Foamy viral vector integration sites in SCID-repopulating cells after MGMTP140K-mediated in vivo selection.

Foamy virus (FV) vectors are promising for hematopoietic stem cell (HSC) gene therapy but preclinical data on the clonal composition of FV vector-transduced human repopulating cells is needed. Human CD34(+) human cord blood cells were transduced with an FV vector encoding a methylguanine methyltransferase (MGMT)P140K transgene, transplanted into immunodeficient NOD/SCID IL2Rγ(null) mice, and selected in vivo for gene-modified cells. The retroviral insertion site profile of repopulating clones was examined using modified genomic sequencing PCR. We observed polyclonal repopulation with no evidence of clonal dominance even with the use of a strong internal spleen focus forming virus promoter known to be genotoxic. Our data supports the use of FV vectors with MGMTP140K for HSC gene therapy but also suggests additional safety features should be developed and evaluated.

Femoral neck shaft angle in men with fragility fractures.

Introduction. Femoral neck shaft angle (NSA) has been reported to be an independent predictor of hip fracture risk in men. We aimed to assess the role of NSA in UK men. Methods. The NSA was measured manually from the DXA scan printout in men with hip (62, 31 femoral neck and 31 trochanteric), symptomatic vertebral (91), and distal forearm (67) fractures and 389 age-matched control subjects. Age, height, weight, and BMD (g/cm(2): lumbar spine, femoral neck, and total femur) measurements were performed. Results. There was no significant difference in mean NSA between men with femoral neck and trochanteric hip fractures, so all further analyses of hip fractures utilised the combined data. There was no difference in NSA between those with hip fractures and those without (either using the combined data or analysing trochanteric and femoral neck shaft fractures separately), nor between fracture subjects as a whole and controls. Mean NSA was smaller in those with vertebral fractures (129.2° versus 131°: P = 0.001), but larger in those with distal forearm fractures (129.8° versus 128.5°: P = 0.01). Conclusions. The conflicting results suggest that femoral NSA is not an important determinant of hip fracture risk in UK men.

Doses to critical organs following radiotherapy and concomitant imaging of the larynx and breast.

The development of conformal radiotherapy carries with it the implication of an increased number of imaging procedures at various stages throughout the overall treatment, principally for verification at some, or all, of the treatment fractions. This raises the issue of the balance between the benefit of these additional imaging exposures and the associated risk of radiocarcinogenesis arising from them. As such, it is necessary to appreciate the doses to critical organs for which individual carcinogenic risks have been estimated. In this study, doses to these organs have been measured with lithium fluoride thermoluminescence dosimetry loaded in anthropomorphic phantoms and subjected to realistic radiotherapy treatments of the larynx and breast, including concomitant CT and electronic portal imaging exposures associated with localization and verification of these treatments. Even for large numbers of concomitant images of either modality, arising from imaging at every fraction, the leakage and scatter from the radiotherapy itself is shown to dominate the overall organ dose, with imaging procedures generally contributing 5-20% of the total organ dose.

Organ doses from prostate radiotherapy and associated concomitant exposures.

In addition to the therapeutic exposure, a course of radiotherapy will involve the additional (concomitant) irradiation of the patient using CT, simulator or portal imaging systems, for localization of the target volume and subsequent verification of treatment delivery. The number of concomitant exposures is likely to increase as the developing technical capabilities for conformal, image-guided radiotherapy make target and critical organ definition an increasingly important aspect of radiotherapy. Estimation of doses and risks to critical organs in the body from all sources is thus necessary to provide the basis for adequate justification of the exposures as required by ICRP. In this paper, doses to selected organs and tissues for which ICRP have identified fatal cancer probabilities have been measured using a realistic anthropomorphic phantom loaded with thermoluminescent dosemeters and irradiated using a treatment protocol for radical radiotherapy of the prostate. Independently, doses to the same organs and tissues have been measured from concomitant CT and portal imaging exposures given for localization and verification purposes. Although negligible in comparison with the target dose, realistic numbers of concomitant exposures give a small but significant contribution to the total dose to most organs and tissues outside the target volume. Generally, this is in the range 5-10% of the total organ dose, but can be as high as 20% for bone surfaces. These data may be used to estimate concomitant doses from any combination of CT and portal imaging and may help in the justification process, especially when additional verification exposures may be required during treatment.

