Search

Background A lack of standardization in chemotherapy patient education practices can lead to decreased efficiency and satisfaction for nurse educators and uncertainty and anxiety for patients. Objectives The goal was to determine whether standardizing chemotherapy education practices improved nurse and patient satisfaction. Methods An interprofessional team was formed to standardize the chemotherapy education process and reduce variation in teaching. Anonymous, self-administered questionnaires assessed satisfaction in chemotherapy education among nurses and patients pre- and postimplementation. Findings Significant improvement in nursing staff satisfaction postimplementation was observed across all individual construct measures, with the average overall score increasing from 3.4 to 4.3. Patient satisfaction scores were high in the pre- and postimplementation phases (average overall score of 4.3 and 4.1, respectively).

The origin of pathogenic autoantibodies remains unknown. Idiopathic pulmonary alveolar proteinosis is caused by autoantibodies against granulocyte–macrophage colony-stimulating factor (GM-CSF). We generated 19 monoclonal autoantibodies against GM-CSF from six patients with idiopathic pulmonary alveolar proteinosis. The autoantibodies used multiple V genes, excluding preferred V-gene use as an etiology, and targeted at least four nonoverlapping epitopes on GM-CSF, suggesting that GM-CSF is driving the autoantibodies and not a B-cell epitope on a pathogen cross-reacting with GM-CSF. The number of somatic mutations in the autoantibodies suggests that the memory B cells have been helped by T cells and re-entered germinal centers. All autoantibodies neutralized GM-CSF bioactivity, with general correlations to affinity and off-rate. The binding of certain autoantibodies was changed by point mutations in GM-CSF that reduced binding to the GM-CSF receptor. Those monoclonal autoantibodies that potently neutralize GM-CSF may be useful in treating inflammatory disease, such as rheumatoid arthritis and multiple sclerosis, cancer, and pain.

Human S100A7 (psoriasin) is considered a marker for specific stages of breast cancer. hS100A15 is almost identical to hS100A7 and difficult to discriminate. We developed specific probes to distinguish hS100A7 and hS100A15, and demonstrate their differential distribution in normal breast tissue. Further, hS100A7 and S100A15 transcripts are elevated in ER/PR negative breast cancers, but hS100A15 protein is detected in all cancer specimens while hS100A7 protein is sporadically expressed. The differential regulation, expression and distribution of hS100A7 and hS100A15 and their reported distinct functions are compelling reasons to discriminate among these proteins in normal breast and breast cancers.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a glycoprotein that is part of the carcinoembryonic antigen and the immunoglobulin superfamilies. We have shown that it functions as a tumor suppressor and that this function depends upon the presence of the longer CEACAM1 cytoplasmic domain. In this report, we describe the generation of a Ceacam1-/- mouse. The Ceacam1-/- colon exhibits increased in vivo proliferation relative to the wild-type counterpart with a corresponding decreased expression of the p21(Cip1) and p27(Kip1) Cyclin D kinase inhibitors. The colonic villi undergo decreased apoptosis. Out of 35 litters of mice, no spontaneous tumors in any tissues normally expressing CEACAM1 were found over the lifespan of the animals, suggesting that CEACAM1 may not be involved in initiation of tumor development. However, when mice are treated with azoxymethane to induce colonic tumors, we find that Ceacam1-/- mice developed a significantly greater number of tumors than their littermate controls. Moreover, the tumor size was greater in the knockout mice relative to that in the wild-type mice. These results indicate that deletion of CEACAM1 favors progression of colon tumorigenesis.

CEACAM1 is an intercellular adhesion glycoprotein. As CEACAM1 plays an important role in epithelial cell signaling and functions, we have examined its localization in epithelial cells. We have observed that distribution at cell contacts is not always seen in these cells, suggesting that CEACAM1 localization might be regulated. In Swiss 3T3 cells, the targeting of CEACAM1 at cell-cell boundaries is regulated by the Rho GTPases. In the present study, we have used the MDCK epithelial cells to characterize the effects of the Rho GTPases and their effectors on CEACAM1 intercellular targeting. Activated Cdc42 and Rac1 or their downstream effector PAK1 targeted CEACAM1 to sites of cell-cell contacts. On the other hand, neither activated RhoA nor activated Rho kinase directed CEACAM1 to cell boundaries, resulting in a condensed distribution of CEACAM1 at the cell surface. Interestingly, inhibition of this pathway resulted in CEACAM1 intercellular localization suggesting that a tightly regulated balance of Rho GTPase activities is necessary to target CEACAM1 at cell-cell boundaries. In addition, using CEACAM1 mutants and chimeric fusion constructs containing domains of the colony-stimulating factor receptor, we have shown that the transmembrane domain of CEACAM1 is responsible for the Cdc42-induced targeting at cell-cell contacts.

