Inherited mutations in the helicase RTEL1 cause telomere dysfunction and Hoyeraal-Hreidarsson syndrome.

Telomeres repress the DNA damage response at the natural chromosome ends to prevent cell-cycle arrest and maintain genome stability. Telomeres are elongated by telomerase in a tightly regulated manner to ensure a sufficient number of cell divisions throughout life, yet prevent unlimited cell division and cancer development. Hoyeraal-Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and a broad range of pathologies, including bone marrow failure, immunodeficiency, and developmental defects. HHS-causing mutations have previously been found in telomerase and the shelterin component telomeric repeat binding factor 1 (TRF1)-interacting nuclear factor 2 (TIN2). We identified by whole-genome exome sequencing compound heterozygous mutations in four siblings affected with HHS, in the gene encoding the regulator of telomere elongation helicase 1 (RTEL1). Rtel1 was identified in mouse by its genetic association with telomere length. However, its mechanism of action and whether it regulates telomere length in human remained unknown. Lymphoblastoid cell lines obtained from a patient and from the healthy parents carrying heterozygous RTEL1 mutations displayed telomere shortening, fragility and fusion, and growth defects in culture. Ectopic expression of WT RTEL1 suppressed the telomere shortening and growth defect, confirming the causal role of the RTEL1 mutations in HHS and demonstrating the essential function of human RTEL1 in telomere protection and elongation. Finally, we show that human RTEL1 interacts with the shelterin protein TRF1, providing a potential recruitment mechanism of RTEL1 to telomeres.

Loss of kindlin-3 alters the threshold for NK cell activation in human leukocyte adhesion deficiency-III.

Recent evidence suggests that kindlin-3 is a major coactivator, required for most, if not all, integrin activities. Here we studied the function of kindlin-3 in regulating NK cell activation by studying a patient with kindlin-3 deficiency (leukocyte adhesion deficiency-III). We found that kindlin-3 is required for NK cell migration and adhesion under shear force. Surprisingly, we also found that kindlin-3 lowers the threshold for NK cell activation. Loss of kindlin-3 has a pronounced effect on NK cell-mediated cytotoxicity triggered by single activating receptors. In contrast, for activation through multiple receptors, kindlin-3 deficiency is overcome and target cells killed. The realization that NK cell activity is impaired, but not absent in leukocyte adhesion deficiency, may lead to the development of more efficient therapy for this rare disease.

Kindlin-3 is required for the stabilization of TCR-stimulated LFA-1:ICAM-1 bonds critical for lymphocyte arrest and spreading on dendritic cells.

Kindlin-3 is a key lymphocyte function-associated antigen-1 (LFA-1) coactivator deleted in leukocyte adhesion deficiency-III (LAD-III). In the present study, we investigated the involvement of this adaptor in lymphocyte motility and TCR-triggered arrest on ICAM-1 or on dendritic cells (DCs). Kindlin-3-null primary T cells from a LAD-III patient migrated normally on the major lymph node chemokine CCL21 and engaged in normal TCR signaling. However, TCR activation of Kindlin-3-null T lymphocytes failed to trigger the robust LFA-1-mediated T-cell spreading on ICAM-1 and ICAM-1-expressing DCs that is observed in normal lymphocytes. Kindlin-3 was also essential for cytoskeletal anchorage of the LFA-1 heterodimer and for microclustering of LFA-1 within ventral focal dots of TCR-stimulated lymphocytes spread on ICAM-1. Surprisingly, LFA-1 on Kindlin-3-null lymphocytes migrating over CCL21 acquired normal expression of an epitope associated with the conformational activation of the key headpiece domain, ß I. This activated LFA-1 was highly responsive to TCR-triggered ICAM-1-driven stop signals in normal T cells locomoting on CCL21, but not in their Kindlin-3-null T-cell counterparts. We suggest that Kindlin-3 selectively contributes to a final TCR-triggered outside-in stabilization of bonds generated between chemokine-primed LFA-1 molecules and cell-surface ICAM-1.

