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Heavy chain deposition disease (HCDD) is a comparatively recently described entity characterized by glomerular and tubular basement membrane deposition of monoclonal heavy chains without associated light chains. To our knowledge, review of the literature shows only 24 previously reported cases of HCDD with unequivocal evidence of monoclonal heavy chain deposition in the kidney using immunofluorescence microscopic and electron microscopic studies. The predominant heavy chain subtype was γ. There has been a single case of μ HCDD and 2 previously reported cases of α HCDD. In this report, we describe 3 additional cases of α HCDD, all with a crescentic pattern of injury and one of which was associated with cutis laxa. We compare their clinicopathologic features with all previously reported cases of HCDD., (Copyright © 2011 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

This study aimed to identify the frequency and distribution of developing B cell populations in the kidney of the rainbow trout, using four molecular B cell markers that are highly conserved between species, including two transcription factors, Pax5 and EBF1, recombination-activating gene RAG1, and the immunoglobulin heavy chain mu. Three distinct B cell stages were defined: early developing B cells (CLP, pro-B, and early pre-B cells), late developing B cell (late pre-B, immature B, and mature B cells), and IgM-secreting cells. Developmental stage-specific, combinatorial expression of Pax5, EBF1, RAG1 and immunoglobulin mu was determined in trout anterior kidney cells by flow cytometry. Trout staining patterns were compared to a well-defined primary immune tissue, mouse bone marrow, and using mouse surface markers B220 and CD43. A remarkable level of similarity was uncovered between the primary immune tissues of both species. Subsequent analysis of the entire trout kidney, divided into five contiguous segments K1-K5, revealed a complex pattern of early developing, late developing, and IgM-secreting B cells. Patterns in anterior kidney segment K1 were most similar to those of mouse bone marrow, while the most posterior part of the kidney, K5, had many IgM-secreting cells, but lacked early developing B cells. A potential second B lymphopoiesis site was uncovered in segment K4 of the kidney. The B cell patterns, or "B cell signatures" described here provide information on the relative abundance of distinct developing B cell populations in the trout kidney, and can be used in future studies on B cell development in other vertebrate species., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

B cell autoreactivity is a component of chronic graft versus host (GVH) disease in humans and mice. Chronic GVH driven by I-A disparity results in loss of B cell tolerance in Ig/sHEL tolerant mice. In these mice, B cell anergy is characterized by down-modulation of sIgM mediated by intracellular retention in the endoplasmic reticulum (ER) and/or a block in post-ER processing of IgM receptors. Here, we report that GVH induces a selective increase in post-ER processing of the micro chain and trafficking to the cell surface of IgM receptors in B cells that bind HEL self-antigen. The increase in sIgM was detectable as early as 6 days post-GVH, before the appearance of circulating autoantibodies, and was particularly prominent in B cells that up-regulated surface I-A. A further increase in sIgM was found at later time points, along with circulating anti-HEL autoantibodies and a marked decrease in serum-free HEL, but no significant change in the amounts of HEL bound to B cells in vivo. These findings suggest that (i) abrogation of ER retention of IgM receptors in self-reactive B cells is an early event triggered by allogeneic T cells and (ii) at later stages of GVH disease the appearance of autoantibodies reduces the availability of free autoantigen, which may further escalate anergy escape of self-reactive B cells, and lead to exacerbation and perpetuation of autoimmunity.

Class switch recombination (CSR) and somatic hypermutation (SHM) are mechanistically related processes that share common key factors such as activation-induced cytidine deaminase. We have previously shown a role for ATM (mutated in ataxia-telangiectasia) in CSR. In this paper we show that the frequency, distribution, and nature of base pair substitutions in the Ig variable (V) heavy chain genes in ataxia-telangiectasia patients are largely similar to those in normal donors, suggesting a normal SHM process. Characterization of the third complementarity-determining region in B cells from ataxia-telangiectasia patients also shows a normal V(D)J recombination process. SHM-like mutations could be identified in the switch (S) mu region (up to several hundred base pairs upstream of the S mu -S(alpha) breakpoints) in normal in vivo switched human B cells. In the absence of ATM, mutations can still be found in this region, but at less than half the frequency of that in normal donors. The latter mutations are mainly due to transitions (86% compared with 58% in controls) and are biased to A or T nucleotides. An ATM-dependent mechanism, different from that generating SHM in V genes, is therefore likely to be involved in introducing SHM-like mutations in the S region. ATM may thus be one of the factors that is not shared by the CSR and SHM processes.

We report a case of follicular lymphoma with crystal inclusions. Swollen lymph nodes taken from the left neck of a 53- year-old Japanese woman were replaced by follicular proliferation of atypical centroblastic and centrocytic cells with intracytoplasmic crystal inclusions. The crystals were confined to lymphoma cells and were not found in histiocytes. Lymphoma cells were positively immunostained with lambda light chain and mu heavy chain, but the crystals were only weakly so. In situ hybridization of light chains disclosed a monoclonal expression of lambda light chain mRNA in lymphoma cells. The crystals had a periodic linear substructure with about 5-nm intervals. The worldwide literature reports 8 cases, including the current case of non-Hodgkin's lymphoma with crystals confined to the neoplastic cells. The cases did not accompany paraproteinemia and crystal-storing histiocytosis and appear to follow a favorable clinical outcome., (Copyright 2002, Elsevier Science (USA). All rights reserved.)

