Characterization of an in vitro model system to explore control of tumor invasion of EMT6 and 4THM breast tumors by CD200:CD200R interactions.

BACKGROUND AND PURPOSE: In BALB/c mice with transplantable breast tumors, we showed that CD200R1KO mice were cured of local and metastatic growth of EMT6 cells following surgical resection of localized tumor and immunization with irradiated cells along with CpG as adjuvant. On the other hand, wild-type (WT) animals treated in the same fashion develop pulmonary and liver metastases within 20 days of surgery. To develop an in vitro system which would mimic the in vivo model and allow exploration of factors controlling tumor invasion as a precursor to in vivo metastasis, we have developed and characterized a two-phase culture system. METHODS: Bone marrow mesenchymal stromal cells (BMMSCs) from WT, CD200KO or CD200R1KO mice were admixed with T lymphocytes from tumor-immunized mice and cultured in collagen gels. Tumor cells were subsequently seeded in fresh medium above this gel 1d later. We then investigated the regulation of tumor invasion from the liquid to the gel layer. Tumor cells were measured in the gel layer following collagenase digestion and cultured at limiting dilution-an aliquot of the digest was also analyzed for cytokine levels in ELISA. RESULTS: BMMSCs from WT, CD200KO and CD200R1KO mice all augmented seeding/growth of EMT6 and 4THM tumor cells into the collagen matrix. Inclusion of IL-6 and IL-17 in the gel matrix was associated with increased invasion of tumor cells into this layer. Inclusion of DLN cells from EMT6 immune or 4THM immune mice further modified tumor invasion, with increased tumor numbers seen using stromal elements from CD200 and CD200R1KO mice and DLN from 4THM immune, while CD200R1KO-derived DLN of EMT6 immune mice attenuated tumor invasion, despite inclusion of IL-6/IL-17 in the gel layer. CONCLUSION: Multiple factors can regulate tumor invasion, including micro-environmental stromal elements, IL-6/IL-17, and signals from tumor-derived DLN cells.

Importance of B cells to development of regulatory T cells and prolongation of tissue allograft survival in recipients receiving autologous bone marrow transplantation.

We previously showed that congenic bone marrow transplantation (BMTx) post myeloablation augmented tissue allograft survival in association with increased regulatory T (Treg) cells of both host and bone marrow donor origin. Regulatory B (Breg) cells can also modulate T-cell immunity and B cells may be implicated in the development of Treg cells. Accordingly, we explored the effect of B-cell depletion in vivo on augmented graft survival post BMTx. C57BL/6 mice received BALB/c skin allografts followed 7 days later by myeloablation using cyclophosphamide and busulphan. Mice then received T-cell-depleted bone marrow from CD45.1 congenic donors, and ongoing immunosuppression with rapamycin (to day 28 after BMTx). Control mice received cyclophosphamide and busulphan followed by rapamycin, but not congenic bone marrow. At different times post BMTx, mice received B-cell-depleting antibody treatment, and the effect on both skin graft survival, and induction of Treg cells was assessed. BMTx resulted in significantly prolonged skin graft survival versus control mice, in association with attenuated donor-specific alloreactivity relative to controls, increased splenic Treg cells and significantly diminished anti-donor IgG. In mice receiving infusion of B-depleting antibodies for 12 days from day 15 post BMTx, both graft survival and Treg cell activity were diminished, particularly for functional Treg cells of donor origin. Adoptive transfer of Breg cells from mice harvested at 15 days post BMTx prolonged survival in naive transplanted mice and increased Treg cell levels. Thus, autologous BMTx augmentation of graft survival is dependent in part upon a population of Breg cells that can modulate the function of donor-derived Treg cells.

Assessment of arthroscopic training in U.S. orthopedic surgery residency programs--a resident self-assessment.

BACKGROUND: There has been an increasing number of arthroscopic surgeries performed in general orthopedic surgery practice, as well as a rapid evolution of arthroscopic techniques. The objective of this investigation was to assess the adequacy of arthroscopic training in U.S. orthopedic residency programs from a resident and program director perspective. MATERIALS AND METHODS: The study was performed with a mail-in survey to orthopaedic surgery residents and program directors. Out of 151 programs contacted, we received responses from 24 program directors (15.9%) and 272 residents (11.1% of 2447 possible residents in years 2 through 5 in 2006). Program demographics and resident and program director assessments of arthroscopic surgical training was obtained from the questionnaire. Assessment of open surgical techniques was used as a control. The responses from fifth-year residents (83 of a possible 612 in 2006 (13.6%)) and program directors were used for detailed analysis. RESULTS: Only 32% (27/83) of fifth-year residents felt there was adequate time dedicated to arthroscopic training, compared to 66% (16/24) of program directors (p < 0.01). Thirty-four percent (28/83) of fifth-year residents felt as prepared in arthroscopy as open techniques, in contrast to 58% (14/24) of program directors, who felt fifth-year residents were appropriately prepared in arthroscopic techniques (p = 0.03). The amount of surgery that residents are allowed to perform correlated significantly (p < 0.01) with confidence levels. CONCLUSIONS: Fifth-year residents who were surveyed felt less prepared in arthroscopic training, compared to open surgical procedures. Program directors surveyed over estimated confidence levels in fifth-year residents performing arthroscopic procedures. To ensure that graduating residents are appropriately prepared for the current demands of a clinical setting, it may be necessary to reexamine residency requirements to ensure adequate practice in developing arthroscopic surgical skills.

Neutral buoyancy and sleep-deprived serum factors alter expression of cytokines regulating osteogenesis.

We examined expression of genes associated with cytokine production, and genes implicated in regulating bone metabolism, in bone stromal and osteoblast cells incubated under standard ground conditions and under conditions of neutral buoyancy, and in the presence/absence of serum from normal or sleep-deprived mice. We observed a clear interaction between these two conditions (exposure to neutral buoyancy and serum stimulation) in promoting enhanced osteoclastogenesis. Both conditions independently altered expression of a number of cytokines implicated in the regulation of bone metabolism. However, using stromal cells from IL-1 and TNFx cytokine(r) KO mice, we concluded that the increased bone loss under microgravity conditions was not primarily cytokine mediated.