An SNX10 mutation causes malignant osteopetrosis of infancy.

BACKGROUND: Osteopetrosis is a life-threatening, rare disorder typically resulting from osteoclast dysfunction and infrequently from failure to commitment to osteoclast lineage. Patients commonly present in infancy with macrocephaly, feeding difficulties, evolving blindness and deafness, and bone marrow failure. In ∼70% of the patients there is a molecularly defined failure to maintain an acid pH at the osteoclast-bone interface (the ruffled border) which is necessary for the bone resorptive activity. METHODS AND RESULTS: In eight patients with infantile osteopetrosis which could be cured by bone marrow transplantation, the study identified by homozygosity mapping in distantly related consanguineous pedigrees a missense mutation in a highly conserved residue in the SNX10 gene. The mutation segregated with the disease in the families and was carried by one of 211 anonymous individuals of the same ethnicity. In the patients' osteoclasts, the mutant SNX10 protein was abnormally abundant and its distribution altered. The patients' osteoclasts were fewer and smaller than control cells, their resorptive capacity was markedly deranged, and the endosomal pathway was perturbed as evidenced by the distribution of internalised dextran. CONCLUSIONS: SNX10 was recently shown to interact with vacuolar type H(+)-ATPase (V-ATPase) which pumps protons at the osteoclast-bone interface. Mutations in TCIRG1, the gene encoding a subunit of the V-ATPase complex, account for the majority of cases of osteopetrosis. It is speculated that SNX10 is responsible for the vesicular sorting of V-ATPase from Golgi or for its targeting to the ruffled border. A mutation in SNX10 may therefore result in 'secondary V-ATPase deficiency' with a failure to acidify the resorption lacuna. Determination of the sequence of the SNX10 gene is warranted in molecularly undefined patients with recessive 'pure' osteopetrosis of infancy.

Gene therapy for immunodeficiency due to adenosine deaminase deficiency.

BACKGROUND: We investigated the long-term outcome of gene therapy for severe combined immunodeficiency (SCID) due to the lack of adenosine deaminase (ADA), a fatal disorder of purine metabolism and immunodeficiency. METHODS: We infused autologous CD34+ bone marrow cells transduced with a retroviral vector containing the ADA gene into 10 children with SCID due to ADA deficiency who lacked an HLA-identical sibling donor, after nonmyeloablative conditioning with busulfan. Enzyme-replacement therapy was not given after infusion of the cells. RESULTS: All patients are alive after a median follow-up of 4.0 years (range, 1.8 to 8.0). Transduced hematopoietic stem cells have stably engrafted and differentiated into myeloid cells containing ADA (mean range at 1 year in bone marrow lineages, 3.5 to 8.9%) and lymphoid cells (mean range in peripheral blood, 52.4 to 88.0%). Eight patients do not require enzyme-replacement therapy, their blood cells continue to express ADA, and they have no signs of defective detoxification of purine metabolites. Nine patients had immune reconstitution with increases in T-cell counts (median count at 3 years, 1.07x10(9) per liter) and normalization of T-cell function. In the five patients in whom intravenous immune globulin replacement was discontinued, antigen-specific antibody responses were elicited after exposure to vaccines or viral antigens. Effective protection against infections and improvement in physical development made a normal lifestyle possible. Serious adverse events included prolonged neutropenia (in two patients), hypertension (in one), central-venous-catheter-related infections (in two), Epstein-Barr virus reactivation (in one), and autoimmune hepatitis (in one). CONCLUSIONS: Gene therapy, combined with reduced-intensity conditioning, is a safe and effective treatment for SCID in patients with ADA deficiency. (ClinicalTrials.gov numbers, NCT00598481 and NCT00599781.)

The effect of cyclosporine initiation time on the outcome of matched allogeneic stem-cell transplantation following fludarabine-based conditioning.