The quasi-monoclonal (QM) mouse has a functionally rearranged H chain gene inserted into its natural position in the IgH locus. In this position, the H chain gene is subject to many of the same activities as normally arranged H chain genes, including somatic hypermutation, V(H) gene replacement, and class switch recombination. Here, we have used this mouse strain to determine some of the rules that govern the V(D)J recombination activity of the IgH locus in thymus. We focused on the requirements for V(H) gene replacement. In normal mice, thymic DJ(H) rearrangements are common, but VDJ(H) rearrangements are not. We found intermediate products of V(H) replacement in double-positive CD4(+)CD8(+) cells of the QM thymus, demonstrating that the inserted V(H) gene was accessible and ruling out the possibility that a V(H) gene per se cannot be rearranged in the thymus. We found transcripts from the knocked-in H chain gene of QM, but no mu H chain protein was detectable in thymocytes. Cloning and sequencing of these transcripts revealed that some had been generated by V(H) gene replacement. Corresponding signal joints could also be identified. These results suggest that neither a B cell-specific signal nor an Ig protein are necessary to activate V(H)-to-VDJ(H) joining in thymocytes. Possible mechanisms remaining to account for overcoming the barrier to V(H) joining in thymocytes include the insertion of a transcriptionally active gene segment and/or the inactivation of a silencer.

In many organisms nonsense mutations decrease the level of mRNA. In the case of mammalian cells, it is still controversial whether translation is required for this nonsense-mediated RNA decrease (NMD). Although previous analyzes have shown that conditions that impede translation termination at nonsense codons also prevent NMD, the residual level of termination was unknown in these experiments. Moreover, the conditions used to impede termination might also have interfered with NMD in other ways. Because of these uncertainties, we have tested the effects of limiting translation of a nonsense codon in a different way, using two mutations in the immunoglobulin mu heavy chain gene. For this purpose we exploited an exceptional nonsense mutation at codon 3, which efficiently terminates translation but nonetheless maintains a high level of mu mRNA. We have shown 1) that translation of Ter462 in the double mutant occurs at only approximately 4% the normal frequency, and 2) that Ter462 in cis with Ter3 can induce NMD. That is, translation of Ter462 at this low (4%) frequency is sufficient to induce NMD.

A B-cell hybridoma, TP67.21 that expresses surface anti-trinitrophenyl (TNP) IgM but does not secrete the antibody spontaneously has been reported to differentiate into anti-TNP IgM-secreting cells in response to lipopolysaccharide or engagement of surface IgM. Here, we report isolation and characterization of a subclone, TP67.21E (TP.E) that undergoes isotype switching to IgE in an interleukin (IL)-4-dependent manner. TP.E cells secreted anti-TNP IgE depending on exogenous IL-4 when they were cultured with an anti-IgM antibody for 6-8 days. 8-Mercaptoguanosine, which has been shown to enhance IgE class switching in murine splenic B-cells further augmented the IgE response in TP.E cells. Immunofluorescence microscopy revealed that approximately 1.2% of the cultured cells became positive for intracellular IgE after the stimulation culture. The germline epsilon transcripts were expressed transiently on days 2-4 of the culture, while expression of the productive epsilon transcripts was induced 5 days after the start of the culture, thus suggesting that IgE class switching occurred in TP.E cells under these conditions.

To investigate the regulation of Ig switch recombination, we have constructed mice with a 56 kb VDJmudeltagamma1 transgene. This transgene included an anti-nitrophenyl VDJ segment, Smu, Cmu, Cdelta, Igamma1, Sgamma1, Cgamma1 and the Cgamma1 membrane exons from the murine Igh(a) haplotype. Two founder lines were produced, with very similar characteristics. Transgenic B cells expressed normal amounts of Cmu (which is >95% transgenic), Cdelta and other cell surface markers, and normal amounts of VDJ and Cmu RNA. Gamma1 germline transcription of the transgenes is properly regulated since stable transcripts were not expressed in B cells treated with lipopolysaccharide (LPS) alone, nor in thymus or non-lymphoid tissues, but were expressed after treatment of B cells with LPS + IL-4 or CD40L + IL-4. B cells from both lines of transgenic mice expressed transgenic gamma1a after in vitro culture with CD40L + IL-4, but not after culture with CD40L alone. However, the CD40L + IL-4 induced IgG1 precursor frequency is much lower for VDJmudeltagamma1 transgenic B cells (1:240-760) than for non-transgenic B cells (1:9). Analysis of DNA from transgenic hybridomas indicated that switch recombination can take place in switch (S) regions, but can also take place outside S regions. These results indicate that targeting of switch recombinase to S regions must include regulation beyond the S regions themselves and correct germline transcription. This additional regulation might include cis-acting elements or appropriate spacing or arrangement of the recombining elements.

The t(1;19)(q23;p13) translocation involving the E2A gene on chromosome 19p13.3 is a nonrandom translocation that is often seen in childhood pre-B-cell acute lymphoblastic leukemia (ALL). However, recent studies have demonstrated the presence of immunophenotypic and molecular heterogeneity among patients with the cytogenetically identical chromosome translocation. Here we report a novel pre-B ALL cell line, TS-2, with t(1;19) translocation not involving the E2A gene. The breakpoint of t(1;19) in TS-2 was demonstrated to be at 19p13.3, a region indistinguishable from the locus of the E2A gene, by cytogenetic study and fluorescence in situ hybridization. However, rearrangement of the E2A gene was not detected in TS-2 by Southern blot analysis. Moreover, the expressions of PBX1 or E2A/PBX1 fusion genes were not detected by an extensive study with Northern blot analysis and reverse transcription-polymerase chain reaction. These findings suggest that TS-2 may have a genetic abnormality involving uncharacterized gene(s) at 19p13.3 distinct from the E2A gene and, therefore, may be useful for investigating the heterogeneity of molecular pathogenesis in leukemias with t(1;19)(q23;p13) translocation.