Differences in bone mineral density and geometry in men and women: the Newcastle Thousand Families Study at 50 years old.

In 1947 Sir James Spence initiated the Newcastle Thousand Families study, which recruited all 1142 children born in the city between May and June that year. At the age of 50 years, 832 survivors were traced and invited to attend for measurement of bone mineral density (BMD) by dual energy X-ray absorptiometry (DXA). The aim was to compare BMD measurements of men and women in this cohort, before and after adjustment for skeletal size. The femoral neck shaft angles (NSA) were also measured manually from the DXA scan printouts. A total of 171 men and 218 women agreed to participate. As expected men had greater bone mineral content and bone area at all sites (p<0.0001) and were taller and heavier (p<0.0001) than women. Men also had significantly higher BMD than women at all regions (p<0.0002), except at the femoral neck or lumbar spine. After correction for skeletal size and body weight, men had statistically significantly lower volumetric BMD at all sites. The measurement of NSA had good intra/interobserver errors and precision (coefficient of variations 0.79%, 1.2% and 1.2%). Men had significantly larger NSAs (mean 130 degrees , range 121-138 degrees ) than women (mean 128 degrees , range 119-137 degrees ). We conclude that there are gender differences in BMD, skeletal size and geometry in middle aged men and women, which together with the subsequent rate of bone loss, may influence fracture risk in later life.

A randomized controlled trial of calcium with vitamin D, alone or in combination with intravenous pamidronate, for the treatment of low bone mineral density associated with Crohn's disease.

BACKGROUND: Osteoporosis is a common complication of Crohn's disease. AIM: To study the effect on the bone mineral density of a bisphosphonate (pamidronate) given intravenously, in combination with oral calcium and vitamin D supplements, compared with oral calcium and vitamin D supplements alone. METHODS: Seventy-four patients with Crohn's disease and low bone mineral density at the lumbar spine and/or hip were randomized to receive either a daily dose of 500 mg of calcium with 400 IU of vitamin D alone or in combination with four three-monthly infusions of 30 mg of intravenous pamidronate over the course of 12 months. The main outcome measure was the change in bone mineral density at the lumbar spine and hip, measured by dual X-ray absorptiometry, at baseline and 12 months. RESULTS: Both groups gained bone mineral density at the lumbar spine and hip after 12 months. There were significant (P < 0.05) changes in the pamidronate group, with gains of + 2.6%[95% confidence interval (CI), 1.4-3.0] at the spine and + 1.6% (95% CI, 0.6-2.5) at the hip, compared with gains of + 1.6% (95% CI, - 0.1-3.2) and + 0.9% (95% CI, - 0.4-2.1) at the spine and hip, respectively, in the group taking vitamin D and calcium supplements alone. CONCLUSIONS: In patients with Crohn's disease and low bone mineral density, intravenous pamidronate significantly increases the bone mineral density at the lumbar spine and hip.

PKCbeta modulates antigen receptor signaling via regulation of Btk membrane localization.

Mutations in Bruton's tyrosine kinase (Btk) result in X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. While targeted disruption of the protein kinase C-beta (PKCbeta) gene in mice results in an immunodeficiency similar to xid, the overall tyrosine phosphorylation of Btk is significantly enhanced in PKCbeta-deficient B cells. We provide direct evidence that PKCbeta acts as a feedback loop inhibitor of Btk activation. Inhibition of PKCbeta results in a dramatic increase in B-cell receptor (BCR)-mediated Ca2+ signaling. We identified a highly conserved PKCbeta serine phosphorylation site in a short linker within the Tec homology domain of Btk. Mutation of this phosphorylation site led to enhanced tyrosine phosphorylation and membrane association of Btk, and augmented BCR and FcepsilonRI-mediated signaling in B and mast cells, respectively. These findings provide a novel mechanism whereby reversible translocation of Btk/Tec kinases regulates the threshold for immunoreceptor signaling and thereby modulates lymphocyte activation.

The use of dynamic phantoms in interventional radiology.

