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.

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.

Downregulation of CD1 marks acquisition of functional maturation of human thymocytes and defines a control point in late stages of human T cell development.

We have investigated whether in the human thymus transition of CD4+CD8+ double positive (DP) to CD4+ or CD8+ single positive (SP) cells is sufficient for generation of functional immunocompetent T cells. Using the capacity of thymocytes to expand in vitro in response to PHA and IL-2 as a criterion for functional maturity, we found that functional maturity of both SP and DP thymocytes correlates with downregulation of CD1a. CD1a- cells with a persistent DP phenotype were also found in neonatal cord blood, suggesting that at least a proportion of mature DP cells can emigrate from the thymus. The requirements for generating functional T cells were investigated in a hybrid human/mouse fetal thymic organ culture. MHC class II-positive, but not MHC class II-negative, mouse thymic microenvironments support differentiation of human progenitors into TCR alpha beta+CD4+ SP cells, indicating that mouse MHC class II can positively select TCR alpha beta +CD4+ SP human cells. Strikingly, these SP are arrested in the CD1a+ stage and could not be expanded in vitro with PHA and IL-2. CD1a+CD4+ SP thymocytes do not represent an end stage population because purified CD1a+CD4+ SP thymocytes differentiate to expandable CD1a- cells upon cocultivation with human thymic stromal cells. Taken together these data indicate that when CD1a+ DP TCR alpha beta low cells mature, these cells interact with MHC, but that an additional, apparently species-specific, signal is required for downregulation of CD1a to generate functional mature TCR alpha beta + cells.

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.

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.

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.

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.

[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.

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.

Subject-related risk factors for sports injuries: a 1-yr prospective study in young adults.

The purpose of this study was to investigate the importance of subject-related risk factors for sports injuries, taking exposure time into account. At baseline in 182 healthy males and females (27 yr) the following subject-related risk factors were assessed: body mass index (BMI), maximal oxygen uptake (direct treadmill measurement), seven aspects of neuromotor fitness (MOPER fitness test), strength of the hamstring and quadriceps muscles (CYBEX), having sustained a sports injury in the 12 months preceding the baseline measurement ("previous injury"), and 16 psychological and psychosocial factors (measured with 8 standard, valid, and reliable questionnaires). For 1 yr, subjects were asked to make daily entries on a monthly log concerning all sports activities exceeding an intensity of 4 MET and all sustained sport injuries. Completed logs were returned by 139 subjects (75 males and 64 females). Fifty-one injuries were registered in 41 subjects. The overall incidence rate (IR) was 3.7 sports injuries per 1000 h of sports participation (95% confidence interval 2.8-4.9). For various subcategories, the following IR per 1000 h of sports participation were calculated: contact sports IR = 11.0 (95% CI 7.4-16.3); noncontact sports IR = 2.3% (95% CI 1.6-3.3); competition IR = 13.4 (95% CI 8.7-20.6); and training IR = 2.8 (95% CI 1.6-5.1). Data were analyzed by stepwise multiple logistic regression. The following five variables were independent and significant (P < 0.05) predictors of risk in sustaining a sport injury: dominance (odds ratio (OR) = 1.71; 95% CI = 1.44-2.03), vital exhaustion (OR = 1.85; CI = 1.22-2.86), stressful life events (OR = 1.84; 95% CI = 1.10-311); these ORs were calculated for an increase of 10% of the range of obtained scores, starting at minimum value. For total sporting time, the OR was calculated by taking the group with a total sporting time below the median (4050 h) as a reference (OR = 6.87; 95% CI = 2.09-22.55). For previous injury, subjects that had not sustained a sports injury in the 12 months preceding the baseline measurements served as a reference for the calculation of the OR (OR = 9.41; 95% CI = 2.80-31.58). These findings confirm that both exposure time and previous injury are more important predictors of sports injuries than psychological, psychosocial, physiological, and anthropometrical factors.

[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.

Bone marrow transplantation for aplastic anemia and acute leukemia at Huddinge Hospital.

Eight patients with aplastic anemia were transplanted with marrow from HLA-identical donors. Two patient rejected their grafts and died while 5 patients (71%) show no ill effects 3 months, 10 months, and more than 1, 2 and 4 years after the transplantation. Three of the patients who received unirradiated donor buffy coat after transplantation developed chronic graft-versus-host disease (GVHD) which, however, resolved following treatment with Prednisolone and Azathioprine. One patient with end-stage acute myeloid leukemia, who was transplanted with marrow from an identical twin, died 6 days after the transplantation of bleedings and sepsis. Eight patients with acute non-lymphoblastic leukemia (ANL) were transplanted, while in remission, with marrow from HLA-identical siblings. One patient died of interstitial pneumonia 3 months after transplantation, while another patient recovered from GVHD of the gut at 5 months after the transplantation. Seven out of 8 patients with ANL (88%) are home and well between 2 and 12 months after the transplantation.

Treatment of progressive systemic sclerosis (scleroderma, PSS) with a new drug influencing connective tissue.

Cyclofenil is a new diphenyl ethylene derivative related to stilboestrol without oestrogenicity but with marked effects on connective tissue metabolism. The drug has been tested, in a daily dose of 200mg X3, in six patients with progressive systemic sclerosis (PSS) to analyze the expected beneficial effects on the PSS symptoms. The typical skin hardness, joint and muscle rigidity, and reduced breathing capacity were improved to varying dgrees. The only side-effect was a slight transient liver enzyme elevation in 1 out of 6 patients. A slight increase was found in urinary calcium and hydroxyproline excretion. In several cases serum calcium, cholesterol, triglyceride and in some cases the serum uric acid levels were decreased. The ANF titres diminished to varying degrees in 4 out of 6 patients. These results indicate that further detailed clinical and laboratory studies on the therapeutic potential of cyclofenil in PSS and other diseases affecting connective tissue seen to be justified.

Diagnostic errors using the Short Portable Mental Status Questionnaire with a mixed clinical population.

The validity of the Short Portable Mental Status Questionnaire (SPMSQ) was evaluated using two criteria: clinical diagnosis and neuropsychological diagnosis. The 40 study participants represented a mixed clinical sample of neurologic and psychiatric patients, all of whom were or had been inpatients. Laboratory data (CT, EEG, etc.) were available for 45% of the patients. Neuropsychological diagnosis of organic impairment was based on an extensive test battery. The SPMSQ did not significantly relate to either clinical or neuropsychological diagnosis. It is recommended that a "normal" score on the SPMSQ be regarded as nonspecific regarding organic cognitive impairment rather than suggestive of normal brain functioning.