The CEACAM1 glycoproteins, formerly called biliary glycoproteins (BGP, Bgp, mmCGM1, C-CAM, CD66a, or MHVR), are members of the carcinoembryonic antigen (CEA) family in the immunoglobulin (Ig) superfamily (4, 22, 44, 51, 70). The nomenclature for the CEA gene family has recently been unified (9), and seven genes are now referred to as the CEACAM genes. CEACAM1, the most conserved gene of the CEA gene family, is found as a single copy on human chromosome 19q13.2 (30) and in the rat genome (24). However, mouse chromosome 7, in the 7A2-A3 region near the centromere, a region syntenic to the region encoding CEACAM1 on human chromosome 19q, contains two highly related Ceacam genes, called Ceacam1 and Ceacam2 (formerly called Bgp1 and Bgp2, respectively) (48, 49, 58). The human, rat, and mouse CEACAM1 genes have highly conserved structures (4, 24, 49), 66% identity between their respective promoters, similar but not identical patterns of mRNA splicing, and similar patterns of expression in different tissues (27, 32, 44, 49, 52, 53). Mouse CEACAM1 isoforms are transmembrane glycoproteins that have either two or four Ig domains produced by alternative splicing of the primary transcript (Fig. ​(Fig.1A1A and B) (43, 44). CEACAM1 isoforms with four Ig domains, denoted CEACAM1/D1-4, include the N domain (D1) attached to three constant Ig domains (D2, D3, and D4). Splice isoforms with two Ig domains (CEACAM1/D1,4) link D1 to D4. The exodomains are linked via a transmembrane domain to either of two cytoplasmic tails. The short cytoplasmic tail (CEACAM1-S) contains 10 amino acids (aa) rich in Ser and Gly residues. The long cytoplasmic tail (CEACAM1-L) results from inclusion of 53-bp Ceacam1 exon 7, which shifts the open reading frame (ORF) for the tail at aa 453 to yield a 73-aa tail (Fig. ​(Fig.1A)1A) (43, 49). FIG. 1 Targeting of the Ceacam1 gene. (A) The mouse Ceacam1 gene encodes nine exons. The ATG initiation codon is located in the first exon, whereas two stop codons can be alternatively used, one in exon 8 [TGA(S), used in the translation of isoforms ... The CEACAM1 glycoproteins are abundantly expressed on apical membranes of epithelial cells in the gastrointestinal and respiratory tracts, in bile canaliculi, and on the proximal tubules of the kidneys (44, 50, 71). They are also found on small vascular endothelial cells, in hemopoietic cells (B cells, neutrophils, macrophages, monocytes, platelets, and activated T cells), and in thymic stromal cells (17, 27, 46, 47). CEACAM1 isoforms are also expressed at the apical surfaces of epithelial cells in the reproductive tissues (uterus, ovary, breast, and prostate) (34, 68, 71) and at low levels on glial cells in the nervous system (27, 62). CEACAM1 is abundantly expressed in endodermal and mesenchymal derivatives during early mouse embryonic development (19). CEACAM1 performs many important cellular functions. It is a cell adhesion molecule (44, 52, 59) and a signaling molecule (10, 31, 51) that regulates the growth of tumor cells (34, 40). Recently, CEACAM1 was shown to be a potent angiogenic factor (25). In addition, CEACAM1 is a receptor for bacterial and viral pathogens. The opa surface proteins of pathogenic strains of Neisseria gonorrhoeae and Neisseria meningitidis and membrane proteins of Haemophilus influenzae bind specifically to the human CEACAM1 protein and several other human CEA-related glycoproteins (29, 72–74). In this study, we explored the role in viral pathogenesis of murine CEACAM1a, a receptor for mouse hepatitis virus (MHV). MHV strains are murine coronaviruses that cause respiratory and enteric infections, hepatitis, splenolysis, immune dysfunction, acute encephalitis, and chronic demyelinating disease of the brain and spinal cord (5, 16). MHV infection of mice varies from inapparent and self-limited infection to severe disease and death, depending on the age, immune status, strain, and route of inoculation of the mouse and on the dose and strain of the virus. In some mouse strains, inapparent MHV infection disrupts normal patterns of cytokine expression for 5 months after infection (18). Many, but not all, of the murine tissues that express CEACAM1 are natural targets for MHV infection. Williams (76), Williams et al. (77), and Dveksler et al. (22) identified and cloned the cDNA encoding the CEACAM1/D1-4 MHV receptor. When