The authors have constructed a 2D motor-controlled test object phantom holder to simulate clinical situations in which patient movement could be a cause of image degradation. The PAtient MOvement SImulation Test Object (PAMOSITO) has been constructed with modular parts to use different mobile test objects and static structures. The system allows the programming of different cycles of movement along two axes. PAMOSITO has been used in X ray equipment dedicated to interventional radiology. Those systems usually allow for different values for frame rate, pulse width or weighted frame averaging methods. The influence of selecting different values of the parameters, patient movement and its relation to patient dose and image quality has been studied. Image blurring due to motion has been evaluated with Leeds test objects TO.10 and 18FG. Spatial resolution limits and the threshold contrast detail detectability performance have been studied.

TCL1 oncogene expression in B cell subsets from lymphoid hyperplasia and distinct classes of B cell lymphoma.

Activation of the TCL1 oncogene has been implicated in T cell leukemias/lymphomas and recently was associated with AIDS diffuse large B cell lymphomas (AIDS-DLBCL). Also, in nonmalignant lymphoid tissues, antibody staining has shown that mantle zone B cells expressed abundant Tcl1 protein, whereas germinal center (GC; centrocytes and centroblasts) B cells showed markedly reduced expression. Here, we analyze isolated B cell subsets from hyperplastic tonsil to determine a more precise pattern of Tcl1 expression with development. We also examine multiple B cell lines and B lymphoma patient samples to determine whether different tumor classes retain or alter the developmental pattern of expression. We show that TCL1 expression is not affected by Epstein-Barr virus (EBV) infection and is high in naïve B cells, reduced in GC B cells, and absent in memory B cells and plasma cells. Human herpesvirus-8 infected primary effusion lymphomas (PEL) and multiple myelomas are uniformly TCL1 negative, whereas all other transformed B cell lines tested express moderate to abundant TCL1. This observation supports the hypothesis that PEL, like myeloma, usually arise from post-GC stages of B cell development. Tcl1 protein is also detected in most naïve/GC-derived B lymphoma patient samples (23 of 27 [85%] positive), whereas most post-GC-derived B lymphomas lack expression (10 of 41 [24%] positive). These data indicate that the pattern of Tcl1 expression is distinct between naïve/GC and post-GC-derived B lymphomas (P < 0.001) and that the developmental pattern of expression is largely retained. However, post-GC-derived AIDS-DLBCL express TCL1 at a frequency equivalent to naïve/GC-derived B lymphomas in immune-competent individuals (7 of 9 [78%] positive), suggesting that TCL1 down-regulation is adversely affected by severe immune system dysfunction. These findings demonstrate that TCL1 expression in B cell lymphoma usually reflects the stage of B cell development from which they derive, except in AIDS-related lymphomas.

Primary angiitis of the central nervous system in children: 5 cases.

We describe 5 children who meet criteria for primary angiitis of the central nervous system (PACNS). All patients presented with headache and/or focal neurologic deficits and exhibited clinical and/or radiographic evidence of disease progression. Two patients had disease progression prior to combined treatment with cyclophosphamide and corticosteroids; one progressed while receiving intravenous cyclophosphamide and stabilized after a change to daily oral dosing; one progressed after discontinuing therapy after less than 12 months and improved after retreatment; and one progressed on steroid therapy alone but was lost to followup. Children who have frequent or severe headaches or focal neurologic deficits should be carefully evaluated and those meeting criteria for PACNS should be treated aggressively.

Unrelated umbilical cord stem cell transplantation for X-linked immunodeficiencies.

Banked unrelated umbilical cord blood matched at 5 of 6 human leukocyte antigen loci was used to reconstitute the immune system in 2 brothers with X-linked lymphoproliferative syndrome and 1 boy with X-linked hyperimmunoglobulin-M syndrome. Pretransplant cytoreduction and posttransplant graft-versus-host prophylaxis were given. Hematopoietic engraftment and correction of the genetic defects were documented by molecular techniques. Two years after transplantation, all 3 patients have normal immune systems. These reports support the wider use of banked partially matched cord blood for transplantation in primary immunodeficiencies.

Engagement of the human pre-B cell receptor generates a lipid raft-dependent calcium signaling complex.