Thirty-seven antigenic determinants were identified in the albumins, the immunoglobulin micro- and IgG(Fc) chains, and the C3 proteins of 51 carnivoran (sub)species from 31 genera, and in 12 noncarnivoran mammals. In addition to 19 determinants plesiomorphic for Carnivora as an order, 18 synapomorphic epitopes of carnivoran families revealed nine phylogenetic reaction groups: (1) canids, (2) ursids, (3) the racoon, (4) the Weddell seal, (5) the lesser panda, (6) the harbour seal, (7) mustelids, (8) viverrids and hyaenas, and (9) felids. These data identify Canoidea (Canidae, Ursidae, Phocidae, Procyonidae, Ailuridae, Mustelidae) and Feloidea (Viverridae, Hyaenidae, Felidae) as two fundamentally differentiated lineages of Carnivora, and confirm the inclusion of seals among the former. The Ursidae are the sister group of the Canidae. The antigenic determinants in the studied proteins do not subdivide the Canidae, Ursidae and Felidae into immunologically differentiated lineages.

Leukemic B cells in chronic lymphocytic leukemia (B-CLL) typically exhibit low or undetectable surface Ig. Because the B29 (CD79b and Ig beta) and mb-1 (CD79a and Ig alpha) gene products are required for surface Ig display in the B-cell receptor complex (BCR), we analyzed the expression of these genes in B-CLL cells. The majority (83%) of the randomly selected B-CLL patient samples analyzed exhibited low or undetectable surface BCR measured by mu heavy chain and B29 expression. Levels of mb-1 mRNA in these B-CLL samples with low surface BCR were similar to those in normal B cells. Among those with decreased surface expression, B29 mRNA was not detected in half of these B-CLL samples. The remaining B-CLL samples with diminished surface BCR contained normal levels of B29 mRNA. Further analysis of cDNA clones from the majority of these latter samples contained point mutations, insertions, or deletions that were largely located in the B29 transmembrane and cytoplasmic domains. These results indicate the occurrence of somatic mutations predicted to affect B29 expression and/or function in the majority of B-CLL and suggest that these aberrations underlie the diminished surface BCR display and loss of BCR signaling characteristic of this leukemia.

Transgenic mice carrying mouse interleukin-7 (IL-7) cDNA under the control of MHC class II (E alpha) promoter develop B lymphoid tumors. We have analyzed population dynamics of early precursor B cells and electron microscopic organization of bone marrow (BM) during the prelymphomatous phase. Immunofluorescence labeling of terminal deoxynucleotidyl transferase (TdT), B220 glycoprotein, and mu heavy chains have been used to quantitate three populations of pro-B cells lacking mu chains, cytoplasmic mu-bearing pre-B cells, and surface mu-bearing B lymphocytes. Proliferative activity was assayed by metaphase arrest. In BM of IL-7 transgenic mice, the number and proliferative activity of cells in each of the pro-B and pre-B cell populations were markedly increased. B lymphocytes increased to a lesser extent. The BM cavity was considerably expanded and cortical bone showed focal osteolysis. Immature lymphoid cells compressed the venous sinusoids and exuded through eroded bone. Apoptotic bodies, macrophages, and plasma cells were unusually prominent. B lymphocytes and cells of B precursor phenotype were also much increased in the spleen. These results demonstrate that overexpression of IL-7 causes excessive proliferation of a wide range of precursor B cells in BM. Such prolonged stimulation at early stages of B cell development, prone to genetic errors, may predispose to neoplasia. The bone resorption in these transgenic mice provides a model for bone lesions in BM malignancies.

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.

Two monoclonal antibodies raised against the complex of mu heavy (H) chain and Vpre-B/lambda 5 surrogate light (L) chains recognize surrogate L chain in different conformations on normal pre-B cells. One, LM34 recognizes free lambda 5 protein and free lambda 5/Vpre-B surrogate L chains and binds to surrogate L chains on the surface of early, pro-B and pre-B-I cells where the surrogate L chain is associated with a gp130/gp35-65 complex of proteins. It also recognizes the surrogate L chain associated with the mu H chain on pre-B-II cells. The other monoclonal antibody, SL156, does not recognize free surrogate L chain or its components, nor its complex with gp130/gp35-65 on pro-B and pre-B-I cells. However, it does bind to a conformational epitope on the surrogate light chain/mu H chain complex on a subpopulation of pre-B-II cells and on mu H chain-positive pre-B cell lines. On mouse precursor B cells prepared ex vivo on ice, expression of the surrogate L chain is very low and almost undetectable. Incubation of the precursor cells for 1 h at 37 degrees C up-regulates the surface expression of surrogate L chain associated with gp130/gp35-65 (early complex) as well as the mu H chain/surrogate L chain complex. These results reconcile some of the apparently discrepant results on surface expression of the surrogate L chain obtained with human and mouse bone marrow pre-B cells, and show that a surrogate L chain/mu H chain-containing pre-B cell receptor can be expressed also on the surface of mouse pre-B-II cells.

We have found that an estrogen deficiency causes a marked increase in bone marrow cells. To examine the effect of estrogen on hemopoiesis, we characterized the increased population of bone marrow cells after ovariectomy (OVX). In OVX mice, the percentage of myeloid cells and granulocytes was decreased, whereas that of B220-positive B lymphocytes was selectively increased 2-4 wk after surgery. The total number of myeloid cells and granulocytes did not change appreciably, but that of B220-positive cells was greatly increased by OVX. When OVX mice were treated with estrogen, the increased B lymphopoiesis returned to normal. B220-positive cells were classified into two subpopulations, B220low and B220high. The majority of the B220low cells were negative for the IgM mu chain, whereas most of the B220high cells were mu-positive. OVX selectively increased the precursors of B lymphocytes identified by B220low. mu-negative phenotype, suggesting that an estrogen deficiency stimulates accumulation of B lymphocyte precursors. When bone marrow-derived stromal cells (ST2) were pretreated with estrogen then co-cultured with bone marrow cells in the presence of estrogen, the stromal cell-dependent B lymphopoiesis was greatly inhibited. The present study suggests that estrogen plays an important role in the regulation of B lymphocyte development in mouse bone marrow.