this murine Ceacam1a cDNA was transfected into hamster cell lines, which are not susceptible to MHV, expression of the mouse CEACAM1a glycoprotein rendered the hamster cells susceptible to MHV (22). The sequence of the virus receptor was found to be identical to that of the biliary glycoprotein identified as a CEA-related cell adhesion glycoprotein (44). Adult SJL mice, which are highly resistant to MHV infection (7, 39, 67), are homozygous for CEACAM1b, an allele of CEACAM1a that differs by 27 of 108 aa in the N domain (D1) (21, 78). The spike (S) glycoprotein of MHV attaches to the N domain (D1) of CEACAM1a (23). Most inbred strains of mice (BALB/c, C57BL/6, C3H, 129Sv, and so forth) are susceptible to MHV infection and are homozygous for the CEACAM1a allele, whereas outbred CD1 mice express both CEACAM1a and CEACAM1b and are susceptible to infection. All MHV strains tested to date utilize the murine CEACAM1a proteins as receptors (15, 21). Mutational analyses showed that the virus binds to the B-C-C′ region of domain 1 of the CEACAM1a protein (57, 75). A monoclonal antibody (CC1) directed against D1 of CEACAM1a blocks virus attachment and prevents infection in vitro and in vivo (23, 65). All four isoforms of murine CEACAM1a serve as receptors for MHV A59 when expressed at high levels in hamster cells (21). Interestingly, the expression of high levels of murine CEACAM1b in hamster cells also makes the cells susceptible to MHV-A59 infection (21). When expressed at high levels in BHK cells, murine CEACAM2 also serves as an MHV receptor, although it is a markedly less efficient MHV A59 receptor than CEACAM1a (48). Soluble CEACAM2 also has much less virus neutralization activity than soluble CEACAM1a (80). The surface density of CEACAM1a/D1,4 with the short cytoplasmic tail affects the susceptibility of cells to infection with the MHV JHM strain. HeLa cells that expressed low levels of recombinant murine CEACAM1a/D1,4 were susceptible to infection with MHV JHM but did not exhibit cytopathic effects, such as cell fusion and death (55). In contrast, HeLa cells that expressed high levels of murine CEACAM1a/D1,4 with the short tail on the cell surface were killed within 14 h of infection with MHV JHM. Complexes formed between the CEACAM1a receptor protein and the viral S glycoprotein in the endoplasmic reticulum and Golgi apparatus (56). MHV infection or expression of the viral S glycoprotein on murine cells rapidly led to the selection of cells that expressed reduced levels of CEACAM1a (14, 55, 63). Persistent infection of murine cells in vitro with MHV A59 leads to the selection of cells resistant to wild-type virus and to the selection of small-plaque viral mutants that have mutations in the receptor-binding N-terminal domain of the viral S glycoprotein, and some of these small-plaque mutants have an extended host range (2, 3, 28, 63, 64). The goal of this research was to derive Ceacam1 knockout mice for the study of CEACAM1 functions in normal and infected animals. We therefore disrupted the mouse Ceacam1a gene in 129Sv mouse embryonic stem (ES) cells in order to generate knockout animals and examined the genotypes and phenotypes of the resulting heterozygous and homozygous mice. Normally this technique completely abrogates or knocks out the expression of the targeted gene in homozygous mice. The only line of mice that resulted from this knockout strategy showed only partial, incomplete reduction in CEACAM1a expression; expressed markedly altered ratios of CEACAM1a isoforms in different murine tissues; but had normal phenotype, fertility, and life span. Expression of the CEACAM1a/D1-4 isoforms was reduced by 90 to 95% in these mice, whereas the CEACAM1a/D1,4 isoforms were expressed at levels higher than normal. Following intranasal inoculation with MHV A59, homozygous (p/p) Ceacam1a-targeted mice failed to develop clinical signs of viral infection, developed fewer and smaller lesions in the liver, and produced less virus in the liver than wild-type (+/+) mice. Thus, reducing the level of expression of CEACAM1a/D1-4 proteins and altering the ratios of two- and four-domain CEACAM1a isoforms in vivo resulted in mice with significantly decreased susceptibility to MHV infection. These results also suggest that the four-domain CEACAM1a isoforms are the principal MHV receptors in vivo.

