Faculty Bibliography

Dendritic cells (DC) have vast potential for immunotherapy. Transferring therapeutic genes to DC may enhance their inherent T cell-stimulatory capacity. Recombinant adenovirus is the most efficient vehicle for DC gene transfer and can alone mature DC. We sought to define the parameters of adenovirus infection of murine bone marrow-derived DC (BMDC) and the concomitant impact on BMDC maturation. The efficiency of adenoviral gene transfer to DC depended on the mouse strain, the organ source of DC, and the level of DC maturation. C57BL/6 BMDC consistently had higher transgene expression than BALB/c DC. While BMDC had considerable GFP expression after AdGFP infection, adenovirus was relatively ineffective in accomplishing transgene expression in freshly isolated hepatic or splenic DC. BMDC that were relatively immature because of a shorter duration of culture had higher transgene expression after infection. Nevertheless, pretreatment of DC with exogenous stimulants such as LPS or TNF-alpha resulted in higher transgene expression. Maturation of BMDC depended only on virus entry but not viral gene or transgene expression. Therefore, DC maturation was disproportionately high compared to the percentage of DC that actually expressed the adenoviral transgene. Maturation by adenovirus was only seen in BMDC, but not in liver or splenic DC, and was more pronounced in DC from later in culture (day 12 versus day 6). There was a dose-response relationship, up to a threshold dose, between adenovirus infection and both DC maturation and enhancement of DC activation of antigen-specific T cells. Our findings underscore the importance of optimizing gene transfer to DC in designing strategies for immunotherapy

Human Flt3 ligand can expand dendritic cells (DC) and enhance immunogenicity in mice. However, little is known about the effects of murine Flt3 ligand (mFlt3L) on mouse DC development and function. We constructed a vector to transiently overexpress mFlt3L in mice. After a single treatment, up to 44% of splenocytes became CD11c(+) and the total number of DC increased 100-fold. DC expansion effects lasted for >35 days. mFlt3L DC were both phenotypically and functionally distinct. They had increased expression of MHC and costimulatory molecules and expressed elevated levels of B220 and DEC205 but had minimal CD4 staining. mFlt3L DC also had a markedly altered cytokine profile, including lowered secretion of IL-6, IL-10, IFN-gamma, and TNF-alpha, but had a slightly increased capacity to stimulate T cells in vitro. However, in a variety of in vivo models, DC expanded by mFlt3L induced tolerogenic effects on T cells. Adoptive transfer of Ag-pulsed mFlt3L splenic DC to naive mice actually caused faster rates of tumor growth and induced minimal CTL compared with control DC. mFlt3L also failed to protect against tumors in which human Flt3 ligand was protective, but depletion of CD4(+) T cells restored tumor protection. Our findings 1) demonstrate that mFlt3L has distinct effects on DC development, 2) suggest an important role for mFlt3L in generating DC that have tolerogenic effects on T cells, and 3) may have application in immunotherapy in generating massive numbers of DC for an extended duration

Dendritic cells (DC) are initiators of T cell-mediated immunity. However, less is known about the relationship between DC and natural killer (NK) cells, and direct evidence of their interaction in vivo is scarce. Interleukin (IL)-12 is an activator of both DC and NK cells. We postulated that secretion of IL-12 by DC would enable them to activate NK cells. Bone marrow-derived DC propagated only in granulocyte macrophage colony-stimulating factor did not activate NK cells. In contrast, DC engineered to express IL-12 markedly stimulated NK cells as determined by coculture experiments in vitro, assays of NK cells isolated from treated animals, and survival experiments in a systemic tumor model. Activation depended on both DC-NK cellular interaction and secretion of IL-12. Adoptive transfer of DC expressing IL-12 to mice markedly increased NK cell interferon-gamma production and lytic activity in vivo. Treated mice were also protected against B16 melanoma hepatic metastases. The in vivo effects on NK cells were DC-specific. Administration of IL-12 protein alone or melanoma cells or fibroblasts engineered to secrete IL-12 were only weakly activating. Our findings demonstrate that IL-12 expression by DC enables them to activate NK cells and provide evidence for a substantial DC-NK relationship in vivo

