Tumor immunotherapy using adenovirus vaccines in combination with intratumoral doses of CpG ODN.

The combination of viral vaccination with intratumoral (IT) administration of CpG ODNs is yet to be investigated as an immunotherapeutic treatment for solid tumors. Here, we show that such a treatment regime can benefit survival of tumor-challenged mice. C57BL/6 mice bearing ovalbumin (OVA)-expressing EG.7 thymoma tumors were therapeutically vaccinated with adenovirus type 5 encoding OVA (Ad5-OVA), and the tumors subsequently injected with the immunostimulatory TLR9 agonist, CpG-B ODN 1826 (CpG), 4, 7, 10, and 13 days later. This therapeutic combination resulted in enhanced mean survival times that were more than 3.5× longer than naïve mice, and greater than 40% of mice were cured and capable of resisting subsequent tumor challenge. This suggests that an adaptive immune response was generated. Both Ad5-OVA and Ad5-OVA + CpG IT treatments led to significantly increased levels of H-2 K(b)-OVA-specific CD8+ lymphocytes in the peripheral blood and intratumorally. Lymphocyte depletion studies performed in vivo implicated both NK cells and CD8+ lymphocytes as co-contributors to the therapeutic effect. Analysis of tumor infiltrating lymphocytes (TILs) on day 12 post-tumor challenge revealed that mice treated with Ad5-OVA + CpG IT possessed a significantly reduced percentage of regulatory T lymphocytes (Tregs) within the CD4+ lymphocyte population, compared with TILs isolated from mice treated with Ad5-OVA only. In addition, the proportion of CD8+ TILs that were OVA-specific was reproducibly higher in the mice treated with Ad5-OVA + CpG IT compared with other treatment groups. These findings highlight the therapeutic potential of combining intratumoral CpG and vaccination with virus encoding tumor antigen.

Differential tissue expression of epitopes of the tetraspanin CD151 recognised by monoclonal antibodies.

CD151, a member of the tetraspanin family of cell membrane proteins, is widely expressed in epithelial, endothelial and muscle cells as well as platelets and megakaryocytes. Several monoclonal antibodies recognising CD151 in transfected cells and immunoprecipitating typical bands of 28 and 32 kDa from cell lysates have been produced. Surprisingly, these antibodies show different patterns of staining on tissue sections and on haemopoietic cells. Here we show that these differences are at least in part due to masking of certain epitopes in integrin/CD151 complexes. These data have important implications for the use of monoclonal antibodies in studies of the distribution and function of CD151. Of six monoclonal antibodies from four laboratories, 11B1 was found to be the most reliable for detection of CD151 in different cell and tissue contexts.

Osteoprotegerin inhibits osteoclast formation and bone resorbing activity in giant cell tumors of bone.

Osteolysis is a common complication of tumors that arise in, or metastasize to, bone. The recent discovery of key regulators of osteoclast formation and activity, including receptor activator of nuclear factor of kappaB ligand (RANKL), RANK, and osteoprotegerin (OPG), may facilitate new treatment regimes for certain tumors associated with excessive bone loss. We recently showed that the stromal cells of osteolytic giant cell tumors (GCT) of bone express high levels of mRNA encoding RANKL, relative to mRNA for the RANKL antagonist, OPG, compared with the expression patterns of other lytic and nonlytic bone tumors. In this study, we found that expression of RANKL and OPG mRNA continued by the stromal element of these tumors in a constitutive manner for at least 9 days in the absence of giant cells. Immunostaining of unfractionated GCT cultured in vitro revealed punctate cytoplasmic/membranous staining for RANKL and both cytoplasmic and extracellular matrix staining for OPG in stromal cells. Giant cells (osteoclasts) were negative for RANKL staining, but stained brightly for cytoplasmic OPG protein. We also investigated the functional relevance of these molecules for GCT osteolysis by adding recombinant OPG and RANKL to cultured GCT cells. We found that OPG treatment potently and dose-dependently inhibited resorption of bone slices by GCT, and could also inhibit the formation of multinucleated osteoclasts from precursors within the GCT. These effects of OPG were reversed by stoichiometric concentrations of exogenous RANKL. These data indicate that both the processes of osteoclast formation and activation in GCT are promoted by RANKL. Therefore, GCT represent a paradigm for the direct stimulation of osteoclast formation and activity by tumor stromal cells, in contrast to the mechanisms described for osteolytic breast tumors and multiple myeloma. The demonstration of these relationships is important in developing approaches to limit tumor-induced osteolysis.