Pre-B cell receptor (pre-BCR) expression is critical for B lineage development. The signaling events initiated by the pre-BCR, however, remain poorly defined. We demonstrate that lipid rafts are the major functional compartment for human pre-B cell activation. A fraction of pre-BCR was constitutively raft associated, and receptor engagement enhanced this association. These events promoted Lyn activation and Igbeta phosphorylation and led to the generation of a raft-associated signaling module composed of tyrosine phosphorylated Lyn, Syk, BLNK, PI3K, Btk, VAV, and PLCgamma2. Formation of this module was essential for pre-BCR calcium signaling. Together, these observations directly link the previously identified genetic requirement for the components of this module in B lineage development with theirfunctional role(s) in human preBCR signaling.

Redundant and opposing functions of two tyrosine kinases, Btk and Lyn, in mast cell activation.

Protein-tyrosine kinases play crucial roles in mast cell activation through the high-affinity IgE receptor (FcepsilonRI). In this study, we have made the following observations on growth properties and FcepsilonRI-mediated signal transduction of primary cultured mast cells from Btk-, Lyn-, and Btk/Lyn-deficient mice. First, Lyn deficiency partially reversed the survival effect of Btk deficiency. Second, FcepsilonRI-induced degranulation and leukotriene release were almost abrogated in Btk/Lyn doubly deficient mast cells while singly deficient cells exhibited normal responses. Tyrosine phosphorylation of cellular proteins including phospholipases C-gamma1 and C-gamma2 was reduced in Btk/Lyn-deficient mast cells. Accordingly, FcepsilonRI-induced elevation of intracellular Ca2+ concentrations and activation of protein kinase Cs were blunted in the doubly deficient cells. Third, in contrast, Btk and Lyn demonstrated opposing roles in cytokine secretion and mitogen-activated protein kinase activation. Lyn-deficient cells exhibited enhanced secretion of TNF-alpha and IL-2 apparently through the prolonged activation of extracellular signal-related kinases and c-Jun N-terminal kinase. Potentially accounting for this phenomenon and robust degranulation in Lyn-deficient cells, the activities of protein kinase Calpha and protein kinase CbetaII, low at basal levels, were enhanced in these cells. Fourth, cytokine secretion was severely reduced and c-Jun N-terminal kinase activation was completely abrogated in Btk/Lyn-deficient mast cells. The data together demonstrate that Btk and Lyn are involved in mast cell signaling pathways in distinctly different ways, emphasizing that multiple signal outcomes must be evaluated to fully understand the functional interactions of individual signaling components.

Aberrant B cell receptor signaling from B29 (Igbeta, CD79b) gene mutations of chronic lymphocytic leukemia B cells.

Chronic lymphocytic leukemia (CLL) B cells characteristically exhibit low or undetectable surface B cell receptor (BCR) and diminished responses to BCR-mediated signaling. These features suggest that CLL cells may have sustained mutations affecting one or more of the BCR proteins required for receptor surface assembly and signal transduction. Loss of expression and mutations in the critical BCR protein B29 (Igbeta, CD79b), are prevalent in CLL and could produce the hallmark features of these leukemic B cells. Because patient CLL cells are intractable to manipulation, we developed a model system to analyze B29 mutations. Jurkat T cells stably expressing micro, kappa, and mb1 efficiently assembled a functional BCR when infected with recombinant vaccinia virus bearing wild-type B29. In contrast, a B29 CLL mutant protein truncated in the transmembrane domain did not associate with mu or mb1 at the cell surface. Another B29 CLL mutant lacking the C-terminal immunoreceptor tyrosine activation motif tyrosine and distal residues brought the receptor to the surface as well as wild-type B29 but showed significant impairment in anti-IgM-stimulated signaling events including mitogen-activated protein kinase activation. These findings demonstrate that B29 mutations previously identified in CLL patients can affect BCR-dependent signaling and may contribute to the unresponsive B cell phenotype in CLL. Finally, the features of the B29 mutations in CLL predict that they may be generated by somatic hypermutation.

Hydroxychloroquine inhibits calcium signals in T cells: a new mechanism to explain its immunomodulatory properties.