Cyclosporine (CSA) is the most commonly used medication for GVHD prophylaxis. The initiation time varies from day -4 to day 0. Initially, we gave CSA starting on day -1. However, since 2003 we have changed CSA initiation timing policy in most of our protocols to day -4, to achieve stable and controlled pretransplant CSA levels. Here, we assessed if initiation time impact the outcome of allogeneic stem-cell transplantation (allo-SCT). Data of 261 patients who underwent allo-SCT for hematological malignancies from a fully matched donor, treated with CSA as a single agent for GVHD prophylaxis were prospectively collected. Patients were divided according to CSA initiation time and analyzed for outcome. The acute GVHD severity, cGVHD extent, GVHD-associated mortality were significantly lower in the CSA -4 group. There was no difference in the rate and timing of acute or chronic GVHD. Overall survival did not differ between the groups. We conclude that the initiation of CSA at day -4 reduced the severity of aGVHD, extent of cGVHD, and GVHD-associated mortality without impact on overall survival.

In vivo T-cell dynamics during immune reconstitution after hematopoietic stem cell gene therapy in adenosine deaminase severe combined immune deficiency.

BACKGROUND: Gene therapy (GT) with hematopoietic stem cells is a promising treatment for inherited immunodeficiencies. OBJECTIVES: Limited information is available on the relative contribution of de novo thymopoiesis and peripheral expansion to T-cell reconstitution after GT as well as on the potential effects of gene transfer on hematopoietic stem cells and lymphocyte replicative lifespan. We studied these issues in patients affected by adenosine deaminase severe combined immune deficiency after low-intensity conditioning and reinfusion of retrovirally transduced autologous CD34(+) cells. METHODS: Immunophenotype, proliferative status, telomere length, and T-cell receptor excision circles were investigated at early and late time points (up to 9 years) after GT treatment. Control groups consisted of pediatric healthy donors and patients undergoing allogeneic bone marrow transplantation (BMT). RESULTS: We observed no telomere shortening in the bone marrow compartment and in granulocytes, whereas peripheral blood naive T cells from both GT and BMT patients showed a significant reduction in telomere length compared with healthy controls. This was in agreement with the presence of a high fraction of actively cycling naive and memory T cells and lower T-cell receptor excision circles. CONCLUSION: These data indicate that T-cell homeostatic expansion contributes substantially to immune reconstitution, like BMT, and is not associated with senescence in the stem cell compartment.

Loss of Kindlin-3 in LAD-III eliminates LFA-1 but not VLA-4 adhesiveness developed under shear flow conditions.

Leukocyte adhesion deficiency (LAD)-III is associated with homozygous stop codon mutations in Kindlin-3, the hematopoietic member of the Kindlin family of integrin coactivators. In addition, a subgroup of LAD-III patients has a homozygous splice junction mutation in and reduced expression of the Rap-1 guanine nucleotide exchange factor, CalDAG-GEFI (CDGI). In this study, we compared the adhesive properties of the leukocyte function-associated antigen-1 (LFA-1) and very late activation antigen-4 (VLA-4) integrins in both primary and activated leukocytes derived from these 2 LAD-III subgroups. Primary lymphocytes lacking both Kindlin-3 and CDGI lost all firm T-cell receptor-stimulated LFA-1 adhesiveness, in contrast to LAD-III lymphocytes deficient in Kindlin-3 alone. Effector T cells expanded from all tested LAD-III variants expressed normal CDGI, but lacked Kindlin-3. These Kindlin-3-null effector T cells exhibited total loss of inside-out LFA-1 activation by chemokine signals as well as abrogated intrinsic LFA-1 adhesiveness. Surprisingly, VLA-4 in Kindlin-3-null resting or effector lymphocytes retained intrinsic rolling adhesions to vascular cell adhesion molecule-1 and exhibited only partial defects in chemokine-stimulated adhesiveness to vascular cell adhesion molecule-1. Deletion of the putative beta(1) Kindlin-3 binding site also retained VLA-4 adhesiveness. Thus, our study provides the first evidence that Kindlin-3 is more critical to LFA-1 than to VLA-4-adhesive functions in human lymphocytes.