Vpre-B and lambda-like genes are selectively expressed in B cell precursors and encode polypeptide chains associated in a mu-pseudo light chain (mu-psi L) complex which is thought to regulate some early steps of B cell differentiation. We have generated anti-Vpre-B monoclonal antibodies which allowed us to identify different steps of differentiation from the pro-B to the immature B cells by following surface expression of Vpre-B, mu and light chains in normal adult human bone marrow. Already present at the surface of a small fraction of B cell progenitors (CD34+/CD19+) the Vpre-B molecule was consistently found coexpressed with CD19 and was also found with the sequentially occurring CD10, CD20, CD21, CD22 and CD5 markers. Three discrete cell types were identified: (i) a subpopulation expressing Vpre-B without mu and which represented an early stage of differentiation, (ii) a minor subpopulation co-expressing Vpre-B and mu without the conventional light chains and (iii) a major subpopulation co-expressing Vpre-B, mu and kappa or lambda chains, considered an intermediate pre-B/B stage. The presence of the psi L chain in various cell subpopulations, in possible association with discrete molecules and/or different contexts, suggests its involvement at several steps of early B cell differentiation.

The presenting characteristics and survival of children with the newly recognized transitional cell pre-B immunophenotype of acute lymphoblastic leukemia (ALL) are compared with those of children with pre-B ALL to determine the clinical significance of the new phenotype. Patients with transitional pre-B ALL (n = 17), defined by lymphoblasts expressing cytoplasmic and surface mu heavy chains without kappa or lambda light chains, have lower initial leukocyte counts (p = 0.02) and a higher frequency of DNA indexes > 1.16 (p < 0.001) than patients with pre-B ALL (n = 501), whether or not cases with the unfavorable prognostic (1;19) translocation are included in the analysis. Patients with transitional pre-B ALL lack FAB L3 morphology, bulky extramedullary disease, surface kappa or lambda chains, and the (8;14), (8;22), and (2;8) translocations, features that characterize the syndrome of B-cell ALL. The 4-year relapse-free survival result for children with transitional pre-B ALL appears better than that for children with pre-B ALL (93.3 +/- 17% versus 72.9% +/- 4.6%), but this difference is not statistically significant. We conclude that patients with transitional pre-B ALL have a very favorable prognosis in the context of the therapy used in this study.

The suspension pro granulocyte/macrophage (SPGM1) cell line was established from a transplantable mouse progranulocytic/promacrophage tumor. Surprisingly, SPGM1 cells expressed a typical CD5 pre-B cell phenotype, being positive for Ly-1 (CD5), PB76, B220 (CD45RA), and the pre-B Ig receptor complex (microH chains, lambda 5 and vpre-B surrogate L chains, and the IgM alpha (mb-1) and Ig beta (B29) co-receptor molecules). Southern Blot analysis revealed clonal rearrangement of the microH chain locus and germ-line L chain loci. SPGM1 formed blast cell-, macrophage-, and occasional granulocytic colonies in soft agar in the presence of murine IL-3. IL-3 also induced macrophage differentiation of SPGM1 cells in suspension cultures. The earliest changes were detectable at 24 h by Northern blot analysis. IL-3-treatment increased Mac1 mRNA, induced c-fms mRNA, and down-regulated mRNA for mu, lambda 5, vpre-B and mb-1. After 2 to 4 days the cells were larger, strongly adherent, expressed the macrophage markers Mac1 and F4/80, had lost microH chain and PB76 surface expression, and readily phagocytosed latex beads. Thus SPGM1 has all the characteristic features of a CD5+ pre-B cell line. However, IL-3 predominantly induced SPGM1 to switch its differentiation program from a pre-B cell to a macrophage. This inducible, rapid switch of virtually the entire population provides a unique model for the molecular analysis of mechanisms involved in cell-fate determination.

In M54 mice transgenic for a completely rearranged mu(a) heavy chain there is a decrease in total B cells and the rearrangement of endogenous immunoglobulin genes is partially inhibited. Surprisingly, however, endogenous immunoglobulin gene rearrangement and significant heavy chain polypeptide production does occur. We tested the hypothesis that only CD5+ B cells produce endogenous immunoglobulin by taking advantage of the fact that X-linked immune deficient (xid) mice normally are deficient in CD5+ B cells. We found that the frequency of CD5+ splenic B cells was similar in XxidY transgenic and non-transgenic F1 males, and in XxidX transgenic and non-transgenic F1 females. In both XxidX and XxidY transgenic F1 mice some, but not all, splenic B cells are CD11b+. There was a striking deficit of splenic B cells expressing endogenous immunoglobulin in XxidY transgenic mice, although this was not true for peritoneal cells. Thus, the introduction of the 17.2.25 mu transgene does not prevent the development of CD5- B cells nor does it limit endogenous immunoglobulin gene arrangement and expression solely to CD5+ B cells. However, in mice capable of expressing B cell surface CD5 or CD11 this transgene can lead to expansion of the fraction of B cells positive for these molecules. We conclude that while the introduction of the 17.2.25 mu transgene alters the frequencies of B cell populations, maturation is not limited to one subpopulation.

