE' has been finalized and is available for the whole HLA community. WG2 (HLA typing standards for population genetics analyses) recommends retaining maximal information when reporting HLA typing results. Rather than using the National Marrow Donor Program coding system, all ambiguities should be provided by listing all allele pairs required to explain each genotype, according to the formats proposed in 'HLA-NET GUIDELINES FOR REPORTING HLA TYPINGS'. The group also suggests taking into account a preliminary list of alleles defined by polymorphisms outside the peptide-binding sites that may affect population genetic statistics because of significant frequencies. WG3 (Bioinformatic strategies for HLA population data storage and analysis) recommends the use of programs capable of dealing with ambiguous data, such as the 'gene[rate]' computer tools to estimate frequencies, test for Hardy-Weinberg equilibrium and selective neutrality on data containing any number and kind of ambiguities. WG4 (Ethical issues) proposes to adopt thorough general principles for any HLA population study to ensure that it conforms to (inter)national legislation or recommendations/guidelines. All HLA-NET guidelines and tools are available through its website http://hla-net.eu.

Association of IL23R and ERAP1 genes with ankylosing spondylitis in a Portuguese population.

OBJECTIVE: Association between ankylosing spondylitis (AS) and two genes, ERAP1 and IL23R, has recently been reported in North American and British populations. The population attributable risk fraction for ERAP1 in this study was 25%, and for IL23R, 9%. Confirmation of these findings to ERAP1 in other ethnic groups has not yet been demonstrated. We sought to test the association between single nucleotide polymorphisms (SNPs) in these genes and susceptibility to AS among a Portuguese population. We also investigated the role of these genes in clinical manifestations of AS, including age of symptom onset, the Bath Ankylosing Spondylitis Disease Activity, Metrology and Functional Indices, and the modified Stoke Ankylosing Spondylitis Spinal Score. METHODS: The study was conducted on 358 AS cases and 285 ethnically matched Portuguese healthy controls. AS was defined according to the modified New York Criteria. Genotyping of IL23R and ERAP1 allelic variants was carried out with TaqMan allelic discrimination assays. Association analysis was performed using the Cochrane-Armitage and linear regression tests of genotypes as implemented in PLINK for dichotomous and quantitative variables respectively. A meta-analysis for Portuguese and previously published Spanish IL23R data was performed using the StatsDirect Statistical tools, by fixed and random effects models. RESULTS: A total of 14 nsSNPs markers (8 for IL23R, 5 for ERAP1, 1 for LN-PEP) were analysed. Three markers (2 for IL23R and 1 for ERAP1) showed significant single-locus disease associations, confirming that the association of these genes with AS in the Portuguese population. The strongest associated SNP in IL23R was rs1004819 (OR=1.4, p=0.0049), and in ERAP1 was rs30187 (OR=1.26, p=0.035). The population attributable risk fractions in the Portuguese population for these SNPs are 11% and 9.7% respectively. No association was seen with any SNP in LN-PEP, which flanks ERAP1 and was associated with AS in the British population. No association was seen with clinical manifestations of AS. CONCLUSION: These results show that IL23R and ERAP1 genes are also associated with susceptibility to AS in the Portuguese population, and that they contribute a significant proportion of the population risk for this disease.

[Genetic basis of ankylosing spondylitis]. / Espondilite Anquilosante e sua base genética.

Ankylosing spondylitis (AS) is a common rheumatic condition, highly heritable. Much of the genetic contribution to the disease lies in the major histocompatibility complex (MHC). The association with the allele group HLA-B*27 has been described worldwide for 30 years. On the other hand, genome wide scans have provided some interesting results showing that other MHC and non-MHC genes could be implicated either in disease susceptibility and phenotypic manifestations. Different hypothesis for disease pathophysiology have been investigated which contribute for a better understanding of the genetic basis of AS. This review aims to summarize the status of the knowledge in this exciting area. New data may, in a near future, change the screening of patients and generate new insights for the emergence of novel therapies.

Influence of human leucocyte antigen-DRB1 on the susceptibility to rheumatoid arthritis and on the production of anti-cyclic citrullinated peptide antibodies in a Portuguese population.

