GATA2 haploinsufficient patients lack innate lymphoid cells that arise after hematopoietic cell transplantation.

Innate lymphoid cells (ILC) are important barrier tissue immune regulators. They play a pivotal role in early non-specific protection against infiltrating pathogens, regulation of epithelial integrity, suppression of pro-inflammatory immune responses and shaping the intestinal microbiota. GATA2 haploinsufficiency causes an immune disorder that is characterized by bone marrow failure and (near) absence of monocytes, dendritic cells, B cells and natural killer (NK) cells. T cells develop normally, albeit at lower numbers. Here, we describe the absence of ILCs and their progenitors in blood and bone marrow of two patients with GATA2 haploinsufficiency and show that all subsets of ILCs appear after allogeneic hematopoietic stem cell transplantation, irrespective of the preparative conditioning regimen. Our data indicate that GATA2 is involved in the development of hematopoietic precursor cells (HPC) towards the ILC lineage.

Autoregulatory lentiviral vectors allow multiple cycles of doxycycline-inducible gene expression in human hematopoietic cells in vivo.

The efficient control of gene expression in vivo from lentiviral vectors remains technically challenging. To analyze inducible gene expression in a human setting, we generated 'human immune system' (HIS) mice by transplanting newborn BALB/c Rag2(-/-)IL-2Rgamma(c)(-/-) immunodeficient mice with human hematopoietic stem cells transduced with a doxycycline-inducible lentiviral vector. We compared several methods of doxycycline delivery to mice, and could accurately measure doxycycline in vivo using a new sensitive detection assay. Two different lentiviral vector designs with constitutive (TRECMV-V14) or autoregulatory (TREAuto-V14) expression of an optimized reverse tetracycline transactivator were used to transduce human hematopoietic stem cells. After transplantation into immunodeficient mice, we analyzed the expression of the green fluorescent protein (GFP) reporter gene in the human hematopoiesis-derived cells that develop and accumulate in the generated HIS mice. We show efficient inducible GFP expression in adult HIS mice containing TREAuto-V14-transduced human cells, whereas GFP expression is poor with the TRECMV-V14 vector. Multiple cycles of doxycycline exposure in the TREAuto-V14 group result in repeated cycles of GFP expression with no loss of intensity. These findings are of major interest for gene therapy and basic research settings that require inducible gene expression.

Evaluation of safety and efficacy of RNAi against HIV-1 in the human immune system (Rag-2(-/-)gammac(-/-)) mouse model.

RNA interference (RNAi) gene therapy against HIV-1 by stable expression of antiviral short hairpin RNAs (shRNAs) can potently inhibit viral replication in T cells. Recently, a mouse model with a human immune system (HIS) was developed that can be productively infected with HIV-1. In this in vivo model, in which Rag-2(-/-)gamma(c)(-/-) mice are engrafted with human CD34(+)CD38(-) hematopoietic precursor cells, we evaluated an anti-HIV RNAi gene therapy. Human hematopoietic stem cells were transduced with a lentiviral vector expressing an shRNA against the HIV-1 nef gene (shNef) or the control vector. We observed normal development of the different cell subsets of the immune system. However, although initial transduction efficiencies were similar for both vectors, a reduced percentage of transduced human immune cells was observed for the shNef vector after establishment of the HIS in vivo. Further studies are required to fully evaluate the safety implications. When we infected the mature human CD4(+) T cells from the HIS mouse ex vivo with HIV-1, potent inhibition of viral replication was scored in shNef-expressing cells, confirming efficacy. When challenged with an shNef-resistant HIV-1 variant, equal replication was scored in control and shNef-expressing cells, confirming sequence-specificity of the RNAi therapy. We thus demonstrated that an antiviral RNAi-based gene therapy on blood stem cells leads to HIV-1-resistant T cells in vivo, an important proof of concept in the clinical development of RNAi against HIV-1.

[The relationship between surgical delay for a hip fracture and the complication risk]. / De samenhang tussen uitstel van operatie bij een heupfractuur en het complicatierisico.