Hydroxychloroquine (HCQ), a lysosomotropic amine, is an immunosuppressive agent presently being evaluated in bone marrow transplant patients to treat graft-versus-host disease. While its immunosuppressive properties have been attributed primarily to its ability to interfere with antigen processing, recent reports demonstrate HCQ also blocks T-cell activation in vitro. To more precisely define the T-cell inhibitory effects of HCQ, the authors evaluated T-cell antigen receptor (TCR) signaling events in a T-cell line pretreated with HCQ. In a concentration-dependent manner, HCQ inhibited anti-TCR-induced up-regulation of CD69 expression, a distal TCR signaling event. Proximal TCR signals, including inductive protein tyrosine phosphorylation, tyrosine phosphorylation of phospholipase C gamma1, and total inositol phosphate production, were unaffected by HCQ. Strikingly, anti-TCR-crosslinking-induced calcium mobilization was significantly inhibited by HCQ, particularly at the highest concentrations tested (100 micromol/L) in both T-cell lines and primary T cells. HCQ, in a dose-dependent fashion, also reduced a B-cell antigen receptor calcium signal, indicating this effect may be a general property of HCQ. Inhibition of the calcium signal correlated directly with a reduction in the size of thapsigargin-sensitive intracellular calcium stores in HCQ-treated cells. Together, these findings suggest that disruption of TCR-crosslinking-dependent calcium signaling provides an additional mechanism to explain the immunomodulatory properties of HCQ.

Case-control study of the pathogenesis and sequelae of symptomatic vertebral fractures in men.

To investigate the pathogenesis and sequelae of symptomatic vertebral fractures (VF) in men, we have performed a case-control study, comparing 91 men with VF (median age 64 years, range 27-79 years) with 91 age-matched control subjects. Medical history, clinical examination and investigations were performed in all patients and control subjects, to identify potential causes of secondary osteoporosis, together with bone mineral density (BMD) measurements. BMD was lower at the lumbar spine and all sites in the hip in patients with VF than in control subjects (p < 0.001). Potential underlying causes of secondary osteoporosis were found in 41% of men with VF, compared with 9% of control subjects (OR 7.1; 95% CI 3.1-16.4). Oral corticosteroid and anti-convulsant treatment were both associated with a significantly increased risk of VF (OR 6.1; 95% CI 1.3-28.4). Although hypogonadism was not associated with an increased risk of fracture, the level of sex hormone binding globulin was higher (p < 0.001) and the free androgen index lower (p < 0.001) in men with VF than control subjects. Other factors associated with a significantly increased risk of VF were family history of bone disease (OR 6.1; 95% CI 1.3-28.4), current smoking (OR 2.8; 95% CI 1.2-6.7) and alcohol consumption of more than 250 g/week (OR 3.8; 95% CI 1.7-8.7). Men with VF were more likely to complain of back pain (p < 0.001) and greater loss of height (p < 0.001) than control subjects, and had poorer (p < 0.001) scores for the energy, pain, emotion, sleep and physical mobility domains of the Nottingham Health Profile. We conclude that symptomatic VF in men are associated with reduced BMD, underlying causes of secondary osteoporosis such as corticosteroid and anti-convulsant treatment, family history of bone disease, current smoking and high alcohol consumption, and that they impair the perceived health of the individual.

Bruton's tyrosine kinase controls a sustained calcium signal essential for B lineage development and function.

Genetic data support a role for Btk during the B lineage development transitions regulated by signaling through both the pre-B and the B cell antigen receptors. Dysregulated signaling at each of these transitions can result in failure of these cell populations to proliferate and subsequent cell death. Btk-dependent IP3 production is crucial for maintaining the sustained calcium signal in response to BCR engagement and is likely to regulate a subset of transcriptional events essential for B lineage growth or survival. Identification of these Btk-dependent signals will be important in understanding B cell activation, differentiation, and cell death. This information may lead to therapies specifically targeting these events in B cell autoimmunity or malignancy and provide a fuller understanding of the appropriate target populations and potential negative consequences of Btk gene therapy in XLA. Identification of Btk/Tec family kinases in an increasing number of vertebrate and invertebrate cell lineages suggests that the link between Btk and the PLC gamma/IP3/calcium signaling pathways may be broadly conserved.

Body composition of preterm infants during infancy.