Coordinated cytokine expression by stromal and hematopoietic cells during human osteoclast formation.

An in vitro culture system to generate human osteoclasts (OC) was recently described in which OC precursors in the human peripheral blood mononuclear cell (PBMC) population differentiate in the presence of murine ST-2 stromal cells. We used this culture system to define the cytokine environment in which human OC form and to determine the separate contributions of the stromal and hematopoietic elements. We designed a panel of reverse transcriptase-polymerase chain reaction (RT-PCR) primers that specifically amplify the respective murine or human mRNA species that correspond to cytokines and their cognate receptors previously shown to promote or inhibit OC differentiation. ST-2 cells were cocultured with human PBMC for up to 21 days in the presence of 1alpha,25(OH)(2) vitamin D(3), dexamethasone, and recombinant human macrophage-colony stimulating factor (M-CSF). OC formation was monitored by the appearance of cells that were positive for tartrate-resistant acid phosphatase (TRAP) and able to form resorption lacunae on slices of dentine. We found that the ST-2 cells in these cultures express messenger RNA (mRNA) encoding a repertoire of many of the reported osteoclastogenic factors (interleukins [IL]-1/IL-1R1, IL-11, IL-6/IL-6R, and IL-17 transforming growth factor [TGF]-beta), as well as the recently described OC differentiation factor (ODF/TRANCE/RANKL). The stromal cells also expressed mRNA encoding two molecules shown to be inhibitory to osteoclastogenesis, osteoprotegerin (OPG) and IL-18. OPG, IL-1, IL-1R1, IL-6, IL-6R, IL-11R, IL-17, IL-18, IL-18R, TGF-beta, and M-CSF were expressed by both the stromal cells and the PBMC. Expression of mRNA encoding RANK, IL-1R2, and c-fms, was specific for the PBMC. In addition, PBMC were found to express sIL-6R, granulocyte macrophage (GM)-CSF, GM-CSFRalpha, and tumor necrosis factor (TNF)-alpha. Whereas this indicated that human OC formation occurs in a complex environment of many positive and negative influences, we identified three apparent features of the cytokine environment that may be a characteristic of normal osteoclast formation. First, the ratio of mouse ODF:OPG mRNA was found to increase during the cocultures, consistent with a key role for ODF in the promotion by stromal cells of OC formation. Second, we found that mRNA encoding IL-1 and IL-17, as well as IL-6 and sIL-6R, were coordinately expressed by the PBMC. Third, analysis of the culture medium showed that the PBMC secreted IL-1, IL-6, and TNF-alpha protein only in coculture with ST-2 cells during the first few days of osteoclast development. We conclude that human OC formation occurs in a complex environment of many positive and negative influences; however, these are likely to be strictly regulated by a coordinated cytokine response of both stromal and hematopoietic cells.

Bidirectional signaling between stromal and hemopoietic cells regulates interleukin-1 expression during human osteoclast formation.