OBJECTIVE: To investigate the factors that contribute to surgical delay and whether this delay can be associated with post-operative complications. DESIGN: Retrospective cohort study. METHOD: Patients admitted with a hip fracture between 1 January 2001-31 December, 2003 to the Onze Lieve Vrouwe Gasthuis, Amsterdam, the Netherlands, were included. The delay before surgery was recorded in 446 patients who underwent surgical treatment for a hip fracture. As possible predictors of delay before surgery, the following factors were investigated: demographic and other patient information, pre-operative medication, co-morbidities, pre-operative acute co-morbidities, classification according to the American Society of Anesthesiologists (ASA) and whether or not the patient had already had surgery to the same hip. To measure the effect of delay before surgery, we investigated post-operative complications like: delirium, decubitus ulcers, urinary tract infections, pulmonary infections, pulmonary embolism, deep vein thrombosis, wound infection, failure ofosteosynthesis and in-hospital mortality. RESULTS: In total, 446 patients, 98 male and 348 female, with a mean age of 82.2 years met the inclusion criteria. Distinct predictors of delay before surgery were: ASA-classification, pre-operative urinary tract infection, pre-operative chest infection, pre-operative delirium, pre-operative anaemia and re-operation. There was no significant association between delay of surgery and the occurrence of post-operative complications. CONCLUSION: Presence of a pre-operative medical condition has an important effect on surgical delay for a hip fracture. The assumption of the Dutch Healthcare Inspectorate that delay of surgery for hip fracture causes more complications could not be confirmed.

[Dobutamine stress magnetic resonance imaging (DS-MRI), a valuable tool for the diagnosis of ischemic heart disease]. / Dobutaminestress-MRI van het hart: een waardevolle onderzoekstechniek voor de diagnostiek van ischemische hartziekten.

OBJECTIVE: Assessment of the clinical applicability of DS-MRI for the detection of myocardial ischemia and myocardial viability. DESIGN: Prospective. METHOD: In the period from 1 November 1999 to 31 October 2000, patients with suspected coronary artery disease who could not be studied by means of conventional bicycle ergometry underwent breath-hold DS-MRI (1 Tesla) 4 days after cessation of anti-ischemic medication. Three left ventricular short-axis planes were examined for the occurrence of disorders in wall movement during infusion of increasing doses of dobutamine (10, 20, 30 and 40 micrograms/kg/min). Temporary recovery of wall thickening in a previously diminished or non-contracting segment under 5 micrograms/kg/min of dobutamine was considered proof of viability. Development of hypo-, a- or dyskinesia at higher doses of dobutamine was taken to indicate ischemia. If the DS-MRI test was positive for ischemia, coronary angiography was performed. If indicated, this was followed by revascularisation. If DS-MRI did not reveal ischemia, the patient was seen at the outpatient department. RESULTS: Of the 100 patients (62 men and 38 women with an average age of 62 years, SD = 12) subjected to DS-MRI, 95 yielded results that were suitable for diagnosis. Of the 42 patients with DS-MRI scans that were considered positive for ischemia and in whom coronary angiography was subsequently performed, 41 had such coronary abnormalities that revascularisation was indicated. One patient was false-positive. All 53 patients with non-ischemic DS-MRI scans were followed-up for 11-23 months (mean 17 months). One patient died suddenly 2 weeks after the MRI-test. The other 52 patients did not experience any coronary events nor sudden cardiac death. The predictive value of a positive DS-MRI scan for ischemia was 98% and the predictive value of a negative DS-MRI scan was also 98%. CONCLUSION: DS-MRI is a safe diagnostic method for the detection or exclusion of myocardial ischemia and viability in patients with suspected coronary artery disease.

Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12.

A novel sequence discovered in a computational screen appears distantly related to the p35 subunit of IL-12. This factor, which we term p19, shows no biological activity by itself; instead, it combines with the p40 subunit of IL-12 to form a novel, biologically active, composite cytokine, which we term IL-23. Activated dendritic cells secrete detectable levels of this complex. IL-23 binds to IL-12R beta 1 but fails to engage IL-12R beta 2; nonetheless, IL-23 activates Stat4 in PHA blast T cells. IL-23 induces strong proliferation of mouse memory (CD4(+)CD45Rb(low)) T cells, a unique activity of IL-23 as IL-12 has no effect on this cell population. Similar to IL-12, human IL-23 stimulates IFN-gamma production and proliferation in PHA blast T cells, as well as in CD45RO (memory) T cells.

