Although interactions with bone marrow stromal cells are essential for multiple myeloma (MM) cell survival, the specific molecular and cellular elements involved are largely unknown, due in large part to the complexity of the bone marrow microenvironment itself. The T-cell costimulatory receptor CD28 is also expressed on normal and malignant plasma cells, and CD28 expression in MM correlates significantly with poor prognosis and disease progression. In contrast to T cells, activation and function of CD28 in myeloma cells is largely undefined. We have found that direct activation of myeloma cell CD28 by anti-CD28 mAb alone induces activation of PI3K and NFB, suppresses MM cell proliferation, and protects against serum starvation and dexamethasone (dex)-induced cell death. Coculture with dendritic cells (DCs) expressing the CD28 ligands CD80 and CD86 also elicits CD28-mediated effects on MM survival and proliferation, and DCs appear to preferentially localize within myeloma infiltrates in primary patient samples. Our findings suggest a previously undescribed myeloma/DC cell-cell interaction involving CD28 that may play an important role in myeloma cell survival within the bone marrow stroma. These data also point to CD28 as a potential therapeutic target in the treatment of MM. IntroductionMultiple myeloma (MM) remains an incurable clonal B lymphoid neoplasm of plasma cells, second only to non-Hodgkin lymphoma in incidence. 1 Despite significant initial responses to chemotherapy, more than 90% of patients with MM relapse with resistant disease, 2 underscoring the need to identify novel therapeutic targets that affect myeloma survival and resistance pathways. Given that MM cells are critically dependent on normal elements of the bone marrow stroma for cell growth and survival, these interactions are attractive targets. One such interaction is stromal production of soluble growth factors, such as IL-6 and TRANCE. 1,3 Another important set of interactions involves direct myeloma cell contact with extracellular matrix (ECM) and/or stromal cells. Such direct contact up-regulates stromal cell IL-6 and VEGF production, induces NFB signaling, drops myeloma cells out of cell cycle, and enhances resistance to chemotherapy. [4][5][6] However, the specific molecular (eg, integrins 4,7 ) and cellular components (eg, osteoclasts 8 ) of these direct interactions within the complex bone marrow microenvironment are only beginning to be described. As important, the characteristic progression of myeloma to stromal independence marks a clinically worse disease, 9 yet the mechanisms that underlie this transition are also poorly understood.Identification of prosurvival receptors typically expressed on myeloma cells may point to the stromal cells expressing the receptor ligands. One potential receptor is CD28. CD28 has a restricted lineage expression, found predominantly on T cells but also on normal plasma cells, primary myeloma isolates, and myeloma cell lines at levels comparable with T cells. [10][11][12][13] In T cells, CD28 recep...

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The E2A-encoded transcription factor E47 is crucial to B lymphopoiesis. Senescent BALB/c mice (∼2 years old) had reduced pre-B cells ex vivo. Pro-B/early pre-B cells from these aged mice, both ex vivo and in vitro, were deficient in E47 protein. In vitro, IL-7 expanded pro-B/early pre-B cells from young BALB/c mice expressed E47 protein that was relatively stable over a 5-h period. Cultured senescent pro-B/early pre-B cells exhibited reduced E47 protein stability with ∼50–90% loss of E47 over the same time period. Degradation of E47 was effectively blocked by the proteasome inhibitor lactacystin as well as calpain I and II inhibitors; E2A proteins were also shown to undergo ubiquitination. Although senescent B cell precursors expressed less E47 protein, E47 mRNA levels and turnover were normal. Therefore, E47 protein levels are reduced relatively early in B lineage differentiation in senescence and the decline in E47 protein occurs via increased protein degradation by proteasome and, possibly, calpain pathways. In contrast, normal E47 protein levels were observed within the highly reduced pre-B cell pool in aged mice. This suggests that pre-B cells in senescence undergo selection based on E47 expression. Increased degradation rates and lower steady-state levels were also observed for the transcription factors Pax-5/BSAP, Bob-1, and Ikaros, but this was not a general property of all proteins in aged B cell precursors. Therefore, altered turnover of multiple, select proteins crucial to B cell development may contribute to diminished B lymphopoiesis in old age.

