Electron Crescent Distributions as a Manifestation of Diamagnetic Drift in an Electron-Scale Current Sheet: Magnetospheric Multiscale Observations Using New 7.5 ms Fast Plasma Investigation Moments.

We report Magnetospheric Multiscale observations of electron pressure gradient electric fields near a magnetic reconnection diffusion region using a new technique for extracting 7.5 ms electron moments from the Fast Plasma Investigation. We find that the deviation of the perpendicular electron bulk velocity from E × B drift in the interval where the out-of-plane current density is increasing can be explained by the diamagnetic drift. In the interval where the out-of-plane current is transitioning to in-plane current, the electron momentum equation is not satisfied at 7.5 ms resolution.

Quantifying the Effect of Non-Larmor Motion of Electrons on the Pressure Tensor.

In space plasma, various effects of magnetic reconnection and turbulence cause the electron motion to significantly deviate from their Larmor orbits. Collectively these orbits affect the electron velocity distribution function and lead to the appearance of the "non-gyrotropic" elements in the pressure tensor. Quantification of this effect has important applications in space and laboratory plasma, one of which is tracing the electron diffusion region (EDR) of magnetic reconnection in space observations. Three different measures of agyrotropy of pressure tensor have previously been proposed, namely, A∅ e , Dng and Q. The multitude of contradictory measures has caused confusion within the community. We revisit the problem by considering the basic properties an agyrotropy measure should have. We show that A∅ e , Dng and Q are all defined based on the sum of the principle minors (i.e. the rotation invariant I 2) of the pressure tensor. We discuss in detail the problems of I 2-based measures and explain why they may produce ambiguous and biased results. We introduce a new measure AG constructed based on the determinant of the pressure tensor (i.e. the rotation invariant I 3) which does not suffer from the problems of I 2-based measures. We compare AG with other measures in 2 and 3-dimension particle-in-cell magnetic reconnection simulations, and show that AG can effectively trace the EDR of reconnection in both Harris and force-free current sheets. On the other hand, A∅ e does not show prominent peaks in the EDR and part of the separatrix in the force-free reconnection simulations, demonstrating that A∅ e does not measure all the non-gyrotropic effects in this case, and is not suitable for studying magnetic reconnection in more general situations other than Harris sheet reconnection.

Immunoglobulin heavy chain expression shapes the B cell receptor repertoire in human B cell development.

Developing B cells must pass a series of checkpoints that are regulated by membrane-bound Ig(mu) through the Igalpha-Igbeta signal transducers. To determine how Ig(mu) expression affects B cell development and Ab selection in humans we analyzed Ig gene rearrangements in pro-B cells from two patients who are unable to produce Ig(mu) proteins. We find that Ig(mu) expression does not affect V(H), D, or J(H) segment usage and is not required for human Igkappa and Iglambda recombination or expression. However, the heavy and light chains found in pro-B cells differed from those in peripheral B cells in that they showed unusually long CDR3s. In addition, the Igkappa repertoire in Ig(mu)-deficient pro-B cells was skewed to downstream Jkappas and upstream Vkappas, consistent with persistent secondary V(D)J rearrangements. Thus, Ig(mu) expression is not required for secondary V(D)J recombination in pro-B cells. However, B cell receptor expression shapes the Ab repertoire in humans and is essential for selection against Ab's with long CDR3s.

Quantitative trait loci analysis of powdery mildew disease resistance in the Arabidopsis thaliana accession kashmir-1.

Powdery mildew diseases are economically important diseases, caused by obligate biotrophic fungi of the Erysiphales. To understand the complex inheritance of resistance to the powdery mildew disease in the model plant Arabidopsis thaliana, quantitative trait loci analysis was performed using a set of recombinant inbred lines derived from a cross between the resistant accession Kashmir-1 and the susceptible accession Columbia glabrous1. We identified and mapped three independent powdery mildew quantitative disease resistance loci, which act additively to confer disease resistance. The locus with the strongest effect on resistance was mapped to a 500-kbp interval on chromosome III.

Circulating human B cells that express surrogate light chains and edited receptors.

