Karen Ferrer

Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo

OBJECTIVE Mesenchymal stem cells (MSCs), multipotential cells that reside within the bone marrow, can be induced to differentiate into various components of the marrow microenvironment, such as bone, adipose, and stromal tissues. The bone marrow microenvironment is vital to the development, differentiation, and regulation of the lymphohematopoietic system. We hypothesized that the activities of MSCs in the bone marrow microenvironment might also include immunomodulatory effects on lymphocytes. METHODS Baboon MSCs were tested in vitro for their ability to elicit a proliferative response from allogeneic lymphocytes, to inhibit an ongoing allogeneic response, and to inhibit a proliferative response to potent T-cell mitogens. In vivo effects were tested by intravenous administration of donor MSCs to MHC-mismatched recipient baboons prior to placement of autologous, donor, and third-party skin grafts. RESULTS MSCs failed to elicit a proliferative response from allogeneic lymphocytes. MSCs added into a mixed lymphocyte reaction, either on day 0 or on day 3, or to mitogen-stimulated lymphocytes, led to a greater than 50% reduction in proliferative activity. This effect could be maximized by escalating the dose of MSCs and could be reduced with the addition of exogenous IL-2. In vivo administration of MSCs led to prolonged skin graft survival when compared to control animals: 11.3 +/- 0.3 vs 7 +/- 0. CONCLUSIONS Baboon MSCs have been observed to alter lymphocyte reactivity to allogeneic target cells and tissues. These immunoregulatory features may prove useful in future applications of tissue regeneration and stem cell engineering.

Mesenchymal stem cells are capable of homing to the bone marrow of non-human primates following systemic infusion

OBJECTIVE The human bone marrow contains mesenchymal stem cells capable of differentiating along multiple mesenchymal cell lineages. Using a non-human primate model, we sought to determine whether the systemic infusion of baboon-derived mesenchymal stem cells was associated with toxicity and whether these cells were capable of homing to and persisting within the bone marrow. MATERIALS AND METHODS Five baboons (Papio anubis) were administered lethal irradiation followed by intravenous autologous hematopoietic progenitor cells combined with either autologous (n = 3) or allogeneic (n = 2) mesenchymal stem cells that had been expanded in culture. In four of these baboons, the mesenchymal stem cells were genetically modified with a retroviral vector encoding either the enhanced green fluorescent protein gene (n = 3) or the human placental alkaline phosphatase gene (n = 1) for tracking purposes. A sixth animal received only intravenous gene marked autologous mesenchymal stem cells but no hematopoietic stem cells or conditioning irradiation. RESULTS Following culture, baboon mesenchymal stem cells appeared morphologically as a homogeneous population of spindle-shaped cells that were identified by the monoclonal antibodies SH-3 and SH-4. These cells did not express the hematopoietic markers CD34 or CD45. Baboon mesenchymal stem cells isolated from primary culture were capable of differentiating along both adipogenic and osteogenic lineages. There was no acute or chronic toxicity associated with the intravenous infusion of mesenchymal stem cells. In all five recipients of gene marked mesenchymal stem cells, transgene was detected in post-transplant bone marrow biopsies. In two animals receiving autologous mesenchymal stem cells, including the one non-conditioned recipient, transgene could be detected over 1 year following infusion. In one recipient of allogeneic gene marked mesenchymal stem cells, transgene was detected in the bone marrow at 76 days following infusion. CONCLUSION These data demonstrate that baboon mesenchymal stem cells: 1) are not associated with significant toxicity when administered intravenously, 2) are capable of homing to the bone marrow following intravenous infusion, and 3) have the capacity to establish residence within the bone marrow for an extended duration following systemic administration.

Baboon Mesenchymal Stem Cells Can Be Genetically Modified to Secrete Human Erythropoietin In Vivo

Human mesenchymal stem cells (MSCs) are capable of differentiating into multiple mesenchymal lineages including chondrocytes, osteocytes, adipocytes, and marrow stromal cells. Using a nonhuman primate model, we evaluated nonhuman primate MSCs as targets for gene therapy. Baboon MSCs (bMSCs) cultured from bone marrow aspirates appeared as a homogeneous population of spindle-shaped cells. bMSCs were capable of differentiating into adipocytes and osteocytes in vitro and chondrocytes in vivo. bMSCs were genetically modified with a bicistronic vector encoding the human erythropoietin (hEPO) gene and the green fluorescent protein (GFP) gene. Transduction efficiencies ranged from 72 to 99% after incubation of MSCs with retroviral supernatant. Transduced cells produced from 1.83 x 10(5) to 7.12 x 10(5) mIU of hEPO per 10(6) cells per 24 hr in vitro before implantation. To determine the capacity of bMSCs to express hEPO in vivo, transduced bMSCs were injected intramuscularly in NOD/SCID mice. In a separate experiment, transduced bMSCs were loaded into immunoisolatory devices (IIDs) and surgically implanted into either autologous or allogeneic baboon recipients. Human EPO was detected in the serum of NOD/SCID mice for up to 28 days and in the serum of five baboons for between 9 and 137 days. NOD/SCID mice experienced sharp rises in hematocrit after intramuscular injection of hEPO-transduced bMSCs. The baboon that expressed hEPO for 137 days experienced a statistically significant (p < 0.04) rise in its hematocrit. These data demonstrate that nonhuman primate MSCs can be engineered to deliver a secreted and biologically active gene product. Therefore, human MSCs may be an effective target for future human gene therapy trials.

