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Bioinspired synthesis of hierarchically structured calcium phosphate (CaP) material is a highly promising strategy for developing improved bone substitute materials. However, synthesis of CaP materials with outstanding mechanical properties still remains an ongoing challenge. Inspired by the formation of lamellar structure in nacre, we designed an organic matrix composed of chitosan and cis-butenediolic acid (maleic acid, MAc) that could assemble into a layered complex and further guide the mineralization of monetite crystals, resulting in the formation of organized and parallel arrays of monetite platelets with a brick-and-mortar structure. Using the layered monetite-chitosan composite as a precursor, we were able to synthesize hydroxyapatite (HAp) with multiscale hierarchically ordered structure via a topotactic phase transformation process. On the nanoscale, needlelike HAp crystallites assembled into organized bundles that aligned to form highly oriented plates on the microscale. On the large-scale level, these plates with different crystal orientations were stacked together to form a layered structure. The organized structures and composite feature yielded CaP materials with improved mechanical properties close to those of bone. Our study introduces a biomimetic approach that may be practical for the design of advanced, mechanically robust materials for biomedical applications., Competing Interests: Notes The authors declare no competing financial interest.

Purpose: With cancer survivors now numbering over 13 million in the United States, and expected to continue to increase, it is important to consider the needs of this growing population. In the literature, one of the most common complaints by cancer survivors is perceived cognitive dysfunction. Since the preponderance of the research has focused on breast cancer survivors, the purpose of the present study was to explore the prevalence and correlates of perceived cognitive dysfunction in a large sample of cancer survivors with representation across a wide range of different types of cancer., Methods: A sample of 3108 post-treatment cancer survivors completed the 2010 LIVESTRONG survey as part of a larger study of cancer survivorship. Respondents completed standardized questions regarding current and past perceived cognitive dysfunction, as well as depressive symptoms, and demographic and medical variables., Results: Current perceived cognitive dysfunction was reported by nearly half of respondents (45.7%), across a wide range of cancer types, with the highest prevalence among survivors of central nervous system cancers. Receiving chemotherapy and current report of depressive symptoms were both strongly associated with current perceived cognitive dysfunction., Conclusion: These findings contribute to a growing appreciation of the high prevalence of perceived cognitive dysfunction in survivors of a wide range of cancer types and the potential interactive effect of concurrent symptoms of depression. These findings highlight a need to develop more effective means of preventing or reducing cognitive dysfunction in cancer survivors., Implications for Cancer Survivors: Perceived cognitive dysfunction was reported in a wide range of cancer survivors. The potential interactive effect of symptoms of depression suggests the need to develop interventions targeting both cognitive dysfunction and depression to achieve improvements in cognitive functioning.

Purpose: Benefit finding has been shown to be beneficial for people with cancer and may be an indication that one is coping adequately with the stress of cancer. This study evaluated the psychometric properties of a four-item benefit finding measure from the cancer survivorship supplement of the Medical Expenditure Panel Survey (MEPS)., Methods: Long-term survivors (5-10 years post-diagnosis) of breast, prostate, colorectal or lung cancer or melanoma (n = 594) completed the MEPS cancer supplement survey in 2013. Four items asked about benefit finding after the cancer: stronger person, coping better, positive changes and having healthier habits. Information on sociodemographics, disease and activity limitations after the cancer was also collected. We examined factor structure, reliability (Kuder-Richardson 20) and validity., Results: The four benefit finding items did not appear to measure one factor. Three of the benefit finding items (stronger person, coping better, positive changes) were related to gender, receipt of chemotherapy and activity limitations but not cancer stage, time since diagnosis or income. Having healthier habits was unrelated to any sociodemographic or disease variable., Conclusions: Three of the items (stronger person, coping better, positive changes) appeared to have validity as they were related to variables that literature has shown are related to benefit finding. However, having healthier habits is likely measuring a separate but related construct. This short instrument may be used in future studies assessing benefit finding post cancer; however, the four items should be analyzed separately., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The rising medical costs associated with cancer have led to considerable financial hardship for patients and their families in the United States. Using data from the LIVESTRONG 2012 survey of 4,719 cancer survivors ages 18-64, we examined the proportions of survivors who reported going into debt or filing for bankruptcy as a result of cancer, as well as the amount of debt incurred. Approximately one-third of the survivors had gone into debt, and 3 percent had filed for bankruptcy. Of those who had gone into debt, 55 percent incurred obligations of $10,000 or more. Cancer survivors who were younger, had lower incomes, and had public health insurance were more likely to go into debt or file for bankruptcy, compared to those who were older, had higher incomes, and had private insurance, respectively. Future longitudinal population-based studies are needed to improve understanding of financial hardship among US working-age cancer survivors throughout the cancer care trajectory and, ultimately, to help stakeholders develop evidence-based interventions and policies to reduce the financial hardship of cancer., (Project HOPE—The People-to-People Health Foundation, Inc.)

Background: Some cancer diagnoses and treatments can place patients at risk for infertility. The American Society of Clinical Oncology recommends that health care providers address the possibility of infertility with cancer patients who are treated during their reproductive years; however, research suggests that many providers do not disclose the risk of infertility to their patients. This study examines adolescent/young adult (AYA) cancer survivors' use of and costs for fertility preservation (FP) over time., Methods: The study included 550 AYA cancer survivors diagnosed at the ages of 15 and 39 years between 2006 and 2012. Logistic regression analyses and chi-squared tests were conducted to identify factors associated with FP use, barriers, and expenses., Results: One hundred eighty two (33%) of the AYA survivors took steps to preserve their fertility. Men, survivors who did not have children, those who received chemotherapy, and those who lived in the Northeast (vs. the South) were more likely to have FP. The majority of men using FP used sperm banking (99%), whereas women used egg preservation (40%), embryo preservation (40%), and other methods (37%). On average, women paid more for FP than men (p < 0.001); however, costs for women significantly declined over time (p = 0.021)., Conclusions: The study points to other areas for research in women's health, including the development of educational interventions with patients and providers to reduce gender disparities in FP and ensure timely patient-provider discussions related to fertility issues.