Interleukin-1 (IL-1) has been shown to promote osteoclast (OC) differentiation, in addition to acting as a survival factor for mature osteoclasts. In this study, we investigate the expression of IL-1 during human osteoclast formation, taking advantage of a recently reported in vitro culture system that generates human OC from precursors in the peripheral blood mononuclear cell (PBMC) fraction, in the presence of murine stromal cells. This system enabled us to use species-specific probes and immunoassays to determine the respective cytokine contributions of the stromal cell and hemopoietic cell populations. Formation of functional osteoclasts occurred in cocultures of human PBMC and ST-2 cells for up to 21 days in the presence of 1alpha,25(OH)2-vitamin D3, dexamethasone, and recombinant human macrophage colony-stimulating factor (rhM-CSF). Total RNA was prepared at intervals during the cocultures and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using primers designed to amplify specifically the mRNA species corresponding to the respective murine or human IL-1alpha and IL-1beta isoforms. Using human-specific primers, it was found that the hemopoietic cell component expressed both IL-1alpha and IL-1beta mRNA. Specific measurement of secreted human IL-1beta protein showed greatly augmented levels in coculture compared with hemopoietic cells grown in the absence of ST-2 cells, consistent with the known signaling from stromal cells to hemopoietic cells during osteoclastogenesis. Specific detection of mouse mRNA products showed that the ST-2 stromal cells in the coculture also expressed mRNA corresponding to IL-1alpha and IL-1beta. The expression of both mouse and human IL-1 mRNA was found to decline over the course of the coculture, although the level of IL-1alpha mRNA relative to IL-1beta mRNA remained constant, indicating that the two isoforms were coregulated in both cell populations under these conditions. Importantly, the hemopoietic cells were found to influence strongly the IL-1 mRNA levels in ST-2 cells, such that mouse IL-1alpha and IL-1beta mRNA levels were greatly enhanced in coculture, compared with ST-2 cells alone. Secreted mouse IL-1beta protein was upregulated in coculture in parallel with mRNA levels. However, the absolute levels of mouse IL-1beta achieved were more than 20-fold lower than the human IL-1beta levels. Prostaglandin estradiol (PGE2) levels were measured and found to be greatly increased in the coculture compared with ST-2 cells or hemopoietic cells alone, consistent with evidence that IL-1 action in osteoclastogenesis is mediated by PGE2. These results provide novel evidence that bidirectional signaling between stromal and hemopoietic cells may be important in the generation of human osteoclasts.

CA125 phosphorylation is associated with its secretion from the WISH human amnion cell line.

Evidence is presented suggesting that CA125 is a serine and/or threonine phosphoprotein and that its secretion from the human amnion WISH cell line is closely linked to its phosphorylation. It is also indicated that regulation of CA125 secretion requires protein(s) tyrosine phosphorylation. WISH cells treated with a tyrosine phosphatase inhibitor, vanadate/ H2O2, resulted in increased levels of CA125 secretion. Exposure of vanadate-treated cells to epidermal growth factor further enhanced this secretory activity. Immunohistochemistry of vanadate-treated cells resulted in a substantial increase in not only cytoplasmic tyrosine phosphoproteins but also in membrane-associated CA125 when stained with the PY20 anti-phosphotyrosine and M11 anti-CA125 monoclonal antibodies, respectively. M11 immunoprecipitation of CA125 from cells labelled with [32P]-orthophosphate was analyzed by SDS-PAGE and autoradiography. Immunoprecipitates from cell lysates demonstrated that a phosphoprotein of > 200 kD was isolated and immunoreacted with both the OC125 and M11 anti-CA125 monoclonal antibodies by Western blotting. CA125 immunoprecipitated from vanadate-treated cells showed a marked increase in cell-associated CA125 phosphorylation. Although CA125 could be immunoprecipitated from WISH cell media incubated with [32P]-orthophosphate in the presence or absence of vanadate as detected by Western blotting, autoradiographic analysis of the Western blots revealed no [32P]-labelled CA125 co-migrating with the 200-kD plus molecule detected by M11. When the PY20 anti-phosphotyrosine monoclonal antibody was used as the probe, no tyrosine-phosphorylated CA125 was detected in cell lysates.