Generation of interferon alpha-producing predendritic cell (Pre-DC)2 from human CD34(+) hematopoietic stem cells.

Upon viral stimulation, the natural interferon (IFN)-alpha/beta-producing cells (IPCs; also known as pre-dendritic cells (DCs 2) in human blood and peripheral lymphoid tissues rapidly produce huge amounts of IFN-alpha/beta. After performing this innate antiviral immune response, IPCs can differentiate into DCs and strongly stimulate T cell-mediated adaptive immune responses. Using four-color immunofluorescence flow cytometry, we have mapped the developmental pathway of pre-DC2/IPCs from CD34(+) hematopoietic stem cells in human fetal liver, bone marrow, and cord blood. At least four developmental stages were identified, including CD34(++)CD45RA(-) early progenitor cells, CD34(++)CD45RA(+) late progenitor cells, CD34(+)CD45RA(++)CD4(+)interleukin (IL)-3Ralpha(++) pro-DC2, and CD34(-)CD45RA(++) CD4(+)IL-3Ralpha(++) pre-DC2/IPCs. Pro-DC2s have already acquired the capacity to produce large amounts of IFN-alpha/beta upon viral stimulation and to differentiate into DCs in culture with IL-3 and CD40 ligand. CD34(++)CD45RA(-) early progenitor cells did not have the capacity to produce large amounts of IFN-alpha/beta in response to viral stimulation; however, they can be induced to undergo proliferation and differentiation into IPCs/pre-DC2 in culture with FLT3 ligand.

Down-regulation of the macrophage lineage through interaction with OX2 (CD200).

OX2 (CD200) is a broadly expressed membrane glycoprotein, shown here to be important for regulation of the macrophage lineage. In mice lacking CD200, macrophage lineage cells, including brain microglia, exhibited an activated phenotype and were more numerous. Upon facial nerve transection, damaged CD200-deficient neurons elicited an accelerated microglial response. Lack of CD200 resulted in a more rapid onset of experimental autoimmune encephalomyelitis (EAE). Outside the brain, disruption of CD200-CD200 receptor interaction precipitated susceptibility to collagen-induced arthritis (CIA) in mice normally resistant to this disease. Thus, in diverse tissues OX2 delivers an inhibitory signal for the macrophage lineage.

DAP12-deficient mice fail to develop autoimmunity due to impaired antigen priming.

DAP12 is an ITAM-bearing membrane adaptor molecule implicated in the activation of NK and myeloid cells. In mice rendered DAP12 deficient by targeted gene disruption, lymphoid and myeloid development was apparently normal, although the activating Ly49 receptors on NK cells were downregulated and nonfunctional. To analyze the consequences of DAP12 deficiency in vivo, we examined the susceptibility of DAP12-/- mice to experimental autoimmune encephalomyelitis (EAE). DAP12-/- mice were resistant to EAE induced by immunization with myelin oligodendrocyte glycoprotein (MOG) peptide. Resistance was associated with a strongly diminished production of IFNgamma by myelin-reactive CD4+ T cells due to inadequate T cell priming in vivo. These data suggest that DAP12 signaling may be required for optimal antigen-presenting cell (APC) function or inflammation.

Prevalence, classification, and severity of epilepsy in children in western Norway.

PURPOSE: To determine prevalence of active epilepsy in school children in a defined area and assess the usefulness of International League Against Epilepsy classification of seizures and epileptic syndromes, with special emphasis on frequency, additional handicaps, and therapeutic problems of severe cases. METHODS: The latest International League Against Epilepsy International Classification of Epileptic Seizures (ICES, 1981) and Epilepsies and Epileptic Syndromes (ICE, 1989) were used for determination of prevalence rates, seizure types, epilepsies and epileptic syndromes, and additional neurological deficits in all 6-to 12-year-old children with epilepsy in a Norwegian county. Children had neuropediatric and EEG examination, intelligence evaluation, and, when necessary, additional investigations. RESULTS: Prevalence of active epilepsy on January 1, 1995, was 5.1 per 1,000. Main seizure type and epilepsy syndrome could be classified in 98% and 90% of patients, respectively. Seizure types/epileptic syndromes were more often partial/localization related than generalized. Among generalized epilepsies, idiopathic forms were more frequent in girls, and cryptogenic and symptomatic forms more frequent in boys. Epileptogenic EEG activity was most often generalized or localized to one or two areas of the brain and was never found in 14% of patients. Symptomatic etiology was found in 46% of all children and in 81% of therapy-resistant cases, respectively. Over the years, 11% of children had never used antiepileptic drugs (AED), 62% had tried one or two AEDs, and 26% had tried from three to 15 AEDs. Twenty-five percent of children were without present AED treatment. Complementary/alternative medicine had been tried by 12% of children. CONCLUSIONS: Although most epilepsies could be classified, the number of cases in non-specific categories was relatively high. Symptomatic etiology was frequent, especially in therapy-resistant cases. Multidisciplinary therapeutic and habilitation approaches are often needed in childhood epilepsy.