We recently reported an amelogenin-chitosan (CS-AMEL) hydrogel as a promising biomimetic material for future in situ human enamel regrowth. To further optimize the necessary conditions for clinical applicability of CS-AMEL hydrogel, herein we studied the effects of viscosity and supersaturation degree on the size and orientation of synthetic crystals by means of scanning electron microscopy (SEM) and X-ray diffraction (XRD). Raising the hydrogel viscosity by increasing chitosan concentration from 1% to 2% (w/v) improved the orientation of the crystals, while a higher supersaturation (σ(HAp) >10.06, [Ca(2+)] >5 mM) resulted in the formation of random crystals with larger sizes and irregular structures. We conclude that optimal conditions to produce organized enamel-like crystals in a CS-AMEL hydrogel are: 2% (w/v) chitosan, 2.5 mM calcium, and 1.5 mM phosphate (degree of supersaturation = 8.23) and 200 µg/ml of amelogenin.

Adolescent and young adult cancer survivors-those who were ages 15-39 at their first cancer diagnosis-have important health limitations. These survivors are at risk for higher health care expenditures and lost productivity, compared to adults without a history of cancer. Using Medical Expenditure Panel Survey data, we present nationally representative estimates of the economic burden among people who were diagnosed with cancer in adolescence or young adulthood. Our findings demonstrate that surviving cancer at this age is associated with a substantial economic burden. Compared to adults without a history of cancer, adolescent and young adult cancer survivors had excess annual medical expenditures of $3,170 per person and excess annual productivity losses of $2,250 per person. Multifaceted prevention strategies, including education and sustained intervention programs to ensure access to lifelong risk-based follow-up care, may be effective ways to improve the economic outcomes associated with cancer survivorship in this population., (Project HOPE—The People-to-People Health Foundation, Inc.)

Purpose: Treatment summaries (TSs), a critical component of survivorship care plans, have been identified as a tool to improve outcomes for the 14 million cancer survivors in the United States., Methods: In 2010, the LIVESTRONG Foundation fielded the LIVESTRONG Survey for People Affected by Cancer. The survey was designed to assess the physical, emotional, and practical concerns after cancer as well as receipt of treatment summaries. Participants were recruited online and through national partners., Results: Over a 9-month period, > 12,000 people completed the survey, including 3,682 post-treatment cancer survivors (PTCSs). PTCSs who received a TS reported that they were closer to time since diagnosis or end of treatment (P < .01), more likely to have received chemotherapy (P < .01), more likely to have received the majority of their health care from a medical oncologist (P < .05), experiencing significantly fewer post-treatment emotional concerns (P < .05), and significantly less likely to say that they had learned to live with their concerns (P < .05). PTCSs who received a TS more often reported that their needs had been met, including receiving information about possible late effects, care they received during treatment, and care they received after treatment., Conclusions: Receipt of a TS was associated with a variety of positive outcomes; however, only approximately one third of PTCSs received one. Future studies focused on patient perspectives on care planning tools can help to improve optimal survivorship care delivery. Possible solutions for improving access to a TS are included.

Biomimetic reconstruction of tooth enamel is a significant topic of study in materials science and dentistry as a novel approach to the prevention, restoration, and treatment of defective enamel. We have developed a new amelogenin-containing chitosan hydrogel for enamel reconstruction that works through amelogenin supramolecular assembly, stabilizing Ca-P clusters and guiding their arrangement into linear chains. These amelogenin Ca-P composite chains further fuse with enamel crystals and eventually evolve into enamel-like co-aligned crystals, anchored to the natural enamel substrate through a cluster growth process. A dense interface between the newly grown layer and natural enamel was formed and the enamel-like layer improved the hardness and elastic modulus compared with etched enamel. We anticipate that this chitosan hydrogel will provide effective protection against secondary caries because of its pH-responsive and antimicrobial properties. Our studies introduce an amelogenin-containing chitosan hydrogel as a promising biomaterial for enamel repair and demonstrate the potential of applying protein-directed assembly to biomimetic reconstruction of complex biomaterials., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

By taking advantage of design and construction of strong graphene-matrix interfaces, we have prepared modified graphene/polyimide (MG/PI) nanocomposites via a two-stage process consisting of (a) surface modification of graphene and (b) in situ polymerization. The 2 wt % MG/PI nanocomposites exhibited a 20-fold increase in wear resistance and a 12% reduction in friction coefficient, constituting a potential breakthrough for future tribological application. Simultaneously, MG also enhanced thermal stability, electrical conductivity, and mechanical properties, including tensile strength, Young's modulus, storage modulus, and microhardness. Excellent thermal stability and compatibility of interface, strong covalent adhesion interaction and mechanical interlocking at the interface, as well as homogeneous and oriented dispersion of MG were achieved here, contributing to the enhanced properties observed here. The superior wear resistance is ascribed to (a) tribological effect of MG, including suppression effect of MG in the generation of wear debris and protective effect of MG against the friction force, and (b) the increase in mechanical properties. In light of the relatively low cost and the unique properties of graphene, the results of this study highlight a pathway to expand the engineering applications of graphene and solve wear-related mechanical failures of polymer parts.