Immunoglobulin gene recombination can result in the assembly of self-reactive antibodies. Deletion, anergy or receptor editing normally silence B cells that produce these autoantibodies. Receptor editing is highly efficient in mouse B cells that carry pre-recombined autoantibody transgenes or gene "knock-ins". However, it has been difficult to identify cells that have edited receptors in unmanipulated mice and humans. To try to identify such cells we isolated and characterized B cells that coexpress surrogate and conventional light chains (V-preB+L+) from the blood of normal human donors. V-preB+L+ B cells express RAG mRNA, display an unusual heavy and light chain antibody repertoire consistent with antiself reactivity, and show evidence of receptor editing. These cells accumulate in the joints of patients with rheumatoid arthritis, consistent with a role for V-preB+L+ B cells and receptor editing in autoimmune disease.

Fine characterization of childhood and adult acute lymphoblastic leukemia (ALL) by a proB and preB surrogate light chain-specific mAb and a proposal for a new B cell ALL classification.

The expression of the surrogate light chain (psiL) - made of the lambda-like (or lambda5) and the VpreB proteins - is a B cell-specific maturation marker. Using an anti-human VpreB mAb (4G7), we recently identified in human normal bone marrows, proB and preB cells that express the psiH-psiL proB (proBCR) and the mu-psiL preB (preBCR) receptors, respectively. In the present study, FACS and biochemical analysis confirm the broad proB and preB reactivity of the 4G7 mAb that contrasts with the narrow specificity of other available anti-psiL reagents for preB cells. This mAb was used to explore intracytoplasmic and cell surface expression of the VpreB protein on a series of 92 precursor B cell ALLs (from 40 child and 52 adult patients), in combination with 24 other mAbs. The major result concerns the identification within proB (or BI) and common (or BII) ALLs, of proBCR and proBCR+ ALLs that express the VpreB in the cytoplasm or at the cell surface, respectively. The percentage of ALLs within these two VpreB sub-groups differ considerably between the ALL origin. In the pediatric series, ALLs present in the majority a proBCR+ phenotype whereas we observed a proBCR+ phenotype for adult ALLs. Based on VpreB expression, and in combination with other published data, we propose a refined classification for precursor B cell ALLs.

Autosomal primary immunodeficiencies affecting human bone marrow B-cell differentiation.

Since the initial report of X-linked agammaglobulinemia by Bruton, numerous autosomal primary immune deficiencies affecting early B-cell differentiation have been described in humans. The identification of these autosomal mutations has been facilitated by phenotype comparison with knockout mice. In mice, defects in B-cell development have been observed after disruption of genes encoding transcription factors, the interleukin-7 pathways as well as structural or signaling components of the pre-B-cell receptor. In general, the phenotypes of primary immune deficiencies in humans correlate with those observed in mutant mice, validating the use of the mouse model approach. In addition, we report a follow-up analysis of an autosomal primary deficiency in a young female patient born from consanguinous parents and characterized by the absence of pre-B and B-cell compartments. The patient's gene defect was identified as a cytosine insertion at the beginning of the CH1 exon of the Ig(mu) gene, resulting in a stop codon at position 48 and the absence of Ig(mu) chain expression. The precise phenotype of this patient is compared to other autosomal primary immunodeficiencies affecting humans and mice.

Stromal cell-derived factor 1 (SDF-1) and antenatal human B cell lymphopoiesis: expression of SDF-1 by mesothelial cells and biliary ductal plate epithelial cells.

