We have identified a population of newly formed bone marrow (BM) B cells that shares multiple characteristics with late transitional B cells in the spleen. Both late splenic transitional B cells and ...cells within this uncharacterized BM population expressed the cell-surface phenotype AA4+ CD23+, yet the developmental kinetics and the renewal rate of AA4+ CD23+ BM B cells mirrored recently formed BM B cells. Further, unlike the least mature B cells in the BM and spleen, AA4+ CD23+ BM B cells expressed the homing receptor CD62L, were dependent on the antiapoptotic cytokine receptor BR3 and the tec family kinase Btk, and proliferated in response to IL-4 plus CD40 stimulation. Finally, frequencies of λ light chain-positive B cells declined among AA4+ CD23+ B cells in both the BM and spleen, suggesting that V-gene selection events correlate with CD23 expression in both compartments. These observations indicate that the first step in B-cell maturation occurs in both the BM and the periphery and suggest that recently formed B cells exit the BM as a heterogeneous pool of immature and semimature B cells.
Selection of recently formed B cells into the follicular or marginal zone (MZ) compartments is proposed to occur by way of proliferative intermediates expressing high levels of CD21/35 and CD23. ...However, we show that CD21/35(high) CD23(+) splenocytes are not enriched for proliferative cells, and do not contribute substantially to the generation of follicular B cells. Instead, ontogenic relationships, steady-state labeling kinetics, and adoptive transfer experiments suggest that CD21/35(high) CD23(+) splenocytes serve primarily as precursors for MZ B cells, although their developmental potential seems to be broader and is influenced by environmental cues that are associated with lymphopenia. Furthermore, CD21/35(high) CD23(+) splenocytes share several key functional characteristics with MZ B cells, including their capacity to trap T-independent antigen and a heightened proliferative response to LPS. These observations challenge previous models of peripheral B cell maturation, and suggest that MZ B cells develop by way of CD21/35(high) CD23(+) intermediates.
During lymphoid development, Notch1 plays a critical role in the T-cell/B-cell lineage decision, while Notch2 is essential for marginal zone B-cell (MZB) development. Notch pathway activation induces ...translocation of intracellular Notch (ICN) to the nucleus, where it interacts with the transcription factor CSL (CBF1/RBP-Jk, Suppressor of Hairless, Lag-1). In vitro, ICN binds Mastermind-like proteins, which act as potent Notch coactivators. Three MAML family members (MAML1-3) have been identified in mammals, but their importance in vivo is unknown. To investigate the function of MAMLs in hematopoietic development, we introduced a dominant negative (DN) mutant of MAML1, capable of inhibiting Notch1-4, in murine hematopoietic stem cells. DNMAML1 resulted in early inhibition of T-cell development and the appearance of intrathymic B cells, phenotypes consistent with Notch1 inhibition. The T-cell differentiation block was as profound as that produced by enforced expression of the Notch modulator Deltex1. In DNMAML1-transduced spleen cells, a dramatic decrease in MZB cells was present, consistent with Notch2 inhibition. In contrast, Deltex1 did not decrease MZB cell numbers. These results suggest a critical role for MAMLs during Notch-mediated cell fate decisions in vivo and indicate that DNMAML1, but not Deltex1, can be used to interfere with the function of multiple Notch family members. (Blood. 2004;104:1696-1702)
The gene encoding the BCL-6 transcriptional repressor is frequently translocated and mutated in diffuse large cell lymphoma. Mice with a disrupted BCL-6 gene developed myocarditis and pulmonary ...vasculitis, had no germinal centers, and had increased expression of T helper cell type 2 cytokines. The BCL-6 DNA recognition motif resembled sites bound by the STAT (signal transducers and activators of transcription) transcription factors, which mediate cytokine signaling. BCL-6 could repress interleukin-4 (IL-4)-induced transcription when bound to a site recognized by the IL-4-responsive transcription factor Stat6. Thus, dysregulation of STAT-responsive genes may underlie the inflammatory disease in BCL-6-deficient mice and participate in lymphoid malignancies.