In the USA, cancer is a leading cause of morbidity and premature death among the Hispanic/Latino population. It is estimated that one in two Hispanic men and one in three Hispanic women will be diagnosed with cancer during their lifetime (American Cancer Society 2010). Despite this significant cancer burden, few innovative strategies for communication and outreach to this population currently exist. In 2009, LIVESTRONG launched a national outreach campaign, which utilized social marketing, specifically targeting Hispanics with the goal of increasing awareness and usage of LIVESTRONG's Spanish-language cancer navigation resources. This campaign, one of the first undertaken by a national cancer-related organization, led to increased awareness and utilization of resources, including a 238% increase in traffic over traditional marketing campaigns which focused on radio alone. The success of this campaign highlights the use of social media as a cost-effective method to raise awareness of cancer resources among Hispanics.

Purpose: The Health Information Technology for Economic and Clinical Health (HITECH) Act has placed an emphasis on electronic health information exchange (EHIE). Research on needs of patient, especially those touched by cancer, has been sparse. Here, we present data on preferences for EHIE among those touched by cancer compared with a nationally representative sample of American adults., Methods: TWO SURVEYS WERE USED: an online survey designed by LIVESTRONG (the Lance Armstrong Foundation) and a dual-frame, nationally representative sample of adults collected through the National Cancer Institute's Health Information National Trends Survey (HINTS)., Results: The LIVESTRONG EHIE survey yielded a sample of 8,411 respondents, including 433 currently receiving cancer treatment, 298 living with cancer as a chronic disease, 2,343 post-treatment survivors, and 5,337 with no history of cancer. The HINTS sample consisted of 7,674 respondents representative of the general adult population. Comparisons revealed a strong positive view of the value of EHIE within the cancer-relevant groups, especially among those living with cancer as a chronic disease. Only about half of the general population showed a similar degree of enthusiasm for EHIE. When asked about specific functions for EHRs, respondents valued privacy and security above all, followed by improving care coordination and data sharing between providers., Conclusion: These data suggest that the EHIE needs among those touched by cancer may be greater than in the general population. This is particularly important because people affected by cancer are among those who access our health care system most frequently and who have the most at stake.

Genetically primed adult C57BL mice were deleted of exon 5 of the gene encoding the transcription factor PU.1 by IFN activation of Cre recombinase. After a 13-week delay, conditionally deleted (PU.1(-/-)) mice began dying of myeloid leukemia, and 95% of the mice surviving from early postinduction death developed transplantable myeloid leukemia whose cells were deleted of PU.1 and uniformly Gr-1 positive. The leukemic cells formed autonomous colonies in semisolid culture with varying clonal efficiency, but colony formation was enhanced by IL-3 and sometimes by granulocyte-macrophage colony-stimulating factor. Nine of 13 tumors analyzed had developed a capacity for autocrine IL-3 or granulocyte-macrophage colony-stimulating factor production, and there was evidence of rearrangement of the IL-3 gene. Acquisition of autocrine growth-factor production and autonomous growth appeared to be major events in the transformation of conditionally deleted PU.1(-/-) cells to fully developed myeloid leukemic populations.

Morpholino (MO) based inhibition of translational initiation represents an attractive methodology to eliminate gene function during Xenopus development (Heasman et al., 2000). However, the degree to which a given target protein can be eliminated and the longevity of this effect during embryogenesis has not been documented. To examine the efficacy of MOs, we have used transgenic Xenopus lines that harbour known numbers of integrations of a GFP reporter under the control of the ubiquitous and highly expressed CMV promoter (Fig. 1a). In addition we have investigated the longevity of the inhibitory effect by using transgenic lines expressing GFP specifically in the lens of tadpoles. These transgenic lines represent the ideal control for the technique as the promoters are highly expressed and GFP can be easily detected by fluorescence and immunoblotting. Moreover, as GFP has no function in development, the levels of inhibition can be tested in an otherwise normal individual. Here we report that MOs are able to efficiently and specifically inhibit the translation of GFP in transgenic lines from Xenopus laevis and Xenopus tropicalis and the inhibitory effect is long-lived, lasting into the tadpole stages. genesis 30:110--113, 2001., (Copyright 2001 Wiley-Liss, Inc.)

Despite being one of the most intensively studied cell types, the molecular basis of B cell specification is largely unknown. The Pax5 gene encoding the transcription factor BSAP is required for progression of B-lymphopoiesis beyond the pro-B cell stage. Pax5-deficient pro-B cells are, however, not yet committed to the B-lymphoid lineage, but instead have a broad lymphomyeloid developmental potential. Pax5 appears to mediate B-lineage commitment by repressing the transcription of non-B-lymphoid genes and by simultaneously activating the expression of B-lineage-specific genes. Pax5 thus functions both as a transcriptional repressor and activator, depending on its interactions with corepressors of the Groucho protein family or with positive regulators such as the TATA-binding protein. Once committed to the B-lineage, B cells require Pax5 function to maintain their B-lymphoid identity throughout B cell development.