Well‐differentiated thyroid carcinomas: p53 mutation status and microvessel density

Risk‐stratification schemes exist for well‐differentiated thyroid carcinoma and include prognostic factors such as age, sex, extent of tumor, size of tumor, and presence of metastasis. Controversy continues, however, over the aggressiveness of initial surgical intervention because of anecdotal experiences of poor clinical outcomes in low‐risk patients. Our objective is to determine the prognostic significance of two biologic tumor markers, the p53 gene mutation and CD34 microvessel density (MVD) count, in well‐differentiated tumors of thyroid gland.

Histologic characteristics of laparoscopic argon beam coagulation.

STUDY OBJECTIVES To describe histologic effects of laparoscopic argon beam coagulation and determine the extent of tissue necrosis at various power settings and exposure times. DESIGN Prospective experimental analysis (Canadian Task Force classification II-1). SETTING University animal laboratory. Subjects. Adult female domestic pigs. INTERVENTIONS Various power settings (40, 60, 80 W) at increasing exposure times (1, 3, 5 sec) were used during laparoscopic application of argon beam coagulation to different tissues (uterine horn, bladder, ureter, kidney, bowel, liver). Animals were sacrificed within 1 hour of coagulation for histologic tissue preparation. MEASUREMENTS AND MAIN RESULTS Histologic measurements of both depth and lateral extent of electrosurgical tissue effects (mm +/- SD) were ascertained and evaluated statistically by one-way repeated measures analysis of variance. Depth of tissue necrosis was confined to 1 mm or less in uterine horn, bladder, and ureter. Even at highest power settings, bowel had tissue necrosis no greater than 2 mm. Both liver and kidney showed a deeper histologic effect (4-5 mm). The lateral extent of tissue necrosis ranged from 2 mm (ureter) to 15 mm (liver). CONCLUSION Laparoscopic argon beam coagulation results in tissue effects that are dependent on both low power setting and duration of application, as well as on electrical and physical characteristics of target tissue. Thermal tissue penetration can be expected to be less than 2 mm in bowel, bladder, and ureter, and less than 5 mm in kidney and liver, even at 5 seconds of exposure time and at a power setting as high as 80 W. As with all thermal modalities used for hemostasis and tissue coagulation, laparoscopic argon beam coagulation can be performed safely as long as the potential for inadvertent thermal injury is understood.

Abstract 859: Race-based survival and prognosis among lower SES women with ER/PR negative breast cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Heterogeneity exists in survival and prognosis among women of different race with ER/PR negative (-) breast cancer. Minority women often have lower SES, which may be a confounding factor. We evaluated survival and prognosis in a cohort of lower SES non-Hispanic White (NHW), African-American (AA), and Hispanic (HIS) women, in an attempt to disaggregate the effects of race and SES in ER/PR- breast cancer. Methods: Chi-square test was used to examine relationship significance [odds ratios (OR), 95% confidence intervals (CIs)]; survival function estimates were generated using Kaplan-Meier (KM) method and compared using log-rank test; proportional hazards regression models [hazard ratios (HR), 95% CIs] were used to select and evaluate factors prognostic for all-cause mortality, in 213 consecutive [30 NHW, 135 AA & 48 HIS] women treated at an urban hospital [44 months median follow-up] with ER/PR- disease. Results: HIS women were younger than NHW [OR, 0.36; 95% CI, 0.14-0.94; p =0.0368] and AA [OR, 0.50; 95% CI, 0.26-0.96; p =0386]. Compared to NHW and HIS, AA women had more comorbid disease [ORs: 3.53; 95% CI, 1.43-8.66; p =0.0053; & 2.12; 95% CI, 1.04-4.33; p =0.0392], and worse poverty status level (PL) [ORs: 5.43; 95% CI, 2.17-13.69; p =0.0001; & 2.79; 95% CI 1.17-6.65; p =0.0192]. No significant differences were noted between groups for stage at diagnosis, grade, p53 or HER2 status, and chemotherapy use. Baseline prognostic factors were: age [HR, 0.99/yr; 95% CI, 0.98-1.02; p =0.822]; stage [(II-IV/I) HR, 2.45; 95% CI, 1.85-3.24; p /≤ census mean) HR, 2.43; 95% CI, 1.12-5.28; p =0.025]; and chemotherapy [(+/-) HR, 0.51; 95% CI, 0.29-0.89; p =0.017]. Race was not associated with greater hazard mortality [(Other/AA) HR, 0.83; 95% CI, 0.60-1.14; p =0.255], and unadjusted 5-yr survival for NHW, AA and HIS women was 60.9%, 52.4%, and 64.4%. 5-yr survival by race was also not different for women aged <50 yrs (p =0.3287) or ≥50 yrs (p =0.6217). Multivariable models indicated that only stage [HR, 2.45; 95% CI, 1.61-3.74; p <0.001] and chemotherapy [HR, 0.31; 95% CI, 0.10-0.95; p =0.041] remained significant prognostic factors when considered together with the other above-mentioned factors. Further, models for Triple Negative [i.e. ER-, PR-, & HER2- (TN)] phenotype (without HER2 covariate) showed similar results: stage [HR, 2.32; 95% CI, 1.45-3.70; p <0.001]; chemotherapy [HR, 0.23; 95% CI, 0.07-0.75; p =0.015]. Conclusion: Survival is not significantly different among lower SES women with ER/PR- breast cancer of different race. Stage and chemotherapy use, but not race, remained independent prognostic factors in Cox models for ER/PR- and TN disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 859.