A 78-year-old woman complaining of a neck mass underwent right hemithyroidectomy. The 7 x 6 cm thyroid tumor consisted predominantly of mildly atypical, epithelial membrane antigen-positive plasma cells and scattered lymphoid follicles. Features of follicular colonization (plasma cell infiltration into germinal centers) were noted. Numerous CD45RO-positive reactive T cells and a smaller number of CD20-positive blast-like B cells were also distributed among the plasma cell infiltrate. IgG, kappa-type monoclonality with J-chain reactivity was identified in the plasma cells, including those in the lymphoid follicles. The association of pre-existing lymphocytic thyroiditis was confirmed histologically in the non-tumorous thyroid tissue. The tumor exhibited deposition of reticulin fiber-rich, amorphous eosinophilic substances, provoking pronounced foreign body reactions. The deposit, polytypically immunoreactive for immunoglobulin gamma-, mu-, kappa- and lambda-chains, beta 2-microglobulin and HLA-DR, was scarcely reactive upon amyloid staining, and consisted ultrastructurally of electron-dense, non-fibrillar material and entrapped collagen fibers. Multiple myeloma was ruled out by laboratory, histologic and clinical examinations. The possible categorization of this extramedullary plasmacytoma of the thyroid within low-grade B cell lymphoma of the mucosa-associated lymphoid tissue (MALT) is discussed.

A highly aggressive human CALLA+C mu+ pre-B acute lymphoblastic leukemia (ALL) cell line (NALM-6-UM1) causes disseminated and invariably fatal leukemia in CB.17 mice with severe combined immunodeficiency (SCID). We used this SCID mouse model of human pre-B ALL to evaluate and compare, in a total of 434 SCID mice, the antileukemic efficacy of B43 (anti-CD19)-pokeweed antiviral protein (PAP) immunotoxin and cyclophosphamide (CPA) as individual reagents and as combined immunochemotherapeutic regimens. B43-PAP plus CPA was superior to either the immunotoxin or drug alone, and combined immunochemotherapy markedly improved the event-free survival (EFS) of SCID mice challenged with NALM-6-UM1 pre-B ALL cells. Notably, 90% to 100% of SCID mice challenged with 1 x 10(6) leukemia cells and then treated with B43-PAP plus CPA combined immunochemotherapy regimens became long-term survivors, a result not achieved with B43-PAP alone or CPA alone. The advantage was particularly evident in mice inoculated with 5 x 10(6) leukemia cells. While neither 15 micrograms B43-PAP (median survival, 58 days) nor 1 mg CPA (median survival, 49 days) resulted in long-term EFS of SCID mice challenged with 5 x 10(6) NALM-6-UM1 pre-B ALL cells, the probability of EFS at 6 months was 50% +/- 16% for SCID mice treated with 15 micrograms B43-PAP plus 1 mg CPA (median survival, greater than 180 days) (P less than .0001). The probability of long-term EFS was only 14% +/- 7% for mice treated with 30 micrograms B43-PAP and 0% +/- 0% for mice treated with 1 mg CPA, but 40% +/- 16% for mice treated with 30 micrograms B43-PAP plus 1 mg CPA (P less than .0001). Similarly, the probability of EFS at 6 months was 40% +/- 16% for mice treated with 2 mg CPA alone, 70% +/- 15% for mice treated with 2 mg CPA plus 15 micrograms B43-PAP, and 70% +/- 15% for mice treated with 2 mg CPA plus 30 micrograms B43-PAP. Ten SCID mice in the B43-PAP plus CPA combined immunochemotherapy arms surviving long term after the inoculation of 5 x 10(6) NALM-6-UM1 pre-B ALL cells were electively killed at 174 to 181 days to assess their leukemia burden. We found no evidence of leukemia in any of the bone marrow specimens by two-color immunofluorescence and multiparameter flow cytometry.(ABSTRACT TRUNCATED AT 400 WORDS)

Among acute lymphoblastic leukemias derived from the B-cell lineage, the subset of cases expressing cytoplasmic mu heavy chain proteins (C mu) in the absence of surface immunoglobulin has been designated pre-B-cell acute lymphoblastic leukemia. This group, traditionally identified using immunologic smear techniques, has been associated with a poor prognosis in some series. In a comparative study, 25 cases of B-lineage acute lymphoblastic leukemia were analyzed for C mu expression using molecular and immunologic techniques. RNA derived from cryopreserved blast cells was hybridized in both Northern and slot-blot analyses using a probe (pBZ311) containing four exons of the human immunoglobulin heavy chain mu constant region gene. Expression of C mu proteins was assessed simultaneously by slide immunofluorescence and flow cytometric techniques in all samples. These studies were correlated with immunoglobulin heavy and light chain gene rearrangements, cell-surface immunophenotype, cytogenetics, and other clinicopathologic features. C mu mRNA transcripts were detected in 14 of 25 cases, whereas C mu proteins were detected in only 9 of these cases using flow cytometric techniques. Only four of these nine cases were positive by slide immunofluorescence techniques. These studies imply that molecular and flow cytometric techniques may be a more sensitive means to assess C mu expression. The identification of five cases that expressed C mu mRNA transcripts in the absence of detectable C mu proteins also suggests that molecular techniques may be valuable in identifying a unique subgroup of pre-B-cell acute lymphoblastic leukemia cases that contain C mu mRNA transcripts, but lack C mu proteins.

We have recently reported that on human B lymphocytes, membrane IgM (mIgM) associates with a heterodimer of 47- and 37-kD polypeptides, the 47-kD subunit being encoded by the mb-1 gene. We show here that expression of mb-1, both at the mRNA and the protein level, is not restricted to IgM+ B cells but can also be found in IgM- pre-B cells and mIgM-IgG+ B cells. Membrane forms of IgD and IgG, isolated from freshly isolated human B cells and B cell lines, are expressed together with heterodimeric protein structures biochemically similar to the mIgM-associated polypeptides, and these were shown to comprise the products of the mb-1 and B29 genes, or homologous genes. Finally, all three classes of antigen receptors are linked to protein kinases, capable of phosphorylating the Ig-associated heterodimers. Our findings provide insight in the structural organization of the different antigen receptors on human B cells and have implications for their function.