During B-lymphocyte development in mouse fetal liver and bone marrow, a pre-B I cell stage is reached in which the cells express B-lineage-specific genes, such as CD19, Ig alpha and Igbeta and VpreB and lambda5, which encode the surrogate light (SL) chain. In these pre-B I cells both alleles of the immunoglobulin heavy (IgH) chain locus are D(H)J(H) rearranged. Transplantation of pre-B I cells from wild-type (e.g. C57Bl/6) mice in histocompatible RAG-deficient hosts leads to long-term reconstitution of some of the mature B-cell compartments and to the establishment of normal IgM levels, a third of the normal serum IgA levels, and IgG levels below the detection limit. Neither T-lineage nor myeloid cells of donor origin can be detected in the transplanted hosts, indicating that the pre-B I cells are committed to B-lineage differentiation. Consequently, the B-cell-reconstituted hosts respond to T-cell-independent antigens but not to T-cell-dependent antigens. Responses to T-cell-dependent antigens can be restored in the pre-B I-cell-transplanted, RAG-deficient hosts by the concomitant transplantation of mature CD4+ T cells. The transplanted wild-type pre-B I cells do not home back to the bone marrow and become undetectable shortly after transplantation. B-lymphocyte development in Pax-5-deficient mice becomes arrested at the transition of pre-B I to pre-B II cells i.e. at the stage when V(H) to D(H)J(H) rearrangements occur and when the pre-B-cell receptor, complete with muH chains and SL chains, is normally formed. T-lineage and myeloid cell development in these mice is normal. Pre-B I cells of Pax-5-deficient mice have a wild-type pre-B I-cell-like phenotype: while they do not express Pax-5-controlled CD19 gene, and express Ig alpha to a lesser extent, they express Igbeta, VpreB and lambda5, and proliferate normally in vitro on stromal cells in the presence of interleukin (IL)-7. Clones of these pre-B I cells carry characteristic D(H)J(H) rearrangements on both IgH chain alleles. However, removal of IL-7 from the tissue cultures, unlike wild-type pre-B I cells, does not induce B-cell differentiation to surface IgM-expressing B cells, but induces macrophage differentiation. This differentiation into macrophages requires either the presence of stromal cells or addition of macrophage colony-stimulating factor (M-CSF). Addition of M-CSF followed by granulocyte-macrophage colony-stimulating factor induces the differentiation to MHC class II-expressing, antigen-presenting dendritic cells. In vitro differentiation to granulocytes and osteoclasts can also be observed in the presence of the appropriate cytokines. Moreover, transplantation of Pax-5-deficient pre-B I clones into RAG-deficient hosts, while not allowing B-cell differentiation, leads to the full reconstitution of the thymus with all stages of CD4-CD8- and CD4+CD8+ thymocytes, to normal positive and negative selection of thymocytes in the thymus, and to the development of normal, reactive mature CD4+ and CD8+ T-cell compartments in the peripheral lymphoid tissues, all carrying the clone-specific D(H)J(H) rearrangements. On the other hand, Ig alpha, Igbeta, VpreB and lambda5 are turned off in the thymocytes, demonstrating that the expression of these genes does not commit cells irreversibly to the B lineage. Further more, Pax-5-deficient pre-B I cells are long-term reconstituting cells. They home back to the bone marrow of the RAG-deficient host, can be reisolated and regrown in tissue culture, and can be retransplanted into a secondary RAG-deficient host. This again develops thymocytes and mature T cells and allows the transplanted clonal pre-B I cells to home to the bone marrow.

The mechanisms controlling the commitment of hematopoietic progenitor cells to the lymphoid lineages are still mostly unknown. Recent findings indicate that the earliest phase of B cell development may proceed in two steps. At the onset of B-lymphopoiesis, the transcription factors E2A and EBF coordinately activate the B-cell-specific gene expression program. Subsequently, Pax5 appears to repress the promiscuous transcription of lineage-inappropriate genes and thus commits progenitor cells to the B-lymphoid pathway by suppressing alternative cell fates. B-lineage commitment by Pax5 seems to occur in a stochastic manner in the bone marrow, as indicated by the random activation of only one of the two Pax5 alleles in early pro-B cells. In contrast, loss- and gain-of-function analyses have implicated the Notch1 receptor in the specification of the T cell fate, which may thus be controlled by instructive signals in the thymus.

The Pax5 gene encoding the B-cell-specific activator protein (BSAP) is expressed within the haematopoietic system exclusively in the B-lymphoid lineage, where it is required in vivo for progression beyond the pro-B-cell stage. However, Pax5 is not essential for in vitro propagation of pro-B cells in the presence of interleukin-7 and stromal cells. Here we show that pro-B cells lacking Pax5 are also incapable of in vitro B-cell differentiation unless Pax5 expression is restored by retroviral transduction. Pax5-/- pro-B cells are not restricted in their lineage fate, as stimulation with appropriate cytokines induces them to differentiate into functional macrophages, osteoclasts, dendritic cells, granulocytes and natural killer cells. As expected for a clonogenic haematopoietic progenitor with lymphomyeloid developmental potential, the Pax5-/- pro-B cell expresses genes of different lineage-affiliated programmes, and restoration of Pax5 activity represses this lineage-promiscuous transcription. Pax5 therefore plays an essential role in B-lineage commitment by suppressing alternative lineage choices.

The mechanisms controlling the commitment of haematopoietic progenitors to the B-lymphoid lineage are poorly understood. The observations that mice deficient in E2A and EBF lack B-lineage cells have implicated these two transcription factors in the commitment process. Moreover, the expression of genes encoding components of the rearrangement machinery (RAG1, RAG2, TdT) or pre-B-cell receptor (lambda5, VpreB, Igalpha, Igbeta) has been considered to indicate B-lineage commitment. All these genes including E2A and EBF are expressed in pro-B cells lacking the transcription factor Pax5. Here we show that cloned Pax5-deficient pro-B cells transferred into RAG2-deficient mice provide long-term reconstitution of the thymus and give rise to mature T cells expressing alpha/beta-T-cell receptors. The bone marrow of these mice contains a population of cells of Pax5-/- origin with the same phenotype as the donor pro-B cells. When transferred into secondary recipients, these pro-B cells again home to the bone marrow and reconstitute the thymus. Hence, B-lineage commitment is determined neither by immunoglobulin DJ rearrangement nor by the expression of E2A, EBF, lambda5, VpreB, Igalpha and Igbeta. Instead, our data implicate Pax5 in the control of B-lineage commitment.