Multilineage hematopoietic reconstitution without clonal selection in ADA-SCID patients treated with stem cell gene therapy.

Gene transfer into HSCs is an effective treatment for SCID, although potentially limited by the risk of insertional mutagenesis. We performed a genome-wide analysis of retroviral vector integrations in genetically corrected HSCs and their multilineage progeny before and up to 47 months after transplantation into 5 patients with adenosine deaminase-deficient SCID. Gene-dense regions, promoters, and transcriptionally active genes were preferred retroviral integrations sites (RISs) both in preinfusion transduced CD34(+) cells and in vivo after gene therapy. The occurrence of insertion sites proximal to protooncogenes or genes controlling cell growth and self renewal, including LMO2, was not associated with clonal selection or expansion in vivo. Clonal analysis of long-term repopulating cell progeny in vivo revealed highly polyclonal T cell populations and shared RISs among multiple lineages, demonstrating the engraftment of multipotent HSCs. These data have important implications for the biology of retroviral vectors, the dynamics of genetically modified HSCs, and the safety of gene therapy.

A retrospective review of the outcome after second or subsequent allogeneic transplantation.

The failure of allogeneic stem cell transplant (allo-SCT) is cumbersome. We analyzed our experience in a second allo-SCT. Between the years 1981 and 2007, 144 patients underwent 2 or more allo-SCT. The first to second transplant interval ranged from 18 days to 13.25 years (median 98 days). The most frequent indications for the second SCT were activity of the basic disease (78), rejection (37), and engraftment failure (25). Twenty-nine of the 144 (20%) patients transplanted survived more then a year with treatment-related mortality of 45.5% as the leading cause of death. Interestingly, despite the low rate of graft-versus-host disease (GVHD) prophylaxis used, only 51 and 16 of the patients developed acute and chronic GVHD (aGVHD, cGVHD), respectively. Factors indicating higher likelihood for survival were nonmalignant disease, a nonrelapse indication for the second SCT, full HLA-matching, and the use of reduced-intensity conditioning (RIC). Age at transplantation, time interval between transplants, the development of GVHD, conditioning regimen, GVHD prophylaxis, or graft source were not shown to influence the prognosis. With a median follow-up of 4.5 years, 25 patients (17.2%) are alive, and 18 are disease-free. We conclude that although toxic, a second allo-SCT can lead to long-term survival.

A new induction protocol for the control of steroid refractory/dependent acute graft versus host disease with alefacept and tacrolimus.

BACKGROUND AIMS: We have shown previously that alefacept is effective in acute steroid resistant/dependent and chronic extensive graft versus host disease (GvHD) with a protocol using timings similar to those used for psoriasis treatment. In this study, we describe the use of an alefacept induction (e.g. for 7 consecutive days) followed by a bi-weekly maintenance treatment in combination with tacrolimus for acute steroid resistant/dependent GvHD 1, 3. METHODS: Sixteen patients were treated in this cohort, most with refractory GvHD. The pre-treatment GvHD grade ranged from 2 to 4 (median 3), involving the skin 16, gut 11 and liver 5. RESULTS: Twelve out of the 16 patients showed a response. As with the first protocol, the response of GvHD in the skin was fastest. In contrast to our previous protocol, however, the gastro-intestinal (GI) GvHD response was faster (P=0.05 compared with the first cohort). A hepatic response was seen in 4/6 patients and was complete in three. All responses were durable, including mucocutaneous, gut and liver GvHD. In all responding patients we were able to decrease the steroid dose significantly and in seven it was completely withdrawn. CONCLUSION: Alefacept induction is safe in acute steroid resistant/dependent GvHD and may be more effective therapeutically than our previous alefacept protocol. We speculate that alefacept initiates an allo-versus-allo cellular effect through its Fc receptor.