The chemokine stromal cell-derived factor 1 (SDF-1) stimulates the growth of pre-B cells in vitro, and mice with a disrupted SDF-1 gene have abnormal fetal liver B cell lymphopoiesis. The origin of SDF-1 production has not been determined yet. Using an anti-SDF-1 mAb, we performed immunohistochemical studies in four human embryos and five fetuses to define which cells express the SDF-1 protein at sites of antenatal B cell lymphopoiesis. All mesothelial cells contained SDF-1 at all stages of development, including in the intraembryonic splanchnopleuric mesoderm early into gestation. In fetal lungs and kidneys, SDF-1 was expressed by epithelial cells, and a few B lymphoid precursors, expressing V pre-B chains, were also detected. In the fetal liver, in addition to mesothelial cells, biliary epithelial cells were the only cells to contain SDF-1. Pre-B cells expressing V chains were abundant and exclusively located around the edge of portal spaces, in close contact with biliary ductal plate epithelial cells. They did not colocalize with biliary collecting ducts. Biliary ductal plate epithelial cells and liver B cell lymphopoiesis display a parallel development and disappearance during fetal life. These results indicate that early B cell lymphopoiesis in the splanchnopleura may be triggered by mesothelial cells producing SDF-1. Later into gestation, biliary ductal plate epithelial cells may support B cell lymphopoiesis, thus playing a role similar to that of epithelial cells in the avian bursa of Fabricius, and of thymic epithelial cells for T cell lymphopoiesis.

The human (PsiL+mu-) proB complex: cell surface expression and biochemical structure of a putative transducing receptor.

The surrogate light chain (PsiL) associates with mu and Igalpha-Igbeta chains to form the preB-cell receptor that plays a critical role in early B-cell differentiation. Discrepancies exist in human concerning the existence of PsiL+mu- proB cells and the biochemical structure of such a proB-cell complex remains elusive. Among new antihuman VpreB monoclonal antibodies (MoAbs), 5 of the gamma kappa isotype bound to recombinant and native VpreB protein with high affinity. They recognized 4 discrete epitopes, upon which 2 were in the extra-loop fragment. Such MoAbs detected the PsiL at the cell surface of either preB or on both proB and preB cells. The previously reported SLC1/SLC2 MoAbs recognize a conformational epitope specific for the mu/PsiL association in accordance with their preB-cell reactivity. Using the proB/preB 4G7 MoAb, PsiL cell surface expression was detected on normal bone marrow, not only on CD34(-)CD19(+) preB but also on CD34(+)CD19(+) proB cells. Futhermore, this MoAb identified PsiL+mu- fresh proB leukemic cells of the TEL/AML1 type. Biochemical studies showed that, at the proB stage, the PsiL is associated noncovalently with two proteins of 105 and 130 kD. Triggering of this complex induces intracellular Ca2+ flux, suggesting that the PsiL may be involved in a new receptor at this early step of the B-cell differentiation.

Mu-surrogate light chain physicochemical interactions of the human preB cell receptor: implications for VH repertoire selection and cell signaling at the preB cell stage.

The surrogate light chain (SL) composed of the A-like and VpreB polypeptides is organized as two Ig domains and an extra-loop structure. It associates to the mu-chain in preB cells. We have produced human VpreB, SL, two Fdmu (VH-CH1), and the two corresponding Fab-like (Fdmu-SL) recombinant proteins in baculovirus. The correctness of the general conformation of the proteins was assessed by epitope mapping and affinity measurements using a new batch of anti-VpreB mAbs. Plasmon resonance analysis showed that both VpreB and the entire SL associated with the Fdmu fragments, with Kd values of 3x10(-8) M for VpreB-Fdmu and of 10(-9) to 10(-10) M, depending upon the V(H), for SL-Fdmu. These results indicate that the A-like chain, in addition to be covalently bound to the Cmu1 domain, also interacts with the VH domain. Therefore, a dual role of the SL emerges: 1) interaction of the C-domain of A-like would release the mu-chain from its interaction with binding protein in the endoplasmic reticulum, and 2) interaction of a part of A-like and most of VpreB would bind to VH, ensuring a "quality control" of the native heavy chain that represents the first step of selection of the B cell repertoire. We also demonstrated that two Fab-like fragments did not interact with each other, suggesting that activation of the cell surface preB receptor does not involve aggregation neither in cis nor in trans of the Fab-like structures.

VPREB3: cDNA characterization and expression in human and chromosome mapping in human and mouse.

The pre-B cell receptor (pre-BCR) regulates pre-B cell expansion and allelic exclusion at the immunoglobulin (Ig) heavy chain locus and mediates the selection of Ig heavy chain variable gene segments. During the early phase of pre-BCR assembly in the mouse, the membrane Ig mu heavy chain transiently associates with the VPREB3 protein in the endoplasmic reticulum. Here, we present the human VPREB3 cDNA sequence and its B cell-specific expression in hematopoietic cell lines. We have localized this gene to chromosome 22q11 close to IGLL genes in human and to chromosome 10C in mouse.