It is generally assumed that most mammalian genes are transcribed from both alleles. Hence, the diploid state of the genome offers the advantage that a loss-of-function mutation in one allele can be compensated for by the remaining wild-type allele of the same gene. Indeed, the vast majority of human disease syndromes and engineered mutations in the mouse genome are recessive, indicating that recessiveness is the 'default' state. However, a minority of genes are semi-dominant, as heterozygous loss-of-function mutation in these genes leads to phenotypic abnormalities. This condition, known as haploinsufficiency, has been described for five of the nine mammalian Pax genes, which are associated with mouse developmental mutants and human disease syndromes. Recently we have reported that the Pax5 gene is subject to allele-specific regulation during B cell development. Pax5 is predominantly transcribed from only one of its two alleles in early B-lymphoid progenitors and mature B cells, while it transiently switches to a biallelic mode of transcription in pre-B and immature B cells. As a consequence, B-lymphoid tissues are mosaic with regard to the transcribed allele, and heterozygous mutation of Pax5 therefore results in deletion of B lymphocytes expressing only the mutant allele. The allele-specific regulation of Pax5 raises the intriguing possibility that monoallelic expression may also be the mechanism causing the haploinsufficiency of other Pax genes. In this review, we discuss different models accounting for the haploinsufficiency of mammalian Pax genes, provide further evidence in support of the allele-specific regulation of Pax5 and discuss the implication of these findings in the context of the recent literature describing the stochastic and monoallelic activation of other hematopoietic genes.

The developmental control genes of the Pax family are frequently associated with mouse mutants and human disease syndromes. The function of these transcription factors is sensitive to gene dosage, as mutation of one allele or a modest increase in gene number results in phenotypic abnormalities. Pax5 has an important role in B-cell and midbrain development. By following the expression of individual Pax5 alleles at the single-cell level, we demonstrate here that Pax5 is subject to allele-specific regulation during B-lymphopoiesis. Pax5 is predominantly transcribed from only one allele in early progenitors and mature B cells, whereas it switches to a biallelic transcription mode in immature B cells. The allele-specific regulation of Pax5 is stochastic, reversible, independent of parental origin and correlates with synchronous replication, in contrast with imprinted and other monoallelically expressed genes. As a consequence, B-lymphoid tissues are mosaics with respect to the transcribed Pax5 allele, and thus mutation of one allele in heterozygous mice results in deletion of the cell population expressing the mutant allele due to loss of Pax5 function at the single-cell level. Similar allele-specific regulation may be a common mechanism causing the haploinsufficiency and frequent association of other Pax genes with human disease.

The formation of the pre-B cell receptor (BCR) corresponds to an important checkpoint in B cell development that selects pro-B (pre-BI) cells expressing a functionally rearranged immunoglobulin mu (Igmu) heavy chain protein to undergo the transition to the pre-B (pre-BII) cell stage. The pre-BCR contains, in addition to Igmu, the surrogate light chains lambda5 and VpreB and the signal transducing proteins Igalpha and Igbeta. The absence of one of these pre-BCR components is known to arrest B cell development at the pre-BI cell stage. Disruption of the Pax5 gene, which codes for the B cell-specific activator protein (BSAP), also blocks adult B lymphopoiesis at the pre-BI cell stage. Moreover, expression of the mb-1 (Igalpha) gene and VH-to-DHJH recombination at the IgH locus are reduced in Pax5-deficient B lymphocytes approximately 10- and approximately 50-fold, respectively. Here we demonstrate that complementation of these deficiencies in pre-BCR components by expression of functionally rearranged Ig mu and chimeric Igmu-Igbeta transgenes fails to advance B cell development to the pre-BII cell stage in Pax5 (-/-) mice in contrast to RAG2 (-/-) mice. Furthermore, the pre-BCR is stably expressed on cultured pre-BI cells from Igmu transgenic, Pax5-deficient bone marrow, but is unable to elicit its normal signaling responses. In addition, the early developmental block is unlikely to be caused by the absence of a survival signal, as it could not be rescued by expression of a bcl2 transgene in Pax5-deficient pre-BI cells. Together, these data demonstrate that the absence of Pax5 arrests adult B lymphopoiesis at an early developmental stage that is unresponsive to pre-BCR signaling.

The Pax-5 gene codes for the transcription factor BSAP which is essential for the progression of adult B lymphopoiesis beyond an early progenitor (pre-BI) cell stage. Although several genes have been proposed to be regulated by BSAP, CD19 is to date the only target gene which has been genetically confirmed to depend on this transcription factor for its expression. We have now taken advantage of cultured pre-BI cells of wild-type and Pax-5 mutant bone marrow to screen a large panel of B lymphoid genes for additional BSAP target genes. Four differentially expressed genes were shown to be under the direct control of BSAP, as their expression was rapidly regulated in Pax-5-deficient pre-BI cells by a hormone-inducible BSAP-estrogen receptor fusion protein. The genes coding for the B-cell receptor component Ig-alpha (mb-1) and the transcription factors N-myc and LEF-1 are positively regulated by BSAP, while the gene coding for the cell surface protein PD-1 is efficiently repressed. Distinct regulatory mechanisms of BSAP were revealed by reconstituting Pax-5-deficient pre-BI cells with full-length BSAP or a truncated form containing only the paired domain. IL-7 signalling was able to efficiently induce the N-myc gene only in the presence of full-length BSAP, while complete restoration of CD19 synthesis was critically dependent on the BSAP protein concentration. In contrast, the expression of the mb-1 and LEF-1 genes was already reconstituted by the paired domain polypeptide lacking any transactivation function, suggesting that the DNA-binding domain of BSAP is sufficient to recruit other transcription factors to the regulatory regions of these two genes. In conclusion, these loss- and gain-of-function experiments demonstrate that BSAP regulates four newly identified target genes as a transcriptional activator, repressor or docking protein depending on the specific regulatory sequence context.