OBJECTIVE: To clarify the influence of the HLA-DRB1 locus on the susceptibility to rheumatoid arthritis and the production of anti-cyclic citrullinated peptide antibodies (anti-CCP) in a Portuguese population. METHODS: 141 patients with rheumatoid arthritis fulfilling the American College of Rheumatology 1987 revised criteria for rheumatoid arthritis were compared with 150 healthy controls. Human leucocyte antigen (HLA)-DRB1 locus genotyping was assessed by polymerase chain reaction reverse probing assays and sequence-specific primers. Anti-CCP antibodies were quantified by ELISA in patients with rheumatoid arthritis. Frequencies between groups were compared by the two-sided Fisher's exact test and considered significant if p<0.05. RESULTS: The HLA-DRB1*04 and HLA-DRB1*10 groups were highly associated with rheumatoid arthritis (p<0.001 and p = 0.031, respectively). High titres of anti-CCP antibodies were largely associated with the presence of HLA-DRB1*04/10. CONCLUSION: The well-recognised susceptibility alleles to rheumatoid arthritis, HLA-DRB1*04, were associated with rheumatoid arthritis in Portuguese patients. The relatively rare DRB1*10 was also associated with rheumatoid arthritis, as was described previously in other southern European countries. Both groups were associated with high anti-CCP titres, reinforcing its relevance to disease onset.

Impact of donor and recipient cytokine genotypes on renal allograft outcome.

Allelic differences in gene promoter or codifying regions have been described to affect regulation of gene expression, consequently increasing or decreasing cytokine production and signal transduction responses to a given stimulus. This observation has been reported for interleukin (IL)-10 (-1082 A/G; -819/-592 CT/CA), transforming growth factor (TGF)-beta (codon 10 C/T, codon 25 G/C), tumor necrosis factor (TNF)-alpha (-308 G/A), TNF-beta (+252 A/G), interferon (IFN)-gamma (+874 T/A), IL-6 (-174 G/C), and IL-4R alpha (+1902 G/A). To evaluate the influence of these cytokine genotypes on the development of acute or chronic rejection, we correlated the genotypes of both kidney graft recipients and cadaver donors with the clinical outcome. Kidney recipients had 5 years follow-up, at least 2 HLA-DRB compatibilities, and a maximum of 25% anti-HLA pretransplantation sensitization. The clinical outcomes were grouped as follows: stable functioning graft (NR, n = 35); acute rejection episodes (AR, n = 31); and chronic rejection (CR, n = 31). The cytokine genotype polymorphisms were defined using PCR-SSP typing. A statistical analysis showed a significant prevalence of recipient IL-10 -819/-592 genotype among CR individuals; whereas among donors, the TGF-beta codon 10 CT genotype was significantly associated with the AR cohort and the IL-6 -174 CC genotype with CR. Other albeit not significant observations included a strong predisposition of recipient TGF-beta codon 10 CT genotype with CR, and TNF-beta 252 AA with AR. A low frequency of TNF-alpha -308 AA genotype also was observed among recipients and donors who showed poor allograft outcomes.

Haploidentical stem cell transplantation with purified CD34 cells after a chemotherapy-alone conditioning regimen.

We investigated whether a novel chemotherapy-alone conditioning regimen would permit durable engraftment of standard doses of CD34+ purified stem cell grafts from full-haplotype mismatched related donors. We also examined the role of infusing limited doses of donor leukocytes for prevention of leukemia relapse. Our conditioning regimen consisted of thiotepa, fludarabine, rabbit antithymocyte globulin, melphalan, cyclosporin, and prednisolone. Since October 1998, 14 patients with high-risk leukemia were treated; 13 donor-patient pairs shared 3 of 6 HLA antigens, and 1 pair shared 5 of 6 HLA antigens. A median of 5.4 x 10(6) CD34+ cells per kilogram, 1.62 x 10(4) CD3+ cells per kilogram, and 9.32 x 10(4) CD19+ cells per kilogram were infused. T-cell depletion was the only graft-versus-host disease (GVHD) prophylaxis. All patients had prompt engraftment, and no late graft rejections were observed. All surviving patients received at least 1 infusion of donor whole blood containing 5, 7, 10, 25, or 50 x 10(3) CD3+ cells per kilogram between days 25 and 95 after transplantation, after which 8 developed acute GVHD (3 grade I, 2 grade II, 2 grade III, and 1 grade IV) and 2 developed a bronchiolitis obliterans-like syndrome. After attaining complete remission, 5 patients relapsed and died with active leukemia. The estimated relapse-related mortality at 4 years is 38.1%. As of June 15, 2003, 6 of 14 patients have survived a median of 43.5 months after transplantation with 100% donor cells. All 6 surviving patients developed acute GVHD and had a natural killer cell mismatch with their donors in the direction of graft versus host. The estimated overall survival and event-free survival for the 14 patients at 4 years is 41.7% +/- 13.5%.