Besides the major alteration of T lymphocytes, B-cell anomalies have been reported in HIV infection, related to late stages of B-cell maturation, and considered to result from the dysregulation of T/B interactions. Because T cells are also involved in the control of lymphopoiesis and/or because of specific alterations of the B lineage, anomalies of B-cell maturation could occur in HIV-infected patients. We investigated the presence of immature pre-B lymphocytes, characterized by cytoplasmic mu chains, in 35 peripheral blood samples from healthy controls, 82 from HIV-positive/non-AIDS patients, and 45 from AIDS patients. Significant numbers of such cells were observed in 48% of HIV-seropositive patients and in 40% of the patients with AIDS disease. The presence of pre-B cells correlated with higher numbers of CD8+ and/or CD57+ cells and of peripheral lymphocytes. These data suggest that B-cell dysregulation in HIV infection may lead to the abnormal release of immature B cells in the peripheral blood. This observation may be interpreted as a sign of bone marrow activity.

A case of early gastric plasmacytoma in a 51-year-old woman is reported. The demonstration of monotypic IgM-kappa immunoglobulin in biopsy specimens by an immunoperoxidase technique contributed to the histological diagnosis. The patient had non-ulcerative lesions (of approximately 15 x 16 cm) of a superficially spreading type of gastric plasmacytoma. Extensive histological analysis of the resected stomach showed the IgM-kappa monoclonal proliferation of plasmacytic tumor cells to infiltrate merely into the lamina propria of the gastric mucosa and not to extend deeply into the submucosa, proper muscle and serosa of the stomach.

To analyze in vivo the induction of B cell tolerance against a T cell surface antigen, we generated transgenic mice expressing an anti-CD8.2 mu heavy chain gene. We show that self-specific B cells are efficiently tolerized if they express the membrane-bound form of the transgenic mu chain on their surface but that they can escape tolerization if they express only the secreted form. In the latter, we find an enhanced expression of anti-CD8.2 antibodies after polyclonal B cell activation. As a result, transgenic anti-CD8.2 antibodies bind to the CD8+ T cells but they did not induce their elimination. Furthermore, we observed the preferential expression of a limited subset of endogenous light chains with the transgenic mu chain. This suggests a positive or negative selection for particular heavy and light chain combinations in B lymphocytes.

Wild-type p53 protein is a growth modulator whose inactivation has been found to be a key event in malignant transformation. Reconstitution of wild-type p53 in the p53-nonproducer, Abelson murine leukemia virus-transformed pre-B-cell line L12 gave rise to stably growing clones. Wild-type p53-producer derived cell lines exhibit an altered cell cycle, however. More cells with an extended G0/G1 phase were found than in the p53-nonproducer parental cell line. Furthermore, when injected into syngeneic mice, these cells induced a lower incidence of tumors and these tumors were less aggressive. Analysis of immunoglobulin expression revealed that wild-type p53 induced the expression of cytoplasmic immunoglobulin mu heavy chain. In addition, these derived cells lines exhibited increased levels of a B-cell-specific surface marker, B220. These results suggest that wild-type p53 may function as a cell differentiation factor that can induce development of pre-B cells into a more advanced stage in the pathway of B-cell maturation. In these pre-B cells, wild-type p53 may induce cell differentiation without terminal growth arrest of the cell population.

Precursors of B cells, which constitute a subpopulation of the lymphocytes in bone marrow, can be identified by their surface expression of nonimmunoglobulin markers and the absence of immunoglobulin kappa and lambda light chains. Most pre-B cells synthesize mu heavy chains but, without light-chain partners, these undergo rapid cytoplasmic degradation. In the present study, we demonstrate that late stage pre-B cells, like their neoplastic counterparts, express low levels of a surface receptor composed of mu chains paired with a surrogate light-chain complex formed by Vpre-B and lambda 5-like proteins. The data define a previously suspected but unrecognized stage in normal pre-B-cell differentiation. Expression of a clonally diverse receptor renders this population of immature B-lineage cells potentially vulnerable to clonal selection by antigens and idiotypic interactions.

Pre-B cells carrying DJH rearrangements in an appropriate reading frame produce a short mu protein consisting of a DJH and the C mu & sequence (D mu protein). We analyzed a D mu-producing Abelson-murine leukemia virus-transformed murine pre-B line, 300-19, and demonstrated that D mu proteins are expressed on the cell surface in association with surrogate L chain proteins (lambda 5 and VpreB). However, when we introduced an expression vector coding for the D mu protein into the null pre-B line P17-27, which produce lambda 5 and VpreB but no Ig molecules, most of the cells did not express D mu proteins on the cell surface although D mu proteins were produced intracellularly. On the other hand, P17-27 brings intact mu chains on the cell surface, when a vector coding for the intact mu chain is introduced. Thus, cell surface expression of the D mu protein has different requirements from that of the intact mu chain. A possible role of the VH protein encoded by germ-line VH transcripts is discussed.

A group of 9 patients with pre-B cell acute lymphoblastic leukaemia (pre-B ALL) was identified prospectively within 209 patients with ALL. This variant of leukaemia was defined by the presence of heavy mu-chains in the cytoplasm of the malignant cells, and no surface immunoglobulins. Four patients displayed the CD10 (CALLA) antigen, in addition to cytoplasmic mu chains. A G-0 acute leukaemia (no blast cells in S-phase) was identified in two cases. Response to treatment of the patients was poor: Only four achieved complete remission; median survival was 24 weeks and the 40-week disease free survival was 20%. These figures are significantly worse than those obtained in patients with early-pre-B (CD10+) ALL. We conclude that the prevalence of pre-B ALL is low in our experience (4.6% of all patients with ALL) and that a poor outcome of treatment was related to it.