Pax-5 codes for the transcription factor BSAP which is expressed throughout B cell development except in terminally differentiated plasma cells. Gene targeting experiments in the mouse revealed a differential dependency of fetal and adult B-lymphopoiesis on this transcription factor. BSAP is required for B-lineage commitment in the fetal liver and for progression beyond an early pro-B cell stage in adult bone marrow. The characterization of Pax-5-deficient pro-B cells demonstrated an important role of BSAP in the regulation of the CD19, mb-1 (Ig alpha) and N-myc genes as well as in the developmental pathway controlling VH-to-DHJH recombination at the immunoglobulin heavy-chain (IgH) locus. The human PAX-5 gene was recently shown to participate together with the IgH locus in the chromosomal translocation t(9;14)(p13;q32). This translocation is characteristic of a small subset of non-Hodgkin lymphomas exhibiting plasmacytoid differentiation. The translocated PAX-5 gene is deregulated by the insertion of IgH regulatory elements into its 5' region, which may contribute to tumorigenesis by interfering with the shut-down of PAX-5 transcription and thus with the completion of plasma cell differentiation.

Pax-5 codes for the transcription factor BSAP which is expressed in all B-lymphoid tissues in addition to the developing central nervous system and testis. Within the B-lymphoid lineage, Pax-5 expression is already detected in the earliest B cell progenitors and persists up to the mature B cell stage. Targeted inactivation of the Pax-5 gene in the mouse germline revealed a differential dependency of fetal and adult B-lymphopoiesis on this transcription factor. Pax-5 is required for the differentiation of the earliest B-lineage-committed precursor cells in the fetal liver. In contrast, B cell development in the adult bone marrow progresses up to an early pro-B cell stage in the absence of Pax-5 function. The expression of CD19, Ig alpha (mb-1) and N-myc is severely reduced in Pax-5-deficient pro-B cells. These BSAP target genes are, however, unlikely to explain the early developmental block based on their known function in B cell development. Moreover, VH-to-DHJH rearrangements at the immunoglobulin heavy-chain locus are approximately 50-fold reduced in Pax-5-deficient pro B-cells, while the DH-to-JH rearrangements occur at a normal frequency. However, the expression of rearranged mu heavy-chain transgenes does not allow Pax-5-deficient pro-B cells to develop further to the pre-B cell stage. Together these data demonstrate therefore that B cell development in the Pax-5 deficient bone marrow is arrested at an early pro-B cell stage which is not yet responsive to pre-B cell receptor signaling.

The Pax5 gene coding for the transcription factor BSAP has an essential role in B lymphopoiesis and midbrain development. Here we present a detailed analysis of the B-cell phenotype of Pax5 mutant mice that revealed a differential dependency of fetal and adult B lymphopoiesis on this transcriptional regulator. B-cell development is arrested in the bone marrow at the early pro-B (pre-BI) cell stage, which is characterized by expression of the early markers c-kit, CD43, lambda5, VpreB, and HSA and the absence of the later markers CD25 and BP-1. These pre-BI cells fail to express the BSAP target gene CD19 and are capable of long-term proliferation in vitro in the presence of stromal cells and IL-7. B-lymphoid progenitors could not be detected in the fetal liver of Pax5 mutant embryos. However, Pax5-deficient fetal liver cells gave rise to the development of pre-BI cells in bone marrow on transplantation into lethally irradiated mice. These data indicate different functions of Pax5 in the distinctive microenvironments of fetal liver and adult bone marrow. As shown by PCR analyses, the pre-BI cells in Pax5-deficient bone marrow have undergone D(H)-to-J(H) rearrangement of the immunoglobulin heavy-chain locus at normal frequency. In contrast, V(H)-to-D(H)J(H) rearrangements were reduced approximately 50-fold in Pax5-deficient pre-BI cells, suggesting a role for Pax5 in the developmental pathway controlling V-to-DJ recombination.

Objective: To quantify the serum antibody responses to Pasteurella multocida A:3 outer membrane proteins (OMP) for cattle vaccinated with the homologous serogroup and to correlate those responses with the extent of experimentally induced pneumonia., Animals: 29, 5- to 8-month-old beef-type calves., Procedure: Calves were vaccinated SC or by aerosal exposure on days 0 and 7 with live or killed P multocida or phosphate-buffered saline solution (control) and subsequently challenge exposed with virulent P multocida. Antibody responses to P multocida A:3 outer membranes were quantified, using an ELISA. Antibody responses to individual OMP were detected by immunoblot analysis (western blot) and were quantified by densitometry. Antibody responses were compared among groups of calves and for various times after vaccination. Regression analyses were used to determine whether significant correlations existed between lesion scores and antibody responses to either whole outer membranes or to individual OMP., Results: By ELISA, antibody responses to outer membranes for calves aerosol vaccinated with live P multocida were significantly (P < 0.05) greater than those for control calves or for killed P multocida vaccinates. There was a significant (P < 0.05) correlation between lesion score and antibody responses to outer membranes. By western blotting and densitometry, antibodies to 11 prominent OMP (100, 97, 90, 85, 74, 53, 46, 35, 32, 21, and 16 kd) were identified and quantified. In experiment 1, SC vaccination with live P multocida increased antibody binding to all protein bands except 85-, 74-, and 35-kd bands. Aerosol vaccination with live P multocida stimulated increases in antibody binding to all bands except 100 and 16 kd. Antibody responses to the 97-, 90-, 74-, and 35- kd bands were significantly (P < 0.05; greater for live aerosol vaccinates than for control calves. In experiment 2, antibody responses were not different between the killed P multocida vaccinates or control calves Antibody responses for live P multocida aerosol vaccinates were significantly (P < 0.05) greater than those for control calves for the 100-, 90-, 85-, 74-, 53-, 35-, and 16-kd bands. Regression analyses indicated significant correlations (P < 0.05) between lesion score and antibody responses to the 100-, 90-, 53-, 46-, 35-, and 32-kd OMP., Conclusions: Several OMP of P multocida type A:3 may be important for stimulating immunity to the organism in cattle.