AIMS: To examine body composition in preterm infants. METHODS: Body composition was measured by dual energy x-ray absorptiometry (DEXA) at hospital discharge, term, 12 weeks, and at 6 and 12 months corrected age in 125 infants (birthweight < or = 1750 g, gestational age < or = 34 weeks). RESULTS: Body weight derived by DEXA accurately predicted that determined by conventional scales. In both sexes lean mass (LM), fat mass (FM), %FM, bone area (BA), bone mineral mass (BMM), and bone mineral density (BMD) increased rapidly during the study; significant changes were detectable between discharge and term. At 12 months, LM, BA, and BMM, but not FM, %FM, or BMD were greater in boys than in girls. Corrected for age, LM was less than those of the reference term infant; FM and %FM were similar; BMM was greater. Corrected for weight, LM was similar to those of the reference infant, while the FM and %FM of study infants were slightly greater. CONCLUSIONS: DEXA accurately measures body mass. Body composition in preterm boys and girls differs. Interpretation of DEXA values may depend on whether age or body weight are regarded as the appropriate reference.

In situ detection of activated Bruton's tyrosine kinase in the Ig signaling complex by phosphopeptide-specific monoclonal antibodies.

Bruton's tyrosine kinase (Btk) is a critical transducer of signals originating from the B cell antigen receptor (BCR). Dosage, sequential phosphorylation, and protein interactions are interdependent mechanisms influencing Btk function. Phosphopeptide-specific mAbs recognizing two distinct phosphotyrosine modifications were used to quantify Btk activation by immunofluorescent techniques during B cell stimulation. In a population of cultured B cells stimulated by BCR crosslinking and analyzed by flow cytometry, transient phosphorylation of the regulatory Btk tyrosine residues (551Y and 223Y) was detected. The kinetics of phosphorylation of the residues were temporally distinct. Tyrosine 551, a transactivating substrate site for Src-family kinases, was maximally phosphorylated within approximately 30 seconds of stimulation as monitored by flow cytometry. Tyrosine 223, an autophosphorylation site within the SH3 domain, was maximally phosphorylated at approximately 5 minutes. Btk returned to a low tyrosine phosphorylation level within 30 minutes, despite persistent elevation of global tyrosine phosphorylation. Colocalization of activated Btk molecules with the crosslinked BCR signaling complex was observed to coincide with the period of maximal Btk tyrosine phosphorylation when stimulated B cells were analyzed with confocal microscopy. The results of these in situ temporal and spatial analyses imply that Btk signaling occurs in the region of the Ig receptor signaling complex, suggesting a similar location for downstream targets of its activity.

In vitro reconstitution of human B-cell ontogeny: from CD34(+) multipotent progenitors to Ig-secreting cells.

We describe a long-term, in vitro culture system initiated with CD34(+) or CD34(+)CD38(-) umbilical cord blood hematopoietic progenitors that supports normal human B-lineage development, including the production of mature Ig-secreting B cells. In the first stage (human B-progenitor long-term culture [HB-LTC]), CD34(+) hematopoietic progenitors are cultured on the murine stromal cell line, S17, leading to the sustained production of large numbers of CD10(+), CD19(+) early B progenitors. Reverse transcriptase-polymerase chain reaction (RT-PCR) and three-parameter flow cytometry for VpreB (surrogate light chain), cytoplasmic mu chain, and surface IgM expression were used to characterize the CD19(+) B progenitors present within these cultures. This analysis showed distinct B-lineage subpopulations, including pro-B cells, cycling pre-B cells, and IgM+, IgD-/+ immature B cells. The limited expansion of IgM+ B cells and the immature surface phenotype of this population (IgM+, IgD+, CD10(+), CD38(+)) suggested that HB-LTC conditions were unable to provide appropriate signals for further differentiation. A second culture stage was used to determine if these immature B cells were functionally competent. Purified CD19(+) cells were transferred onto fibroblasts expressing human CD40-ligand in the presence of IL-10 and IL-4. This lead to cell proliferation, modulation of the IgM+ cell surface phenotype to one consistent with an activated mature B cell, secretion of Ig, and isotype switching. Notably, IgM and IgG producing B cells were also generated using two-stage cultures established with highly purified multipotent CD34(+)CD38(-) hematopoietic stem cell progenitors. This culture model should permit detailed in vitro analysis and genetic manipulation of the major transition points in human B ontogeny, beginning with commitment to the B lineage and leading to development and activation of mature B cells.