Survival of patients who have been on a waiting list for renal transplantation.

INTRODUCTION: Survival of transplanted patients is generally much better than for those on dialysis. This comparison is, however, incorrect, as in order to be accepted for renal transplantation the patient has to be in a relatively good condition and in addition transplanted patients are usually younger. We compared survival of all renal replacement therapy (RRT) patients who had undergone an identical medical check-up, been accepted, and put on the waiting list for cadaveric-kidney transplantation at Huddinge University hospital. A comparison with patients who were transplanted with a kidney from a living related donor (LD) is also included. METHODS: All patients (n=608) accepted and on the waiting list for renal transplantation between January 1987 and April 1996 formed the basis of the study. Follow-up was terminated on 31 December 1997. Survival was recorded from the date that the patients were accepted and put on the waiting list. As long as the patient was not transplanted and remained on dialysis treatment, survival was considered as 'survival on dialysis', and if transplanted, subsequent survival was defined as 'survival after cadaveric-kidney transplantation'. A patient who had been transplanted remained in that group for the rest of the observation period even if the transplantation had failed and the patient had to go back to dialysis after the surgery. RESULTS: Five-year survival was considerably better after LD-kidney transplantation (94%), than after cadaveric-kidney transplantation (76%) or on chronic dialysis (60%). Cox hazard regression analysis gave an age-adjusted relative risk for death of 0.46 for LD-transplanted and 1.49 for remaining on dialysis compared with cadaveric-transplanted patients. Transplanted patients, however, experienced a higher mortality during the first year after the transplantation than patients still on dialysis. CONCLUSIONS: LD-kidney transplantation is clearly associated with a superior survival. Mortality is relatively high after cadaveric-kidney transplantation, especially during the first months after surgery. Nevertheless, in the long term cadaveric kidney transplanted patients have a better survival than those remaining on dialysis.

PIM1 reconstitutes thymus cellularity in interleukin 7- and common gamma chain-mutant mice and permits thymocyte maturation in Rag- but not CD3gamma-deficient mice.

The majority of lymphomas induced in Rag-deficient mice by Moloney murine leukemia virus (MoMuLV) infection express the CD4 and/or CD8 markers, indicating that proviral insertions cause activation of genes affecting the development from CD4(-)8(-) pro-T cells into CD4(+)8(+) pre-T cells. Similar to MoMuLV wild-type tumors, 50% of CD4(+)8(+) Rag-deficient tumors carry a provirus near the Pim1 protooncogene. To study the function of PIM proteins in T cell development in a more controlled setting, a Pim1 transgene was crossed into mice deficient in either cytokine or T cell receptor (TCR) signal transduction pathways. Pim1 reconstitutes thymic cellularity in interleukin (IL)-7- and common gamma chain-deficient mice. In Pim1-transgenic Rag-deficient mice but notably not in CD3gamma-deficient mice, we observed slow expansion of the CD4(+)8(+) thymic compartment to almost normal size. Based on these results, we propose that PIM1 functions as an efficient effector of the IL-7 pathway, thereby enabling Rag-deficient pro-T cells to bypass the pre-TCR-controlled checkpoint in T cell development.

Disruption of alpha beta but not of gamma delta T cell development by overexpression of the helix-loop-helix protein Id3 in committed T cell progenitors.