Although Notch receptor expression on malignant B cells is widespread, the effect of Notch signaling in these cells is poorly understood. To investigate Notch signaling in B-cell malignancy, we ...assayed the effect of Notch activation in multiple murine and human B-cell tumors, representing both immature and mature subtypes. Expression of constitutively active, truncated forms of the 4 mammalian Notch receptors (ICN1-4) inhibited growth and induced apoptosis in both murine and human B-cell lines but not T-cell lines. Similar results were obtained in human precursor B-cell acute lymphoblastic leukemia lines when Notch activation was achieved by coculture with fibroblasts expressing the Notch ligands Jagged1 or Jagged2. All 4 truncated Notch receptors, as well as the Jagged ligands, induced Hes1 transcription. Retroviral expression of Hairy/Enhancer of Split-1 (Hes1) recapitulated the Notch effects, suggesting that Hes1 is an important mediator of Notch-induced growth arrest and apoptosis in B cells. Among the B-cell malignancies that were susceptible to Notch-mediated growth inhibition/apoptosis were mature B-cell and therapy-resistant B-cell malignancies, including Hodgkin, myeloma, and mixed-lineage leukemia (MLL)–translocated cell lines. These results suggest that therapies capable of activating Notch/Hes1 signaling may have therapeutic potential in a wide range of human B-cell malignancies.
To compare the efficacy and safety of orally administered capecitabine (Xeloda; Roche Laboratories, Inc, Nutley, NJ), a novel fluoropyrimidine carbamate designed to mimic continuous fluorouracil ...(5-FU) infusion but with preferential activation at the tumor site, with that of intravenous (IV) 5-FU plus leucovorin (5-FU/LV) as first-line treatment for metastatic colorectal cancer.
We prospectively randomized 602 patients to treatment with capecitabine 1,250 mg/m(2) administered twice daily days 1 to 14 every 3 weeks, or to the 4-weekly Mayo Clinic regimen (5-FU/LV) until disease progression or unacceptable toxicity.
The primary objective, to demonstrate at least equivalent response rates in the two treatment groups, was met. The overall response rate was 18.9% for capecitabine and 15.0% for 5-FU/LV. In the capecitabine and 5-FU/LV groups, respectively, median time to disease progression was 5.2 and 4.7 months (log-rank P =.65); median time to treatment failure was 4.2 and 4.0 months (log-rank P =.89); and median overall survival was 13.2 and 12.1 months (log-rank P =.33). The toxicity profiles of both treatments were typical of fluoropyrimidines. However, capecitabine led to significantly lower incidences (P <.00001) of stomatitis and alopecia, but a higher incidence of cutaneous hand-foot syndrome (P <.00001). Capecitabine also resulted in lower incidences (P <.00001) of grade 3/4 stomatitis and neutropenia, leading to a lower incidence of grade 3/4 neutropenic fever and sepsis. Only grade 3 hand-foot syndrome (P <.00001) and uncomplicated grade 3/4 hyperbilirubinemia (P <.0001) were reported more frequently with capecitabine.
Oral capecitabine achieved an at least equivalent efficacy compared with IV 5-FU/LV. Capecitabine demonstrated clinically meaningful safety advantages and the convenience of an oral agent.
It is well accepted that Ag-induced B cell differentiation often results in the generation of exceptionally long-lived plasma cells. Much of the work supporting this viewpoint stems from studies ...focused on germinal center-derived plasma cells secreting high-affinity isotype-switched Abs in mice immunized with T cell-dependent Ags. In contrast, less attention has been devoted to understanding Ab responses to T cell-independent Ags and pathogens. In this study, we review recent work showing that T cell-independent Ags consisting of either polysaccharides or LPSs also induce the formation of long-lived plasma cells, despite their general inability to sustain germinal center responses. This new information provides a framework for more fully understanding the forces underlying immunity to pathogens that resist T cell recognition and the extracellular cues governing plasma cell longevity.