Antigen receptor engagement turns off the V(D)J recombination machinery in human tonsil B cells.

The germinal center (GC) is an anatomic compartment found in peripheral lymphoid organs, wherein B cells undergo clonal expansion, somatic mutation, switch recombination, and reactivate immunoglobulin gene V(D)J recombination. As a result of somatic mutation, some GC B cells develop higher affinity antibodies, whereas others suffer mutations that decrease affinity, and still others may become self-reactive. It has been proposed that secondary V(D)J rearrangements in GCs might rescue B cells whose receptors are damaged by somatic mutations. Here we present evidence that mature human tonsil B cells coexpress conventional light chains and recombination associated genes, and that they extinguish recombination activating gene and terminal deoxynucleotidyl transferase expression when their receptors are cross-linked. Thus, the response of the recombinase to receptor engagement in peripheral B cells is the opposite of the response in developing B cells to the same stimulus. These observations suggest that receptor revision is a mechanism for receptor diversification that is turned off when antigen receptors are cross-linked by the cognate antigen.

A non-XLA primary deficiency causes the earliest known defect of B cell differentiation in humans: a comparison with an XLA case.

We report a detailed comparison of B cell defects in two patients, one XLA and one non-XLA. Both had severe agammaglobulinemia with a total absence of CD19+ cells in the periphery. In the non-XLA case, CD19 expression was also highly impaired in the bone marrow, resulting in the absence of both B and preB compartments. Early proB cells were present since CD34+CD10+ and some CD19+CD10+ mostly CD34+ were identified, although diminished. By contrast, in the XLA patient the CD34+CD19+ proB cells were increased whereas the CD34-CD19+ preB cell population was low. Semi-quantitative RT-PCR analysis performed on mononuclear bone marrow cells from the non-XLA patient indicated that lambda-like, VpreB, Rag-1, Rag-2 and TdT transcripts expressed during proB cell stages were found at normal levels whereas E2A, CD10, Syk, Pax-5, CD19, Ig alpha, Ig beta, VH-C mu and V kappa-C kappa transcripts characteristic of later stages were severely depressed. By contrast in the XLA patient most of these transcripts were observed in normal amounts. The phenotype of the non-XLA patient resembles that of Pax-5 or Ig beta knock-out mice, but since the coding sequence of both cDNAs were shown to be normal, the blockage might rather result from an altered regulation of one of these genes or from defect of other genes. All these data indicate that the non-XLA patient suffers from a new genetic defect that results in an arrest of differentiation within the proB cell compartment, before the onset of Ig gene rearrangements. From all agammaglobulinemias reported so far, including XLA cases and those resulting from C mu gene defects, the non-XLA patient exhibits the earliest blockage in the B cell differentiation pathway.

Individual CD34+CD38lowCD19-CD10- progenitor cells from human cord blood generate B lymphocytes and granulocytes.

Identification of human hematopoietic stem cells and analysis of molecular mechanisms regulating their function require biological assays that permit differentiation in all hematopoietic lineages simultaneously. In this study, we established conditions that permit the joint expression of the B-lymphoid and myeloid potential from cord blood-derived CD34+CD38lowCD19-/CD10- primitive progenitors that lack B-specific markers and transcripts. When cocultured during 6 weeks with the murine stromal cells MS-5 in the absence of exogenous human cytokines, CD34+CD38low-CD19-CD10- cells generated a high number of CD19+ B cells. Virtually all of these cells expressed a CD34-CD10+- CD19+cIgM- phenotype of late pro-B cells and transcripts of Pax-5, lambda-like, and mu chain were detected. We further show that 7% of CD34+CD38lowCD19- cells from cord blood, when grown individually with MS-5 cells, generated both CD19+ and CD11b+ cells after 6 weeks. Efficient B-cell differentiation was also observed in vivo after transplantation of human cord blood-derived unfractionated mononuclear cells or CD34+CD19+CD10- cells into immune-deficient mice. In contrast to the in vitro situation, all stages of B-cell differentiation were observed in vivo, including pro-B, pre-B, and sIgM+ B cells. Interestingly, human progenitors with the ability to differentiate along both B-lymphoid and granulocytic pathways were also detected among human CD34+CD38low cells in the marrow of chimeric mice 6 to 7 weeks after transplantation. Both in vitro and in vivo systems will offer an invaluable tool to further identify the lymphoid and myeloid potentialities of primitive progenitor cells isolated from fetal as well as adult human hematopoietic tissues and characterize stromal-derived signals that regulate their function.