We have isolated a new member of the human glutamate receptor family from a fetal brain cDNA library. This cDNA clone, designated EAA3a, shares a 90% nucleotide identity with the previously reported rat GluR5-2b cDNA splice variant and differed from human GluR5-1d in the amino and carboxy terminal regions. Cell lines stably expressing EAA3a protein formed homomeric ligand-gated ion channels responsive, in order of decreasing affinity to domoate, kainate, L-glutamate and (RS)-alpha-amino-3-hydroxy-5- methylisoxazole-propionate (AMPA). Kainate-evoked currents showed partial desensitization that was reduced on incubation with concanavalin A (conA) but not cyclothiazide and were attenuated by the non-N-methyl-D-aspartate (NMDA) receptor antagonist CNQX (6-cyano-7-nitro-quinoxalinedione). Coexpression of EAA3a and human EAA1 cDNAs in HEK 293 cells formed a heteromeric channel with unique properties. Kainate and AMPA activated the heteromeric channel with significantly higher affinities than observed for EAA3a alone. Ligand binding studies with the recombinant EAA3a receptor expressed in mammalian cells indicated a high affinity kainate binding site (Kd = 120 +/- 15.0 nM). The relative potency of compounds in displacing [3H]-kainate binding to EAA3a receptor was: domoate > kainate > L-glutamate = quisqualate > 6,7-dinitroquinoxaline-2,3-dione (DNQX) = CNQX > AMPA > dihydrokainate > NMDA.

A member of the ionotropic family of glutamate receptors, hGluR4, was isolated from a human cDNA library and characterized following expression in mammalian cell lines. Human GluR4 possessed a 99% amino acid and 92% nucleotide homology to that of its rat counterpart with sequence differences restricted to the carboxy and amino terminal regions of the molecule. Transfection of simian kidney cells (COS-1) with an hGluR4 expression plasmid resulted in the transient formation of a membrane protein that possessed high specific binding for [3H](RS)-alpha-amino- 3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA) but not [3H]kainate. Competition studies yielded a displacement profile of AMPA = quisqualate > glutamate > domoate > kainate >> N-methyl-D-aspartate (NMDA) or dihydrokainate. Whole-cell, voltage-clamp recordings from a human embryonic kidney cell line (HEK 293) stably expressing hGluR4 confirmed the presence of constitutively active, ligand-gated ion channels activated by AMPA, glutamate and kainate but not N-methyl-D-aspartate. Kainate-evoked currents were reversibly attenuated by 6-cyano-7-nitro- quinoxaline-2,3-dione (CNQX) but not DL-2-amino-5- phosphonovalerate (DL-AP5). Agonist-evoked currents exhibited inward rectification and ion substitution experiments indicated that hGluR4 receptor-linked ion channels in their homomeric state are permeable to both CA2+ and Na+ ions. In the same cell line antibody to rat GluR4 immunoprecipitated a major protein band at approximately 108 kDa and a minor one at approximately 340 kDa. The immunoblot analysis of membranes chemically crosslinked with dithiobis(succinimidylpropionate) showed a broad band at 550-600 kDa suggesting that the GluR4 receptor forms a pentamer in situ. This is the first report of the cloning of hGluR4 receptor and its stable expression in a human cell line.

RNA editing has been shown to be critical in generating the molecular diversity of rodent kainate receptors. We have examined cDNAs derived from various human brain sources to assess the occurrence and extent of RNA editing in human brain. Comparison of genomic and cDNA sequences revealed extensive editing of the human EAA4 (GluR6) mRNA at the isoleucine/valine, tyrosine/cysteine sites of the transmembrane I region, and the glutamine/arginine site of the transmembrane II region. Of the eight potential molecular variants generated by the nucleotide exchange, five were observed in the tissues examined. The distribution of the various RNA editing combinations were not uniform, and displayed tissue and/or age dependent distribution. Editing of the glutamine/arginine site was also confirmed for EAA3 (GluR5), which displays a significantly higher extent of editing in specific human brain regions compared with rodent whole brain. Hence, it can be concluded that RNA editing is a determinant of the phenotype of human kainate receptor complexes.

Several cDNA clones encoding the human glutamate receptor subunit GluR3 flip and flop isoforms, were isolated from human hippocampus and fetal brain libraries. DNA sequence analysis revealed overlapping clones permitting the reconstruction of full-length GluR3-flip and GluR3-flop cDNAs. The GluR3 cDNAs demonstrated an 94.1-94.7% nucleotide (nt) identity with the corresponding rat cDNAs. The nt sequence of the GluR3 cDNAs would encode 894 amino acid proteins that have a 99.4% identity with the rat GluR3 isoforms. The human GluR3 cDNAs predict an additional 6 amino acid in the N-terminal signal peptide as compared to the rat GluR3.