Localizations of J-chain-positive cells (JPC) were examined in chicken lymphatic tissues before and after hatching. The cells containing J chain were first detected in medullary areas of the bursa of FABRICIUS during the embryonic stage. These positive cells were partly detected in the developing small lymphatic follicles: perhaps on newly differentiating precursor B-cells. In addition to these lymphatic follicles, connective tissue of bursal fold were also detected as J-chain positive. Although similar localizations of JPC were again observed in hatched chickens, positive areas of follicular medulla were strongly stained for fluorescence with corresponding antisera than that of embryonic ones. These data may reflect differences in the physiology of lymphocytes in respect to functional development. JPC localizations were next compared between the B-cell subpopulations, mu-(microPC) and alpha-chain-positive cells (alpha PC). The J-chains detectable in the IgM molecules were also found in follicular medulla. However, these follicles were almost found to be negative for J-chains detectable in the alpha PC before hatching. Any strong stainings for J-chain in the alpha PC were, moreover, not be observed in bursa after hatching. The microPC localizations in hatched chickens were roughly equal with the pattern of JPC localization. These analyses revealed the presence of the cells having the chains of both mu and J. The results together with other recent studies further shown that bursal J-chain can be partly detected in newly differentiated lymphatic follicles lacking IgM-producing and suggest the possible presence of B-cell-differentiation sequence of Ig-J+----IgM+J+----IgA+J+.

A dysregulation of B-cell differentiation and activation has long been evidenced in rheumatoid arthritis (RA). Such analyses have, however, usually focused on the latest stages of B-cell development. Using a classical technique of immunofluorescence labeling on cytospins, we investigated the presence of peripheral pre-B lymphocytes in 92 RA patients and 23 controls. Cells with intracytoplasmic mu chains were evidenced in 58.7% of the RA patients studied, ranging between 0 and 30%, while small numbers of c-mu cells, never exceeding 6% of peripheral blood lymphocytes, were observed in 9 controls. Relationships between this feature and clinical or laboratory data were investigated, showing a negative correlation between the percentage of c-mu + lymphocytes and Ritchie's index (P = 0.05), the number of tender or swollen joints (P = 0.05), erythrocyte sedimentation rate (P = 0.005), seropositivity (P = 0.05), and disease duration (P = 0.01).

Using a polyclonal rabbit antiserum against recombinant mouse lambda 5 protein, we determined that the pre-B cell specific mouse lambda 5 gene encodes a 22-kDa protein. The lambda 5 protein, which is related to conventional Ig lambda L chain proteins forms a complex with Ig mu H chain protein and an as yet unidentified 16-kDa protein (p16) in mu+ pre-B cell lines carrying a functionally rearranged VH-DH-JH allele. In pre-B cell lines which carry DH-JH rearrangements and do not express mu H chain protein, lambda 5 protein is associated with p16. Thus the expression of lambda 5 protein precedes the expression of intact mu H chain protein. This suggests the existence of developmentally regulated protein complexes involving the Ig L chain-like protein lambda 5 and p16 in mu- pre-B cells; lambda 5, p16, and Ig H chain protein in mu+ pre-B cells and Ig H chain and conventional Ig L chain proteins in B cells and plasma cells.

Membrane IgM is expressed on the surface of B lymphocytes. It is not transported to the surface of transfected plasmacytoma or COS cells. Here, we show that mutation of four hydrophilic amino acids in the microm transmembrane is sufficient to overcome the intracellular retention of membrane IgM in non-B cells. This suggests that the B cell-specific IgM-associated proteins that have been postulated to assist the transport of membrane IgM to the cell surface (3) act either by forming a hydrophobic sheath that surrounds the microm transmembrane segment or by displacing an interaction with this segment that would otherwise cause retention. Experiments with a CD8/mu hybrid H chain indicate that the proteins that assist the transport of membrane IgM to the B cell surface at most need the mu CH4 and transmembrane/cytoplasmic portion for interaction.

The population dynamics of granulopoietic cells, B-lineage cells, and T lymphocytes were analyzed by immunofluorescence in mouse hemopoietic tissues as a function of age. Mac-1+ myeloid cells were present on day 11 of gestation in the liver, where they peaked shortly after birth and declined subsequently. Waves of myeloid population growth began in spleen and bone marrow by days 15 and 19, respectively. Mac-1+ cells increased in number to relatively low plateau levels in spleen by the 3rd wk after birth, whereas in the bone marrow higher plateau levels were reached around 3 mo of age. The 14.8 monoclonal antibody was utilized as one marker of B-lineage precursor cells. 14.8+ cells were detected in the liver on day 11 of gestation, reached peak numbers during the first week after birth and decreased thereafter. On day 15 and 19, 14.8+ cells were found in spleen and bone marrow, respectively, and progressively increased in numbers to reach plateau levels in both sites by 3 mo of age. Mu+ pre-B cells appeared in significant numbers in the 13-day fetal liver, reached a peak shortly after birth, and disappeared from the liver by the end of the second postnatal week. Pre-B cells were found in the spleen and bone marrow on days 15 and 19, respectively. In the spleen pre-B cells reached peak values at birth and disappeared 2 wk later. In spite of the sequential appearance of mu+ pre-B cells in fetal liver, spleen, and bone marrow, their sIgM+ B cell progeny appeared in all these hemopoietic tissues on day 17 of gestation. In the liver, sIgM+ B cells reached their peak at birth and declined thereafter. In the spleen and bone marrow, B cells increased to plateau levels between 1 and 4 mo of age. Thy-1.2+ T cells were relatively late acquisitions in all three hemopoietic tissues. Finally, the expression of the 14.8 antigen by mu+ cells was examined as a function of gestational age. While pre-B cells from day-13 fetuses had no detectable 14.8 antigen, the antigen was weakly expressed on the vast majority of the mu+ pre-B cells by day 17 of gestation. Newborn liver cells expressing 14.8 antigen were found to include a small proportion of cells with peroxidase+ granules. Thus, demonstration of rearrangement and expression of immunoglobulin genes may be required for precise identification of cells of B lineage early in ontogeny.