Enforced expression of Id3, which has the capacity to inhibit many basic helix-loop-helix (bHLH) transcription factors, in human CD34(+) hematopoietic progenitor cells that have not undergone T cell receptor (TCR) gene rearrangements inhibits development of the transduced cells into TCRalpha beta and gamma delta cells in a fetal thymic organ culture (FTOC). Here we document that overexpression of Id3, in progenitors that have initiated TCR gene rearrangements (pre-T cells), inhibits development into TCRalpha beta but not into TCRgamma delta T cells. Furthermore, Id3 impedes expression of recombination activating genes and downregulates pre-Talpha mRNA. These observations suggest possible mechanisms by which Id3 overexpression can differentially affect development of pre-T cells into TCRalpha beta and gamma delta cells. We also observed that cell surface CD4(-)CD8(-)CD3(-) cells with rearranged TCR genes developed from Id3-transduced but not from control-transduced pre-T cells in an FTOC. These cells had properties of both natural killer (NK) and pre-T cells. These findings suggest that bHLH factors are required to control T cell development after the T/NK developmental checkpoint.

TCR gene rearrangements and expression of the pre-T cell receptor complex during human T-cell differentiation.

Recent studies have identified several populations of progenitor cells in the human thymus. The hematopoietic precursor activity of these populations has been determined. The most primitive human thymocytes express high levels of CD34 and lack CD1a. These cells acquire CD1a and differentiate into CD4(+)CD8(+) through CD3(-)CD4(+)CD8(-) and CD3(-)CD4(+) CD8alpha+beta- intermediate populations. The status of gene rearrangements in the various TCR loci, in particular of TCRdelta and TCRgamma, has not been analyzed in detail. In the present study we have determined the status of TCR gene rearrangements of early human postnatal thymocyte subpopulations by Southern blot analysis. Our results indicate that TCRdelta rearrangements initiate in CD34(+)CD1a- cells preceding those in the TCRgamma and TCRbeta loci that commence in CD34(+)CD1a+ cells. Furthermore, we have examined at which cellular stage TCRbeta selection occurs in humans. We analyzed expression of cytoplasmic TCRbeta and cell-surface CD3 on thymocytes that lack a mature TCRalphabeta. In addition, we overexpressed a constitutive-active mutant of p56(lckF505) by retrovirus-mediated gene transfer in sequential stages of T-cell development and analyzed the effect in a fetal thymic organ culture system. Evidence is presented that TCRbeta selection in humans is initiated at the transition of the CD3(-)CD4(+)CD8(-) into the CD4(+)CD8alpha+beta- stage.

Cell-fate decisions in early T cell development: regulation by cytokine receptors and the pre-TCR.

During lymphocyte development, cell-fate decisions are determined by a myriad of signals produced by the micro- environment of the thymus and the bone marrow. These yet to be fully defined developmental cues regulate stage-specific gene expression, and the extraordinarily well-characterized stages of T and B cell development have provided attractive model systems for studying regulation of cellular differentiation. In particular, studies on the contribution of both antigen receptors and cytokine receptors to lymphoid development have illuminated essential signalling pathways in early T and B cells. Here, we review investigations supporting an obligatory role for the IL-7 receptor pathway in early T cell development. IL-7 is produced by both thymus and bone marrow stromal cells, and its potential contribution to survival, differentiation and proliferation of pro-T cells is discussed. We also address the contribution of the pre-T cell receptor (pre-TCR) to differentiation past the pro-T cell stage, and recent advances in deciphering the composition and function of the pre-TCR complex are discussed. Finally, we suggest future directions in this field that may serve to reveal whether and how signals initiated by the cytokine receptors and pre-TCR may intersect, and to define which down-stream molecular events are regulated by these receptors.

Early stages in the development of human T, natural killer and thymic dendritic cells.

T-cell development is initiated when CD34+ pluripotent stem cells or their immediate progeny leave the bone marrow to migrate to the thymus. Upon arrival in the thymus the stem cell progeny is not yet committed to the T-cell lineage as it has the capability to develop into T, natural killer (NK) and dendritic cells (DC). Primitive hematopoietic progenitor cells in the human thymus express CD34 and lack CD1a. When these progenitor cells develop into T cells they traverse a number of checkpoints. One early checkpoint is the induction of T-cell commitment, which correlates with appearance of CD1a and involves the loss of capacity to develop into NK cells and DC and the initiation of T-cell receptor (TCR) gene rearrangements. Basic helix-loop-helix transcription factors play a role in induction of T-cell commitment. CD1a+CD34+ cells develop into CD4+CD8 alpha+ beta+ cells by upregulating first CD4, followed by CD8 alpha and then CD8 beta. Selection for productive TCR beta gene rearrangements (beta selection) likely occurs in the CD4+CD8 alpha+ beta- and CD4+CD8 alpha+ beta+ populations. Although the T and NK-cell lineages are closely related to each other, NK cells can develop independently of the thymus. The fetal thymus is most likely one site of NK-cell development.