Excessive mucus production by airway epithelium is a major characteristic of a number of respiratory diseases, including asthma, chronic bronchitis, and cystic fibrosis. However, the signal transduction pathways leading to mucus production are poorly understood. Here we examined the potential role of IB kinase ␤ The airway epithelium is the first layer of cellular interaction with airborne antigens and plays a key role in initiating allergic responses. Research over the last decade has established a critical role for the epithelium not only by providing a physical boundary but also in transducing and integrating signals that help mount optimal responses to a wide variety of insults (1-3). Airway mucus is a highly hydrated glycoprotein exhibiting a gel state and, in combination with the ciliated cells in the airways, constitutes a mucociliary clearance system critical for protection of the respiratory tract (4). The apoprotein component of mucus is the product of a family of mucin genes (generally referred to as MUC

Introduction The aim of the Rural Older Adult Memory pilot study (ROAM) was to evaluate the feasibility of screening and diagnosing dementia in patients aged 75 years or older in six rural primary care practices in a practice—based research network. Methods Clinicians and medical assistants were trained in dementia screening using the ROAM protocol, via distance learning methods. Medical assistants screened patients aged 75 and older. For patients who screened positive, the clinician was alerted to the need for a dementia work-up. Outcomes included change in the proportion of patients screened and diagnosed with dementia or mild cognitive impairment and clinician confidence in diagnosing and managing dementia. Patient, medical assistant, and clinician response to the intervention were also evaluated. Results Results included a substantial increase in screening for dementia, a modest increase in the proportion of cases diagnosed with dementia or mild cognitive impairment, and improved clinician confidence in diagnosing dementia. Although clinicians and medical assistants found the ROAM protocol easy to implement, there was substantial variability in adherence to the protocol in the six practices. Conclusion This study demonstrated the complex issues that must be addressed in implementing a dementia screening process in rural primary care. Further study is needed to develop effective strategies for overcoming the factors that impeded the full uptake of the protocol, including the logistical challenges in implementing practice change and clinician attitudes towards dementia screening and diagnosis.

Proapoptotic Bcl-2 family members alter mitochondrial permeability resulting in the release of apoptogenic factors that initiate a caspase cascade. These changes are well described; however, the effects of caspases on mitochondrial function are less well characterized. Here we describe the consequence of caspase-9 and effector caspase inhibition on mitochondrial physiology during intrinsic cell death. Caspase inhibition prevents the complete loss of mitochondrial membrane potential without affecting cytochrome c release. When effector caspases are inhibited, mitochondria become uncoupled and produce reactive oxygen species. Interestingly, the effector caspase-mediated depolarization of the mitochondria occurs independent of the activity of complexes I-IV of the electron transport chain. In contrast, caspase-9 inhibition prevents mitochondrial uncoupling and ROS production and allows for continued electron transport despite the release of cytochrome c. Taken together, these data suggest that activated caspase-9 prevents the accessibility of cytochrome c to complex III, resulting in the production of reactive oxygen species, and that effector caspases may depolarize mitochondria to terminate ROS production and preserve an apoptotic phenotype.

The transcriptional regulator E47, encoded by the E2A gene, is crucial to B lymphopoiesis. In BALB/c senescent mice (∼2 years old), the incidence of E47-expressing pro-B cells in vivo and E47 protein steady state levels in B cell precursors in vitro were reduced. Poor expression of E47 protein was a consequence of accelerated proteasome-mediated turnover and was associated with heightened ubiquitin modification of E2A-encoded proteins in aged B cell precursors. Both MAPK and Notch activity have been previously associated with E2A-encoded protein stability in lymphocytes. Aged B cell precursors exhibited heightened levels of MAPK activity reflected in increased levels of phospho-ERK proteins. Phosphorylation of E2A-encoded proteins was also increased in aged B cell precursors and pharmacologic inhibition of MEK-1 resulted in a partial restoration of their E47 protein. Both Notch proteins and their Delta-like ligands were detected comparably in young and aged B cell precursors. Either inhibition of Notch activation via gamma-secretase or Ab blockade of Notch-Delta-like ligand interactions partially restored E47 expression in aged B cell precursors. We hypothesize that increased MAPK activity promotes phosphorylation of E2A-encoded protein in aged B cell precursors. Subsequently, E2A-encoded proteins undergo ubiquitination and accelerated degradation in a Notch-dependent process. The dysregulation of E2A-encoded protein expression may contribute to the reductions seen in early B lymphopoiesis during murine senescence.

Objective: To present the survey methods and instruments used to characterize a geographically and professionally diverse rural practice-based research network (PBRN).Methods: A cross-sectional study of Oregon PBRN (ORPRN) member practices and clinicians using a 3-part survey including a survey of the practices, of clinician members, and an anonymous survey of clinician satisfaction.Results: A total of 31 of 32 (97%) participating ORPRN practices completed the practice survey, 96 of 129 (74%) clinicians within these practices completed the clinician member survey, and 81 of 129 (63%) clinicians completed an anonymous survey of clinician satisfaction.The survey provided a detailed description of the structure of member practices, patient and clinician demographics, services provided by the practices, and access to specialty and ancillary services.