ABO incompatibility is associated with increased non-relapse and GVHD related mortality in patients with malignancies treated with a reduced intensity regimen: a single center experience of 221 patients.

The effect of ABO-incompatibility on transplantation outcome remains a controversial issue, with many of the reported studies showing conflicting results. In this study, we evaluate: the association between ABO-incompatibility and myeloid engraftment; the incidence and severity of acute and chronic graft-versus-host disease (GVHD); non-relapse mortality (NRM); GVHD-associated mortality, relapse and overall survival (OS). Our study includes 221 patients with malignant diseases treated in the same institution with the same reduced intensity regimen. Other variables known to affect the transplantation outcome such as age, disease, disease risk, and donor characteristics were well-balanced between ABO-matched and ABO-mismatched transplants. Analysis of our data shows increased incidence of NRM during the first months after transplantation in the groups of patients with major and minor ABO-incompatibility. Although neither incidence nor severity of GVHD differed significantly among the different groups, we found increased mortality associated with GVHD in the major ABO-incompatible groups. Long-term OS and relapse rate were not different, although we observed a trend for decreased OS during the first year post transplantation in the group of patients with major ABO-incompatibility. Our study showed that ABO-incompatibility has an adverse impact on the transplantation outcome.

Dyskeratosis congenita: dental management of a medically complex child.

Dyskeratosis congenita (DKC) is a rare syndrome characterized by cutaneous hyperpigmentation, nail dystrophy, leukoplakia, and pancytopenia. The purpose of this case study was to describe the management of a 7-year-old girl diagnosed with DKC who urgently needed dental treatment under general anesthesia before bone marrow transplantation (BMT). The patient presented normal skin, nails, and hair, but oral examination revealed a number of ulcers, leukoplakia, gingival recessions, alveolar bone loss, and dental caries. Hematologic preparation included raising blood parameters, and the anesthesiologist to had consider pulmonary infection. The alveolar bone loss and the gingival recessions required the consultation of a periodontist. Avoiding stainless steel crowns was necessary due to potential plaque accumulation in the crown margins. The goal of this dental treatment was eliminating potential sources of infection before transplantation was conducted. It is important for the pediatric dentist to recognize the medical aspects associated with dental management prior to BMT, and to incorporate them into the treatment plan.

Intrahepatic arterial administration of low-dose methotrexate in patients with severe hepatic graft-versus-host disease: an open-label, uncontrolled trial.

BACKGROUND: Hepatic grafr-versus-host disease (GVHD) is associated with significant morbidity and mortality. Standard therapy includes systemically administered immunosuppressive drugs. More recent reports have described catheter-directed intrahepatic arterial (IHA) delivery of low-dose methotrexate (MTX) and methylprednisolone in the treatment of corticosteroid-resistant severe hepatic GVHD. OBJECTIVE: This article reports on MTX toxicity and the variability in plasma drug concentrations after IHA administration of low-dose MTX in patients with severe hepatic GVHD. METHODS: In this open-label, uncontrolled pilot study, MTX and methylprednisolone were administered via the hepatic artery in patients with corticosteroid-resistant grade III or IV GVHD of the liver. Patients also received standard therapy. MTX concentrations were measured in the hepatic artery 5 and 10 minutes after injection and in peripheral venous blood at 1, 2, and 24 hours. RESULTS: Six patients (5 males [83.3%], I female [16.7%]; median age, 32 years; range, 8-42 years) were enrolled in the study. No hepatotoxicity was observed after IHA administration of MTX. In 5 patients with normal renal function, plasma drug concentrations 24 hours after administration of MTX ranged from 0.01 to 0.12 micromol/L (mean [SD], 0.043 [0.042] micromol/L). In 1 patient with renal failure, plasma MTX concentrations were 1.0 micromol/L 24 hours after administration and 0.07 micromol/L 5 days after administration. The severe hematologic and renal toxicity observed in this patient may have contributed to his death. Adverse events in patients with GVHD and normal renal function, who had normal plasma MTX concentrations, were comparable to those that have been reported after administration of an intravenous infusion. CONCLUSIONS: In patients with GVHD and normal renal function, IHA administration of low-dose MTX was not associated with liver or bone marrow toxicity. Further study is needed to determine the optimal protocols for treating corticosteroid-resistant hepatic GVHD.