RNA editing in rat brain has been found to control a determinant of cation flow in alpha-amino-3-hydroxy-5-methyl-4-isoxasolepropionic acid (AMPA)-gated channels. Here we provide the first evidence that this RNA editing phenomenon occurs in human brain and is differentially regulated. Sequence analysis of human genomic DNA revealed a Q codon (CAG) in the putative channel-forming segment of human GluR-2, whereas in the majority of cDNA clones an R codon (CGG) was found. Examination of editing in various brain tissues revealed differences in the efficiency of this process. The hippocampus, cerebellum and temporal cortex harbour 100% edited GluR-2, whereas only 72% of substantia nigra, 89% of corpus striatum and 96% of fetal cDNAs have been found to be edited. This new discovery of differential efficiency of RNA editing has important implications in AMPA receptor channel-mediated calcium influx. AMPA receptors are thought to mediate the majority of the fast excitatory synaptic neurotransmission; the RNA editing process may therefore play a critical role in normal brain function and development. Dysfunction of this RNA editing process may have neuropathological consequences and could be related to certain neurodegenerative diseases.

A cDNA encoding an ionotropic glutamate receptor subunit protein humEAA4 (GluR6), has been cloned from a human fetal brain library. This cDNA when expressed in COS or HEK-293 cells is associated with high-affinity kainate receptor binding and ion channel formation. We have successfully established cell lines stably expressing humEAA4 in HEK-293 cells This is the first report of the establishment of stable cell lines expressing a glutamate receptor channel. The relative potency of compounds for displacing [3H]-kainate binding to humEAA4 receptors expressed in COS or HEK-293 cells is domoate > kainate > quisqualate > 6-cyano-7-nitroquinoxaline-2,3-dione > L-glutamate = 6,7- dinitroquinoxaline-2,3-dione > dihydrokainate. Applications of kainate, glutamate, and domoate but not AMPA evoked rapidly desensitizing currents in cells expressing homo-oligomeric humEAA4 in a concentration dependent manner. The order of potency was: domoate > kainate > L-glutamate. Although AMPA did not itself activate humEAA4 receptors it did reduce, to a limited extent, kainate-evoked responses. AMPA may therefore be a weak partial agonist for this receptor. To date this effect has not been demonstrated with rat GluR6. It is possible that subtle species differences may exist in the nature of agonist receptor interaction. Kainate evoked currents were attenuated by the quinoxalinediones CNQX and DNQX but not by DAP5. The receptor desensitization was attenuated on application of concanavalin A. Ion-permeability studies indicated that the receptor-linked ion channel is permeable to both Na+ and Ca2+ ions.

Kainate is a potent neuroexcitatory agent; its neurotoxicity is thought to be mediated by an ionotropic receptor with a nanomolar affinity for kainate. In this report, we describe the cloning of a cDNA encoding a human glutamate ionotropic receptor subunit protein from a human hippocampal library. This cDNA, termed humEAA1, is most closely related to rat and human cDNAs for kainate receptor proteins and, when expressed in COS or Chinese hamster ovary cells, is associated with high-affinity kainate receptor binding. We have successfully established cell lines stably expressing humEAA1. This is the first report of establishment of stable cell lines expressing a glutamate receptor subunit. The relative potency of compounds for displacing [3H]kainate binding of humEAA1 receptors expressed in these stable cell lines was kainate > quisqualate > domoate > L-glutamate >> (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid > dihydrokainate > 6,7-dinitroquinoxaline-2,3-dione > 6-cyano-7-nitroquinoxaline-2,3-dione. Homooligomeric expression of humEAA1 does not appear to elicit ligand-gated ion channel activity. Nevertheless, the molecular structure and pharmacological characterization of high-affinity kainate binding of the humEAA1 expressed in the stable cell line (ppEAA1-16) suggest that the humEAA1 is a subunit protein of a human kainate receptor complex.

Several cDNA clones encoding EAA5 receptor polypeptides were isolated from a human fetal brain library. The EAA5 cDNAs demonstrated an 88.7-90.1% nucleotide identity with rat GluR7 cDNAs. The nucleotide sequence of EAA5 would encode a 919-amino acid protein, that has a 97.7-98.9% identity with the rat GluR7 receptor. Two variation of the EAA5 cDNA were identified which result in amino acid substitutions in the predicted extracellular amino-terminal region; Ser310-->Ala and Arg352-->Gln. These variations can be attributed to RNA editing involving T-->G and G-->A substitutions. Both the location (with respect to glutamate receptors), and the nucleotides involved, in this putative RNA editing are novel and may therefore involve novel mechanisms. Ligand binding studies with membranes of transfected COS-1 cells expressing EAA5 polypeptides demonstrate a rank order of ligand affinity similar to that observed with the rat GluR7 receptor, and a dissociation constant for kainate (2.72 +/- 0.12 nM (n = 3)) that is approximately 20- to 30-fold higher than that observed for the rat GluR7 receptor. All of the ligands tested had a higher affinity for the human EAA5 receptor as compared to the rat GluR7 receptor. This report provides another example of pharmacological differences for similar receptors across species.

Intracranial hemorrhages are a recognized but relatively uncommon complication of brain tumors. Metastatic tumors are more often associated with hemorrhages than are primary tumors. The types of hemorrhages found include intraparenchymal, subarachnoid, subdural, and epidural. Treatment must be directed at both the hemorrhage and the underlying tumor.

In a one-year study of 49 homeless chronic mentally ill patients, the subjects, selected at admission for inpatient treatment, were randomly assigned to one of two groups. One group was placed in an experimental residential treatment program following discharge, and the other group received standard postdischarge care. Subjects were interviewed every four months during the year as well as at index hospitalization and discharge. Although the study remains exploratory due to the small sample size and case attrition, the authors found that compared with the control group, the subjects in the residential treatment program spent significantly more nights in adequate shelter, spent fewer nights in hospitals or undomiciled, and were more satisfied with and committed to their living arrangements.