Human granulocyte macrophage colony-stimulating factor (hGM-CSF) is a haematopoietic growth factor and proinflammatory cytokine. Recombinant hGM-CSF is important not only as a research tool but also as a biotherapeutic. However, rhGM-CSF expressed in E. coli is known to form inclusion bodies of misfolded, aggregated protein. Refolding and subsequent purification of rhGM-CSF from inclusion bodies is difficult with low yields of bioactive protein being produced. Here we describe a method for the isolation, refolding and purification of bioactive rhGM-CSF from inclusion bodies. The method is straightforward, not requiring extensive experience in protein refolding nor purification and using standard laboratory equipment.

Most monoclonal antibodies (mAbs) generated from humans infected or vaccinated with the 2009 pandemic H1N1 (pdmH1N1) influenza virus targeted the hemagglutinin (HA) stem. These anti-HA stem mAbs mostly used IGHV1-69 and bound readily to epitopes on the conventional seasonal influenza and pdmH1N1 vaccines. The anti-HA stem mAbs neutralized pdmH1N1, seasonal influenza H1N1 and avian H5N1 influenza viruses by inhibiting HA-mediated fusion of membranes and protected against and treated heterologous lethal infections in mice with H5N1 influenza virus. This demonstrated that therapeutic mAbs could be generated a few months after the new virus emerged. Human immunization with the pdmH1N1 vaccine induced circulating antibodies that when passively transferred, protected mice from lethal, heterologous H5N1 influenza infections. We observed that the dominant heterosubtypic antibody response against the HA stem correlated with the relative absence of memory B cells against the HA head of pdmH1N1, thus enabling the rare heterosubtypic memory B cells induced by seasonal influenza and specific for conserved sites on the HA stem to compete for T-cell help. These results support the notion that broadly protective antibodies against influenza would be induced by successive vaccination with conventional influenza vaccines based on subtypes of HA in viruses not circulating in humans.

Introduction c-Jun activation domain-binding protein-1 (Jab1) is a multifunctional signaling protein that previously has been shown to be a master regulator of a poor prognostic gene signature in invasive breast cancer and to mediate the action of S100A7. Since epidermal growth factor receptor (EGFR), like S100A7, is often expressed in estrogen receptor-alpha-negative (ERα-) breast cancer, we set out to investigate the role of Jab1 in mediating EGFR signaling, another facet of the ERα- phenotype. Methods MDA-MB-231 and MDA-MB-468 ERα-/EGFR+ cell lines were assessed for localization of Jab1 and levels of downstream genes by immunofluorescence and nuclear protein extract assay following treatment with epidermal growth factor (EGF) and extracellular signal-regulated kinase (ERK) pathway inhibitor. A cohort of 424 human breast tumors was also assessed by immunohistochemistry. Results EGF treatment of cell lines resulted in increased Jab1 nuclear expression. This effect was inhibited by the ERK pathway inhibitor, PD98059. EGF treatment was also associated with colocalization of pERK (phosphorylated ERK) and Jab1 as well as regulation of the Jab1 downstream target gene, p27. When Jab1 activity was knocked down, p27 levels were restored to pre-EGF treatment level. Analysis of EGFR and Jab1 expression in a cohort of invasive breast tumors by tissue microarray and immunohistochemistry confirmed a relationship between EGFR and increased nuclear Jab1 within the ERα- subset (n = 154, P = 0.019). The same association was also confirmed for S100A7 and Jab1 (P = 0.036), and high Jab1 nuclear expression was most frequent in tumors that were positive for both EGFR and S100A7 (P = 0.004). Conclusion Jab1 is a target of EGFR signaling in ERα- cell lines and breast tumors and therefore may be a common central factor and potential therapeutic target for important cell signaling pathways in ERα- breast cancer.

CEACAM1a glycoproteins are members of the immunoglobulin (Ig) superfamily and the carcinoembryonic antigen family. Isoforms expressing either two or four alternatively spliced Ig-like domains in mice have been found in a number of epithelial, endothelial, or hematopoietic tissues. CEACAM1a functions as an intercellular adhesion molecule, an angiogenic factor, and a tumor cell growth inhibitor. Moreover, the mouse and human CEACAM1a proteins are targets of viral or bacterial pathogens, respectively, including the murine coronavirus mouse hepatitis virus (MHV),Haemophilus influenzae,Neisseria gonorrhoeae, andNeisseria meningitidis, as well asMoraxella catarrhalisin humans. We have shown that targeted disruption of theCeacam1a(MHVR) gene resulting in a partial ablation of the protein in mice (p/p mice) led to reduced susceptibility to MHV-A59 infection of the modified mice in the BALB/c background. We have now engineered and produced aCeacam1a−/−mouse that exhibits complete ablation of the CEACAM1a protein in every tissue where it is normally expressed. We report that 3-week-oldCeacam1a−/−mice in the C57BL/6 genetic background are fully resistant to MHV-A59 infection by both intranasal and intracerebral routes. Whereas virus-inoculated wild-type +/+ C57BL/6 mice showed profound liver damage and spinal cord demyelination under these conditions,Ceacam1a−/−mice displayed normal livers and spinal cords. Virus was recovered from liver and spinal cord tissues of +/+ mice but not of −/− mice. These results indicate that CEACAM1a is the sole receptor for MHV-A59 in both liver and brain and that its deletion from the mouse renders the mouse completely resistant to infection by this virus.