During aging, adaptive immunity is severely compromised, due in part to decreased production of B lymphocytes and loss of immunoglobulin (Ig) diversity. However, the molecular mechanisms that ...underlie age-associated diminished B cell production remain unclear. Using in vivo labeling, we find that this reduction in marrow pre-B cells reflects increased attrition during passage from the pro-B to pre-B cell pool. Analyses of reciprocal bone marrow chimeras reveal that the magnitude and production rates of pre-B cells are controlled primarily by microenvironmental factors, rather than intrinsic events. To understand changes in pro-B cells that could diminish production of pre-B cells, we evaluated rag2 expression and V(D)J recombinase activity in pro-B cells at the single cell level. The percentage of pro-B cells that express rag2 is reduced in aged mice and is correlated with both a loss of V(D)J recombinase activity in pro-B cells and reduced numbers of pre-B cells. Reciprocal bone marrow chimeras revealed that the aged microenvironment also determines rag2 expression and recombinase activity in pro-B cells. Together, these observations suggest that extrinsic factors in the bone marrow that decline with age are largely responsible for less efficient V(D)J recombination in pro-B cells and diminished progression to the pre-B cell stage.
To report the use of thoracoscopic thoracic duct ligation (TDL) and pericardectomy for treatment of chylothorax. Case series. Dogs with chylothorax (n=12). Dogs with secondary or idiopathic ...chylothorax had thoracoscopy performed in sternal recumbency through 3 portals in the caudal right hemithorax for TDL and were then repositioned in dorsal recumbency for pericardectomy. Portals were placed in the 5th and 7th intercostal spaces of the right hemithorax with 1 transdiaphragmatic portal in the right paraxiphoid position. Follow-up was performed by recheck examination or telephone interview to determine outcome. Seven dogs (58%) had idiopathic chylothorax; 6 dogs (85.7%) had complete resolution of their effusion, whereas only 2 of the 5 nonidiopathic dogs (40%) had complete resolution. Thoracoscopy is minimally invasive, provides excellent observation, and allows for ligation of the thoracic duct in the caudal thorax. Patients with idiopathic chylothorax may have a better prognosis after TDL and pericardectomy than dogs with nonidiopathic chylothorax. Thoracoscopy for ligation of the thoracic duct and pericardectomy is an acceptable surgical technique for treatment of chylothorax.
Abstract
Plasma cell (PC) differentiation is a highly regulated process that radically changes the morphology and function of a B lymphocyte to that of a professional protein secretor. It is becoming ...clear that this process precedes increase in immunoglobulin synthesis rather than reacting to it. We have shown previously that large segments of the PC transcriptome are initiated in an mTORC1-dependent manner prior to both Blimp-1 and Xbp1s expression. Still, the kinetics of induction of the PC proteome remain largely unknown. We performed a whole proteome kinetic analysis of activated Blimp-1-reporter follicular B cells in PC-inductive conditions every 12 hours for three days and observed distinct patterns in induction of the PC proteome. To elucidate transcriptional networks, we performed single cell RNA-seq with the same conditions and correlated transcription factor expression with genes coding for these proteome modules within single cells. We found distinct subgroups within proteome modules corresponding to transcriptional patterns each with a highly correlated transcription factor set. These included robust, transient, Myc-associated factors required for translation initiation; rapid, sustained expression of protein synthesis machinery; and gradual, sustained increases in proteins associated with ER function and secretion. Each of these preceded the induction of Blimp-1 and increase in immunoglobulin protein. Finally, using inducible deletion of Blimp-1 in conjunction with the reporter allele we show that these unique transcriptional and proteomic programs are initiated independently of Blimp-1 expression. Thus, activated B cells prepare for protein secretion by initiating the PC proteome prior to antibody secretion.
Supported by grants from the NIH: R01 AI113545, AI139123, AI154932, R21 AI133998 (PI:Allman) and BTG was supported by training grant T32 CA009140 (PI:Pear)
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