Abnormal CD40-mediated activation pathway in B lymphocytes from patients with hyper-IgM syndrome and normal CD40 ligand expression.

The CD40-mediated activation pathway of B cells from 10 patients with hyper-IgM syndrome and normal expression of CD40 ligand was studied. In all 10 cases, B cells were found to be defective for IgG, IgA, and IgE production after stimulation by anti-CD40 mAbs and cytokines. In the patients tested, neither B cell proliferation (n = 6) nor CD23 molecule expression (n = 5) were observed in cultures stimulated with anti-CD40 mAb. These results point to an intrinsic B cell deficiency and a defect in the CD40-triggered B cell activation pathway; this conclusion was supported by a lack of detectable germinal centers in the spleen of two patients. CD40-triggered activation events, i.e., phosphatidylinositol 3 (PI3) kinase activation and induction of transcription factors NF-kappaB and AP-1, were next analyzed in B cell lines derived from five patients. Three distinct patterns were observed: an absence of detectable abnormalities (n = 1), defective PI3 kinase activation with normal induction of NF-kappaB and AP-1 (n = 3), and defects in both PI3 kinase activation and induction of NF-kappaB and AP-1 (n = 1). In three B cell lines, each exhibiting one of the CD40-mediated activation patterns, sequences of CD40 and CD40 binding protein coding regions were normal. The coding region of TNF receptor-associated factor 2 (TRAF2), which is known to interact with CD40 for NF-kappaB induction, was also found to be normal in B cell lines deficient in NF-kappaB induction. Altogether, these results suggest that CD40 ligand-positive hyper-IgM syndrome could be genetically heterogeneous, although phenotypic variability is not excluded, and that an early defect in the CD40-triggered activation cascade can account for defective Ig class switching in some patients with CD40 ligand-positive hyper-IgM syndrome.

Bronchiolitis obliterans organizing pneumonia as the first manifestation of polymyositis.

Bronchiolitis obliterans organizing pneumonia (BOOP) preceding polymyositis is rare. In this report, a 51-year-old patient with fever, nonproductive cough, and dyspnea had bilateral basal interstitial infiltrates on chest roentgenogram. Open lung biopsy was consistent with BOOP. Prednisone therapy led to improvement, but 8 weeks later, fever, cough, and weakness of the arms and legs developed because the patient had not been compliant with the prednisone regimen. The creatine kinase (CK), the macrophage inflammatory protein (MIP-1), and the tumor necrosis factor (TNF-alpha) were elevated. Anti-Jo-1 antibody was not present. Quadriceps femoris muscle biopsy was compatible with polymyositis. After a second course of corticosteroid therapy, the patient became afebrile, the dyspnea resolved, the pulmonary infiltrates decreased, and the muscle strength improved. The serum CK, MIP-1, and TNF-alpha levels declined significantly. This is only the second reported case of BOOP preceding polymyositis. Patients with idiopathic BOOP should have follow-up for the possible development of connective tissue disorders including polymyositis.

A human non-XLA immunodeficiency disease characterized by blockage of B cell development at an early proB cell stage.