The immunoglobulins produced by the earliest recognizable B cell precursors (pre-B cells) were characterized in the mouse and human. Immunofluorescent analysis revealed no evidence of surface IgM components, and only mu heavy chains could be detected intracytoplasmically in pre-B cells. Surface IgM components could not be isolated from intact fetal liver cells that lacked sIgM+ B lymphocytes but possessed pre-B cells. Pre-B cells were shown to synthesize and secrete mu heavy chains but not light chains by immunochemical analysis. These mu chains constituted less than 0.01% of TCA precipitable protein synthesized and secreted by fetal liver cells during an 8 hr labelling period. Migration of both intracellular and secreted mu chains on SDS-PAGE suggested that they were smaller than mu chains secreted by mouse and human plasmacytomas. These data indicate that mu chain synthesis precedes light chain expression during B cell ontogeny and suggest a new role for pre-B cells in the generation and expression of a diverse immunoglobulin repertoire.

A long-term in vitro culture system derived from murine bone marrow cells can successfully support the growth of B cell precursors, pre-B cells, and IgM-expressing B cells. Intermediates in the B cell developmental pathway are known to have differential sensitivities to the toxic effects of corticosteroids. We demonstrate here that long term B lineage cultures can be established with the corticosteroid-resistant cell population from bone marrow. Kinetics for the establishment and growth of cultures derived from corticosteroid-treated marrow are similar to those observed with control cultures. Cells obtained from both sets of cultures have similar morphologies and ranges of phenotypic markers. These results indicate that the cell responsible for the outgrowth of the long term B lineage cultures is corticosteroid resistant and is likely to be earlier in the B lymphocyte lineage than steroid-sensitive pre-B cells.

The fraction of blood S-phase (S+) lymphocytes from 41 patients with chronic lymphocytic leukemia of B cell type was determined by flow cytometry. The patients were grouped according to the smig isotype pattern of the leukemic cells. Patients with IgM as the predominant smig had higher numbers of S+ lymphocytes than patients with a leukemic clone co-expressing IgM and IgD (p less than 0.001). High relative as well as total numbers of S+ lymphocytes were associated with short therapy-free and overall survival. T cell proliferation was low although significantly higher in active than in indolent disease.

We report on a patient with a bone marrow plasmacell infiltration, since this case displays some outstanding clinical and laboratory features: the presence of massive splenomegaly and the absence of bone lesions; the cytoplasmic heavy chain type mu, without any light chain detectable by immunofluorescence (IF); the non secretory feature of this plasma cell proliferation. The position of such an entity within the spectrum of B-lymphoproliferative disorders, from mu heavy chain disease to non secretory IgM myeloma, is discussed.

The detection of Igmu chain proteins in the serum of a patient with HCD by a combining of starch gel electrophoresis and immunodiffusion is described. Partial isolation of mu chain protein by immunoadsorbtion on polyacrylamide gel has been acchieved. As no contaminations of mu chain protein with IgM protein could be detected a separate measurement of these proteins using immunoadsorbants of suitable exclusion limits seems to be possible.

A 55-year-old female developed a rapidly fatal, infiltrative, bilateral pulmonary disease. Open lung biopsy and subsequent autopsy revealed diffuse involvement by a malignant lymphoproliferative condition showing a striking angiocentric and angioinvasive pattern. This feature, together with microscopic involvement of hilar lymph nodes, bone marrow, spleen, and other viscera suggested lymphomatous transformation of lymphomatoid granulomatosis (LYG). The paucity of necrosis and of the typical polymorphic infiltrate was at variance with the classical description of that condition; however, the bilaterality of the process and the distinctive angioinvasive growth pattern were unlike the typical primary pulmonary lymphoma. Plasmacytoid cells were observed both by light and electron microscopy. Immunohistochemicl evaluation characterized this disease as a monoclonal lymphoproliferative malignancy.

Snyder-Theilen feline sarcoma virus (ST-FeSV) codes for a protein kinase with specificity for tyrosine residues (Barbacid et al., Proc. Natl. Acad. Sci. U.S.A. 77:5158-5163, 1980), properties analogous to those of the transforming gene product of Abelson murine leukemia virus (Witte et al., Nature (London) 283:826-831, 1980). In the present report, ST-FeSV was demonstrated to transform murine hematopoietic cells under in vitro assay conditions which detect lymphoid cell transformation by Abelson murine leukemia virus. Bone marrow colony formation was shown to require ST-FeSV, follow single-hit kinetics, and require the presence of mercaptoethanol in the agar medium. ST-FeSV-induced colonies could be established in culture as continuous cell lines that demonstrated unrestricted self-renewal capacity and leukemogenicity in vivo. The hematopoietic blast cells transformed by ST-FeSV in culture appeared to be at an early stage of B cell differentiation. They possessed Lyb 2 surface antigens, were dependent on mercaptoethanol for growth, and contained only low levels of terminal deoxynucleotidyl transferase. Moreover, a large fraction of the lines synthesized immunoglobulin mu chain in the absence of light chains. Thus, the phenotype of ST-FeSV hematopoietic transformants was indistinguishable from that of the pre-B lymphoblast transformants induced by Abelson murine leukemia virus. These findings indicate that the in vitro functional similarities in the onc gene products of ST-FeSV and Abelson murine leukemia virus may reflect a common pathway by which they exert their oncogenic potential.

An unusual case of 'non-excretory' myeloma is described in which, using immunofluorescence, only 'mu' heavy chains were detected in almost all bone marrow plasma-cell cytoplasm. Cytoplasmic light chains were completely lacking, and neither monoclonal whole immunoglobulin (Ig) nor free heavy nor light chains were detected in serum and urine, although the clinical and morphological features showed the classical pattern of myeloma. The possible mechanism which could play a role in the disturbance of the Ig-chain secretion observed in this case is discussed.