T cell precursors in man and mice.

The thymus is seeded at week 7-8 of gestation with hematopoietic progenitor cells derived from the liver. By week 15-16 of gestation a fully differentiated thymus with a cortical/medullary junction and Hassal's corpuscles has been formed. The thymus is continuously populated by progenitor cells first from the liver and then from bone marrow. This process continues in childhood after which the thymus starts to involute. Recent information indicates that the cells that populate the thymus are not committed to the T cell lineage. When developing to T cells these progenitor cells traverse a series of cellular stages that can be discriminated on the basis of cell surface and cytoplasmic markers, status of TCR gene rearrangements and precursor cell activities. The early stages of T cell development in the mouse thymus have been described in detail. The recent development of assays to measure the T cell precursor activity of human thymic and extrathymic progenitor cell subsets has led to a rapid accumulation of data on early events in human thymic development as well. The information available now permits a comparison of early cellular stages of T cell development in mice and man. Some of the extrinsic and intrinsic factors that govern T cell differentiation will be discussed. Data on the role of the cytokine, interleukin-7, in human and mouse T cell development will be summarized. Furthermore, recent data on the involvement of transcription factors in early T cell development are reviewed.

PJA-BP expression and TCR delta deletion during human T cell differentiation.

Recombination of deltaRec to psiJalpha will delete the TCR delta gene, which is thought to play an important role in the bifurcation of the TCR alphabeta versus TCR gammadelta differentiation lineages. We recently detected a DNA-binding protein in human thymocytes, the so-called PJA-BP, which recognizes the psiJalpha gene segment and might be one of the factors involved in the regulation of preferential deltaRec-psiJalpha rearrangements. We now investigate PJA-BP expression and its correlation with TCR delta gene deletion in thymocytes. Our electrophoretic mobility shift assay experiments showed that the PJA-BP is evolutionary conserved in human, murine and simian thymocytes. Using a large series of human hematopoietic malignancies (n = 30), we conclude that PJA-BP expression is thymocyte specific and seems to be restricted to thymocytes committed to the TCR alphabeta lineage. Analysis of seven well-defined human thymocyte subpopulations showed that preferential deltaRec-psiJalpha rearrangements as well as PJA-BP expression can be detected from the immature CD34-/CD1+/CD3-/CD4+/CD8alpha+beta- thymocyte differentiation stage onwards. These experiments indicate that expression of PJA-BP in human thymocytes starts simultaneously with preferential deltaRec-psiJalpha rearrangements, which supports our hypothesis that PJA-BP is one of the factors involved in the preferential recombination of deltaRec to psiJalpha.

Inhibition of T cell and promotion of natural killer cell development by the dominant negative helix loop helix factor Id3.

Bipotential T/natural killer (NK) progenitor cells are present in the human thymus. Despite their bipotential capacity, these progenitors develop predominantly to T cells in the thymus. The mechanisms controlling this developmental choice are unknown. Here we present evidence that a member(s) of the family of basic helix loop helix (bHLH) transcription factors determines lineage specification of NK/T cell progenitors. The natural dominant negative HLH factor Id3, which blocks transcriptional activity of a number of known bHLH factors, was expressed in CD34+ progenitor cells by retrovirus-mediated gene transfer. Constitutive expression of Id3 completely blocks development of CD34+ cells into T cells in a fetal thymic organ culture (FTOC). In contrast, development into NK cells in an FTOC is enhanced. Thus, the activity of a bHLH transcription factor is necessary for T lineage differentiation of bipotential precursors, in the absence of which a default pathway leading to NK cell development is chosen. Our results identify a molecular switch for lineage specification in early lymphoid precursors of humans.