Dendritic cells initiate the immune response by presenting antigen in the context of varying levels of costimulation. The maturation state of the dendritic cell determines the quantity and quality (Th1, Th2) of the subsequent T cell response. Members of the NF-kappaB family of transcription factors have previously been implicated in dendritic cell development. Here, we used a mouse with a homozygous c-Rel deletion to investigate the role of c-Rel in the function of bone marrow derived dendritic cells. When direct presentation was evaluated, we found c-Rel(-/-) dendritic cells induce less allogeneic T cell stimulation than c-Rel(+/+) dendritic cells. In addition, T cell encounters with c-Rel(-/-) dendritic cells generate less IFN-gamma and IL-4 when compared to those with c-Rel(+/+) DCs. A similar degree of functional compromise was observed in antigen-specific T cells that were stimulated by c-Rel(-/-) dendritic cells. Functional deficits were not linked to differences in the ability to undergo maturation per se, as LPS exposure induced similar morphologic and cell surface changes in both c-Rel(+/+) and cRel(-/-) DCs. Although LPS induced a compensatory increase in the nuclear activity of fellow NF-kappaB family members, RelB and p65, LPS exposure was unable to negate the deficiencies in autologous T cell proliferation and cytokine production associated with the loss of c-Rel in dendritic cells. Taken together, our study supports a unique and non-redundant role for c-Rel in dendritic cell costimulatory capacity

Dendritic cells (DCs) are rare but ubiquitous antigen-presenting cells situated in lymphoid and nonlymphoid organs throughout the body. The study of DCs located in the liver has been restricted by their relative scarcity and the difficulty of their isolation. Because granulocyte-macrophage colony-stimulating factor (GM-CSF) is a critical growth factor for DCs in vitro, we postulated that it would expand hepatic DCs in vivo. We found that adenoviral-mediated GM-CSF overexpression in normal mice increased the number of liver DCs 400-fold to more than 100 million cells. GM-CSF-recruited DCs were CD11c(+)DEC205(-) and had high expression of major histocompatibility complex (MHC) class II, CD54, and CD80 but low CD40 and CD86 staining. Further maturation occurred after overnight culture. In addition to CD11c(+)DEC205(-) DCs, a population of CD11c(-)DEC205(low/-) cells resembling DC progenitors described previously in normal mice was expanded as serum GM-CSF levels increased. GM-CSF-recruited CD11c(+)DEC205(-) DCs and CD11c(-)DEC205(low/-) cells had different functional capabilities. CD11c(+)DEC205(-) DCs captured far more protein antigen in vivo, produced higher amounts of interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha, and induced greater allogeneic and antigen-specific T-cell stimulation. A proportion of CD11c(-)DEC205(low/-) cells differentiated into CD11c(+) cells and gained T-cell stimulatory ability when cultured in the presence of GM-CSF. In conclusion, our findings show that GM-CSF can profoundly influence recruitment and development of DCs in murine liver

GM-CSF is critical for dendritic cell (DC) survival and differentiation in vitro. To study its effect on DC development and function in vivo, we used a gene transfer vector to transiently overexpress GM-CSF in mice. We found that up to 24% of splenocytes became CD11c+ and the number of DC increased up to 260-fold to 3 x 10(8) cells. DC numbers remained substantially elevated even 75 days after treatment. The DC population was either CD8alpha+CD4- or CD8alpha-CD4- but not CD8alpha+CD4+ or CD8alpha-CD4+. This differs substantially from subsets recruited in normal or Flt3 ligand-treated mice or using GM-CSF protein injections. GM-CSF-recruited DC secreted extremely high levels of TNF-alpha compared with minimal amounts in DC from normal or Flt3 ligand-treated mice. Recruited DC also produced elevated levels of IL-6 but almost no IFN-gamma. GM-CSF DC had robust immune function compared with controls. They had an increased rate of Ag capture and caused greater allogeneic and Ag-specific T cell stimulation. Furthermore, GM-CSF-recruited DC increased NK cell lytic activity after coculture. The enhanced T cell and NK cell immunostimulation by GM-CSF DC was in part dependent on their secretion of TNF-alpha. Our findings show that GM-CSF can have an important role in DC development and recruitment in vivo and has potential application to immunotherapy in recruiting massive numbers of DC with enhanced ability to activate effector cells

Dendritic cells (DCs) are potent antigen-presenting cells with the potential for cancer immunotherapy. Adenoviral-mediated gene transfer is an attractive means to manipulate the immunostimulatory properties of DCs for therapeutic advantage. Because adenovirus induces DC maturation, we postulated that it would significantly alter their immune functions. Infected DCs markedly increased allogeneic and antigen-specific T-cell proliferation, and augmented natural killer cell lytic activity and IFN-gamma production. The enhanced effector cell stimulation by infected DCs was dependent on their secretion of interleukin 12. Immunization with infected DCs pulsed with tumor antigen protected against flank tumors in 78% of mice and induced a memory response. Antitumor immunity was dependent on both T cells and natural killer cells. Antigen-pulsed, mock-infected DCs were nonprotective. The findings that adenoviral vectors alone critically alter DC immune functions and antitumor properties have important implications for the design and interpretation of immunotherapy regimens using vector-based gene transfer to modulate immunity