HL-60 myeloid leukaemia cells acquire immunostimulatory capability upon treatment with retinoic acid: analysis of the responding population and mechanism of cytotoxic lymphocyte activation.

HL-60 myeloid leukaemia cells are ineffective as stimulators of allogeneic lymphocytes in mixed leucocyte culture (MLC). These cells can be induced to differentiate along the monocytic or granulocytic pathways with or without acquisition of major histocompatibility complex (MHC) class II antigen by various agents. Surprisingly, treatment of HL-60 cells with 10 nM all-trans retinoic acid (RA) for 7 days (HL-60-R7) resulted in a marked increase in MLC stimulation although the cells lacked detectable MHC class II antigen expression at the initiation of the MLC. In contrast, treatment with interferon-gamma (IFN-gamma), with or without RA, induced MHC class II antigen expression but failed to enhance MLC stimulation. Lymphocytes responding to HL-60-R7 were predominantly CD8+ and/or CD16+ and displayed enhanced cytolytic capacity for HL-60 and HL-60-R7 cells as well as natural killer (NK)-sensitive K562 cells. Nevertheless, monoclonal antibodies (mAb) to MHC class II antigens substantially inhibited the MLC and some CD4+ lymphocytes in the responding population were required, although this requirement could be replaced by the addition of interleukin-2 (IL-2). HL-60-R7 (and HL-60) cells were shown to acquire detectable MHC class II antigen expression during the first 3 days of the MLC. Thus a low level of activation by MHC class II+ stimulator cells appears to be required for the response. Analysis of the role of cytokines with costimulatory activity for T cells and/or NK cells indicated that tumour necrosis factor-alpha (TNF-alpha) was important in the proliferative response, while interleukins-1, -6 and -12 and stem cell factor did not seem to be involved. Cell interaction molecules lymphocyte function-associated antigen-1 (LFA-1) (CD11a), intracellular adhesion molecule-1 (ICAM-1) (CD54), ICAM-3 (CD50) and B7.2 (CD86) were up-regulated on HL-60-R7. Blocking mAb to LFA-1 and B7.2 potently inhibited the proliferative response indicating a key role for these molecules in the enhanced immunostimulation by HL-60-R7 cells. The results may have implications for the mechanism of the therapeutic effect of RA in acute promyelocytic leukaemia and may also provide valuable information in regard to the immunogenicity of tumour cells in general.

Nursing management of cranial nerve dysfunction.

Cranial nerve dysfunction is frequently seen in neuroscience patients. Deficits range in severity and can present many challenges for nurses. Patients are at risk for complications such as worsening vision, corneal damage, improper nutrition, aspiration and respiratory difficulty. With a proper understanding of how to manage these patients, nurses can perform interventions that will minimize complications and assure better outcomes for the patient. Multidisciplinary care is often needed for these patients but it is the nurse who is in a pivotal role to provide appropriate care, coordinate the multidisciplinary needs and offer patient and family education and support.

Intensive care unit psychosis revisited: understanding and managing delirium in the critical care setting.

Delirium occurs frequently in critically ill patients, yet this syndrome is often unrecognized and poorly understood. Health professionals must recognize delirium and patients at risk because delirium can lead to higher morbidity rates and longer lengths of stay. Various disease states and pathophysiologic disorders cause delirium, as do many commonly used drugs. Other factors, such as age and sleep deprivation, contribute or predispose patients to its development. The clinical presentation of delirium is varied, and delirious patients can have increased or decreased psychomotor activity, impaired attentional and memory systems, disorganized thinking, and hallucinations or delusions. Treatment of delirium focuses on finding the cause and managing the symptoms, often with the use of pharmacologic agents. Critical care nurses need to perform cognitive assessments so that deficits can be recognized and specific interventions for prevention of cognitive impairment can be used. Appropriate nursing care can lessen the severity of delirium, shorten its course, and decrease the morbidity associated with the syndrome.