Hemin augments growth and hemoglobinization of erythroid precursors from patients with diamond-blackfan anemia.

Diamond-Blackfan anemia (DBA) is congenital pure red-cell anemia due to a differentiation block in erythroid precursors. The disease is commonly caused by mutations in genes for ribosomal proteins. Despite the identification of disease causal genes, the disease pathogenesis is not completely elucidated. The ribosomal abnormalities are assumed to inhibit globin translation which may lead to excess free heme, stimulating a generation of free radicals and thereby damaging the precursors. We studied the effect of hemin (heme chloride) on cultured human erythroid precursors and found that contrary to aforementioned hypothesis, although hemin moderately stimulated free radicals, it did not cause apoptosis or necrosis. In erythroid precursors derived from DBA patients, hemin significantly stimulated growth and hemoglobinization. Thus, heme toxicity is unlikely to play a role in the pathophysiology of most DBA cases. Moreover, its beneficial effect in culture suggests a therapeutic potential.

Diminished telomeric 3' overhangs are associated with telomere dysfunction in Hoyeraal-Hreidarsson syndrome.

BACKGROUND: Eukaryotic chromosomes end with telomeres, which in most organisms are composed of tandem DNA repeats associated with telomeric proteins. These DNA repeats are synthesized by the enzyme telomerase, whose activity in most human tissues is tightly regulated, leading to gradual telomere shortening with cell divisions. Shortening beyond a critical length causes telomere uncapping, manifested by the activation of a DNA damage response (DDR) and consequently cell cycle arrest. Thus, telomere length limits the number of cell divisions and provides a tumor-suppressing mechanism. However, not only telomere shortening, but also damaged telomere structure, can cause telomere uncapping. Dyskeratosis Congenita (DC) and its severe form Hoyeraal-Hreidarsson Syndrome (HHS) are genetic disorders mainly characterized by telomerase deficiency, accelerated telomere shortening, impaired cell proliferation, bone marrow failure, and immunodeficiency. METHODOLOGY/PRINCIPAL FINDINGS: We studied the telomere phenotypes in a family affected with HHS, in which the genes implicated in other cases of DC and HHS have been excluded, and telomerase expression and activity appears to be normal. Telomeres in blood leukocytes derived from the patients were severely short, but in primary fibroblasts they were normal in length. Nevertheless, a significant fraction of telomeres in these fibroblasts activated DDR, an indication of their uncapped state. In addition, the telomeric 3' overhangs are diminished in blood cells and fibroblasts derived from the patients, consistent with a defect in telomere structure common to both cell types. CONCLUSIONS/SIGNIFICANCE: Altogether, these results suggest that the primary defect in these patients lies in the telomere structure, rather than length. We postulate that this defect hinders the access of telomerase to telomeres, thus causing accelerated telomere shortening in blood cells that rely on telomerase to replenish their telomeres. In addition, it activates the DDR and impairs cell proliferation, even in cells with normal telomere length such as fibroblasts. This work demonstrates a telomere length-independent pathway that contributes to a telomere dysfunction disease.

NK cytotoxicity mediated by CD16 but not by NKp30 is functional in Griscelli syndrome.