We report a detailed analysis of a B cell defect affecting a patient girl born from first cousin parents, characterized by a severe non-X-linked agammaglobulinemia with a total absence of CD19- cells in the periphery. In the bone marrow, CD19 expression was also highly impaired, resulting in the absence of both B and preB compartments. By contrast, CD34+CD10+, CD34psiL+, and some CD19+CD10+ mostly CD34+ early proB cells were present, although diminished. Semiquantitative RT-PCR analysis performed on mononuclear bone marrow cells indicated that lambda-like, VpreB, Rag-1, Rag-2, and TdT transcripts expressed during proB cell stages were found at normal levels whereas E2A, CD10, Syk, Pax-5, CD19, Igalpha, Igbeta, VH-Cmu, and Vkappa-Ckappa transcripts characteristic of later stages were severely depressed. This phenotype resembles that of Pax-5 knock-out mice, but since the coding sequence of the patient Pax-5 cDNA was shown to be normal, the defect might rather result from an altered regulation of this gene. All these data indicate that the patient suffers from a new genetic defect that results in an arrest of differentiation within the proB cell compartment, i.e., earlier than X-linked agammaglobulinemia, before the onset of Ig gene rearrangements.

Cell surface expression of surrogate light chain (psi L) in the absence of mu on human pro-B cell lines and normal pro-B cells.

Surrogate light chains (psi L) encoded by lambda-like (lambda 5) and VpreB genes play a critical role in controlling the early steps of B cell differentiation. We prepared new anti-VpreB monoclonal antibodies (mAb) (3C7/6F6) which preferentially recognize the VpreB epitope at the cell surface of human cell lines that do not express the mu chain. These mAb provide the first characterization of human pro-B cell lines expressing surface psi L. We demonstrate that surface psi L expression is considerably enhanced upon interleukin-7 stimulation and that the psi L complex is formed independently of the Ig alpha/Ig beta heterodimer. Finally, using these antibodies, we confirm the existence of a normal pro-B cell population in human adult bone marrow. These cells are CD34+ CD38+ psi L+, do or do not express CD19, CD10, or both epitopes, and may represent the earliest cell population committed to B cell differentiation.

Composite exon structure of an unusual Ig lambda-like gene located at human 22q11 position.

The surrogate light chain, composed of the VpreB and the lambda-like proteins, plays a critical role in controlling the early stages of B lymphocyte development. The lambda-like locus, located on the q11. 2-q11.3 region of human Chromosome (Chr) 22, contains three genes (14.1 Flambda-1, and 16.1) among which only the 14.1 is functional. This gene contains three exons, whereas the others lack exon 1. We have isolated in fetal liver a transcript of the Flambda-1 gene that contains the exon 3 sequence and a long non-Ig related sequence upstream. We show that this sequence resulted from the splicing of three new exons located telomeric to the Flambda-1 gene, highly homologous to beta-glucuronidase exon 11 (Chr 7), to the ABR exon 8 (Chr 17), and to an Expressed Sequence Tag (EST), respectively. We also show that this chimeric transcript is expressed in cells or tissues from various origins. This composite gene structure appears to be a new example of human genome flexibility, which can be explained by mechanisms such as exon shuffling and which results in the emergence of new transcription units inserted in regions involved in translocations.

Osteonecrosis of the humeral head in sickle cell disease.

To determine the effects of sickle cell disease on the glenohumeral joint, 28 shoulders in 14 patients with SS sickle cell hemoglobinopathy were studied clinically and roentgenographically. patients were randomly selected; their mean age was 46 years (range, 22 to 63 years). Pain, stability, and function of the shoulders were assessed, and roentgenograms were evaluated for osteonecrosis. All 28 shoulders had some degree of pain with activity, but functional range of motion was maintained despite symptoms. Seventy-one percent of the patients had had total hip arthroplasty and 21% had had total knee arthroplasty for osteonecrosis; there was a mean of 1.5 previous joint implants per patient. Our study results show that, in patients with sickle cell hemoglobinopathy, symptoms of humeral head osteonecrosis are better tolerated than those of osteonecrosis in the lower extremities, delaying the need for surgical intervention. With severe pain and functional limitations, shoulder arthroplasty is the procedure of choice in this patient population. However, the risks are greater for patients with sickle cell disease than for other patients who have humeral head osteonecrosis, and thorough preoperative medical and anesthesia evaluations are necessary. These patients require perioperative transfusion or plasmapheresis and sufficient intraoperative hydration and oxygenation to avoid precipitating a sickle cell crisis; in addition, use of methyl methacrylate should be avoided.