Small bowel obstruction (SBO) is a particularly vexing problem in the postoperative period. The goal of this study was to compare the results of operative versus nonoperative treatment. A secondary goal was to explore risk factors for necessitating reoperation in the immediate postoperative period. We conducted a historical cohort retrospective chart review at a university-affiliated hospital. The medical records of patients treated between 1985 and 2000 at the Sir Mortimer B. Davis Jewish General Hospital (Montreal, Quebec, Canada) who developed SBO after undergoing a laparotomy during that admission were reviewed. Postoperative SBO was defined as cessation of flatus or bowel movements after their resumption following operation. To compare operative versus nonoperative management of early postoperative mechanical SBO we used the following outcome measures: Reoperation rate, time to return of function, length of stay, and mortality. Of 52 patients who developed SBO in the immediate postoperative period 37 were male, 25 had colorectal surgery, and nine had a gastrectomy as the initial operation on admission; five had inflammatory bowel disease, six had a previous SBO, 22 had virgin abdomens before the current operation, and 11 had adhesions noted at the initial operation. The median time to the development of obstructive symptoms was 8 days (range 1-33). The reoperation rate was 42 per cent overall (67% in women and 32% in men, P = 0.02). For operatively treated patients the median time between onset of symptoms and surgery was 5 days [range 1-23, interquartile range (IQR) = 5]. The median time to the return of bowel function was greater in the operatively treated patients compared with nonoperatively treated patients [11.5 days (range 4-37, IQR = 11) vs 6 days (range 1-28, IQR = 7), P < 0.0001] as was median length of stay from onset of obstruction [23 days (range 6-60, IQR = 14) vs 12 days (range 2-45, IQR = 16), P < 0.009]. Operatively treated patients also stayed longer after their obstruction was relieved although not significantly longer [8 days (range 1-35, IQR = 11) vs 4.5 days (range 0-40, IQR = 10), P = 0.15]. There were 11 complications in nine of 22 patients who underwent operative treatment of their SBO. Immediate postoperative SBO can be treated nonoperatively in stable patients resulting in significantly quicker return of bowel function and shorter lengths of hospital stay. Definitive risk factors for immediate SBO could not be identified

OBJECTIVES: To determine the frequency of readmission for early postoperative small-bowel obstruction (SBO), to highlight factors that may predispose to this condition, to define the risks of strangulation and to compare the immediate and long-term risks and benefits of operative versus nonoperative treatment. DESIGN: A chart review. SETTING: The Sir Mortimer B. Davis-Jewish General Hospital, a university-affiliated teaching hospital in Montreal. PATIENTS: Out of a total of 1001 cases of SBO in 552 patients, 30 patients were readmitted within 50 days of a previous laparotomy with the diagnosis of SBO. INTERVENTION: Selective nonoperative management and exploratory laparotomy. MAIN OUTCOME MEASURES: The value of nonoperative management and need for operation. RESULTS: Adhesions were the cause of the obstruction in most cases (24); other causes were Crohn's disease (2), hernia (1), malignant neoplasm (1) and a combination of adhesions and malignant disease (2). Thirteen (43%) of the procedures preceding the obstruction were primary small-bowel operations. There was only 1 episode of strangulated bowel. Of the patients readmitted for SBO, 7 (23%) were treated operatively with a long-term recurrence rate of 57% compared with 63% for those treated nonoperatively for the SBO. The median time to recurrence was 0.1 years (range from 0.02-6 yr) for those whose SBO was managed operatively, compared with 0.7 years (range from 0.08-5 yr) for those managed nonoperatively for the SBO. The median length of stay for patients managed operatively for SBO was 12 days (range from 9-17 d) compared with 6 days (range from 2-33 d) for those managed nonoperatively. CONCLUSIONS: Readmission for SBO within 50 days of a previous laparotomy represents a small percentage of all cases of SBO. They frequently follow small-bowel operations. Cases of strangulation are no more common than in general cases of SBO. Patients treated nonoperatively for SBO did not experience less favourable outcomes with respect to resolution of symptoms, length of stay, risk of recurrence and reoperation. Thus, operative intervention is not necessary in an otherwise stable patient