Griscelli syndrome (GS) type 2 is an autosomal recessive disorder represented by pigment dilution and impaired cytotoxic T lymphocyte (CTL) activity. NK activity has been scarcely investigated in GS patients. Here, we describe a new patient, possessing a hemophagocytic syndrome with a homozygous Q118X nonsense RAB27A mutation. Single specific primer-polymerase chain reaction (SSP-PCR) was developed based on this mutation and is currently used in prenatal genetic analysis. As expected, CTLs in the patient are not functional and NK cytotoxicity against K562 or 721.221 cells is diminished. Surprisingly, however, we demonstrate that CD16-mediated killing is intact in this patient and is therefore RAB27A independent, whereas NKp30-mediated killing is impaired and is therefore RAB27A dependent. We further analyzed the signaling pathways of these 2 receptors and demonstrated phosphorylation of Vav1 after CD16 activation but not after NKp30 engagement. Thus, we identify a novel homozygous mutation in the RAB27A gene of a new GS patient, observe for the first time that some activating NK receptors function in GS patients, and demonstrate a functional dichotomy in the killing mediated by these human NK-activating receptors.

Secondary acute myeloid leukemia after etoposide therapy for retinoblastoma.

Retinoblastoma is the most common eye tumor in children and is highly curable. Patients with hereditary retinoblastoma, have an increased risk of developing additional tumors, predominantly sarcomas. Most chemotherapy regimens used in retinoblastoma include etoposide, an epipodophyllotoxin associated with a risk of secondary myeloid leukemia. The use of etoposide in patients with a cancer predisposition syndrome such as retinoblastoma is potentially harmful, however, reports of secondary acute myeloid leukemia in patients treated with etoposide for retinoblastoma are rare. We report a case of a patient who developed secondary acute myeloid leukemia after etoposide treatment for retinoblastoma.

Intercellular transfer of carcinoembryonic antigen from tumor cells to NK cells.

The inhibition of NK cell killing is mainly mediated via the interaction of NK inhibitory receptors with MHC class I proteins. In addition, we have previously demonstrated that NK cells are inhibited in a class I MHC-independent manner via homophilic carcinoembryonic Ag (CEA) cell adhesion molecules (CEACAM1)-CEACAM1 and heterophilic CEACAM1-CEA interactions. However, the cross-talk between immune effector cells and their target cells is not limited to cell interactions per se, but also involves a specific exchange of proteins. The reasons for these molecular exchanges and the functional outcome of this phenomenon are still mostly unknown. In this study, we show that NK cells rapidly and specifically acquire CEA molecules from target cells. We evaluated the role of cytotoxicity in the acquisition of CEA and demonstrated it to be mostly killing independent. We further demonstrate that CEA transfer requires a specific interaction with an unknown putative NK cell receptor and that carbohydrates are probably involved in CEA recognition and acquisition by NK cells. Functionally, the killing of bulk NK cultures was inhibited by CEA-expressing cells, suggesting that this putative receptor is an inhibitory receptor.

Allogeneic stem cell transplantation for severe acquired aplastic anaemia using a fludarabine-based preparative regimen.

We reviewed our experience in the treatment of 13 patients with severe acquired aplastic anaemia, using a newly developed non-myeloablative regimen consisting of fludarabine (total dose 180 mg/m2), cyclophosphamide (total dose 120 mg/kg), and antithymocyte globulin (total dose 40 mg/kg). All except one patient received multiple transfusions and had failed prior immunosuppressive treatment. Twelve out of 13 patients achieved sustained engraftment. One patient was not evaluable for engraftment because of early death on day +10. None of the patients developed graft failure. Mucositis of mild-to-moderate severity was the only observed regimen-related toxicity. The cumulative incidence of acute graft-versus-host disease (GvHD) grade II-IV and III-IV was 8.3% and 0%, respectively. With a median follow-up period of 45 months, the 5-year overall survival probability was 84%. Eight out of 11 surviving patients have been followed for more than 1 year and only one developed limited chronic GvHD. All patients enjoy a normal life style, with a Karnofsky score of 100%, and all except three, followed for 3, 5 and 6 months respectively, are free of any immunosuppressive medication. The results of this study look promising, while prospective clinical trials may be required to confirm the benefits of this regimen as an alternative to existing protocols.