Anti‐LFA‐1 or rapamycin overcome costimulation blockade‐resistant rejection in sensitized bone marrow recipients

While costimulation blockade‐based mixed chimerism protocols work well for inducing tolerance in rodents, translation to preclinical large animal/nonhuman primate models has been less successful. One recognized cause for these difficulties is the high frequency of alloreactive memory T cells (Tmem) found in the (pre)clinical setting as opposed to laboratory mice. In the present study, we therefore developed a murine bone marrow transplantation (BMT) model employing recipients harboring polyclonal donor‐reactive Tmem without concomitant humoral sensitization. This model was then used to identify strategies to overcome this additional immune barrier. We found that B6 recipients that were enriched with 3 × 10⁷ T cells isolated from B6 mice that had been previously grafted with Balb/c skin, rejected Balb/c BM despite costimulation blockade with anti‐CD40L and CTLA4Ig (while recipients not enriched developed chimerism). Adjunctive short‐term treatment of sensitized BMT recipients with rapamycin or anti‐LFA‐1 mAb was demonstrated to be effective in controlling Tmem in this model, leading to long‐term mixed chimerism and donor‐specific tolerance. Thus, rapamycin and anti‐LFA‐1 mAb are effective in overcoming the potent barrier that donor‐reactive Tmem pose to the induction of mixed chimerism and tolerance despite costimulation blockade.     

Results of a phase I/II trial of recombinant human granulocyte- macrophage colony-stimulating factor in very low birthweight neonates: significant induction of circulatory neutrophils, monocytes, platelets, and bone marrow neutrophils

Neonates, especially those of very low birthweight (VLBW), have an increased risk of nosocomial infections secondary to deficiencies in development. We previously demonstrated that granulocyte-macrophage colony-stimulating factor (GM-CSF) production and mRNA expression from stimulated neonatal mononuclear cells are significantly less than that from adult cells. Recombinant murine GM-CSF administration to neonatal rats has resulted in neutrophilia, increased neutrophil production, and increased survival of pups during experimental Staphylococcus aureus sepsis. In the present study, we sought to determine the safety and biologic response of recombinant human (rhu) GM-CSF in VLBW neonates. Twenty VLBW neonates (500 to 1,500 g), aged < 72 hours, were randomized to receive either placebo (n = 5) or rhuGM-CSF at 5.0 micrograms/kg once per day (n = 5), 5.0 micrograms/kg twice per day (n = 5), or 10 micrograms/kg once per day (n = 5) given via 2-hour intravenous infusion for 7 days. Complete blood counts, differential, and platelet counts were obtained, and tibial bone marrow aspirate was performed on day 8. Neutrophil C3bi receptor expression was measured at 0 and 24 hours. GM-CSF levels were measured by a sandwich enzyme-linked immunosorbent assay at 2, 4, 6, 12, and 24 hours after the first dose of rhuGM-CSF. At all doses, rhuGM-CSF was well tolerated, and there was no evidence of grade III or IV toxicity. Within 48 hours of administration, there was a significant increase in the circulating absolute neutrophil count (ANC) at 5.0 micrograms/kg twice per day and 10.0 micrograms/kg once per day, which continued for at least 24 hours after discontinuation of rhuGM-CSF. When the ANC was normalized for each patient's first ANC, there was a significant increase in the ANC on days 6 and 7 at each dose level. By day 7, all tested doses of rhuGM- CSF resulted in an increase in the absolute monocyte count (AMC) compared with placebo-treated neonates. In those receiving rhuGM-CSF 5.0 micrograms/kg twice per day, there was additionally a significant increase in the day 7 and 8 platelet count. Tibial bone marrow aspirates demonstrated a significant increase in the bone marrow neutrophil storage pool (BM NSP) at 5.0 micrograms/kg twice per day and 10.0 micrograms/kg once per day. Neutrophil C3bi receptor expression was significantly increased 24 hours after the first dose of rhuGM-CSF at 5.0 micrograms/kg once per day. The elimination half-life (T1/2) of rhuGM-CSF was 1.4 +/- 0.8 to 3.9 +/- 2.8 hours.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular analysis of relapse in chronic myeloid leukemia after allogeneic bone marrow transplantation

Four patients with Philadelphia (Ph') positive chronic myeloid leukemia (CML) were studied before, after, and on relapse following allogeneic bone marrow transplantation (BMT). Southern analysis of DNA from cells collected before and at relapse after BMT was performed in order to investigate the origin of the leukemia at relapse. Using minisatellite probes we showed that the relapse occurred in cells of host origin in all four patients and this was confirmed with a Y chromosome specific probe in two male patients who had a female donor. Furthermore, using two probes for the breakpoint cluster region (bcr) on chromosome 22, we showed that leukemic cells at relapse bore identical rearrangements to those in the disease at time of presentation of each patient. We conclude that relapse in all four patients is due to re-emergence of the original leukemic clone.     

Human Papillomavirus Prevalence in Oropharyngeal Cancer before Vaccine Introduction,United States

We conducted a study to determine prevalence of HPV types in oropharyngeal cancers in the United States and establish a prevaccine baseline for monitoring the impact of vaccination. HPV DNA was extracted from tumor tissue samples from patients in whom cancer was diagnosed during 1995–2005. The samples were obtained from cancer registries and Residual Tissue Repository Program sites in the United States. HPV was detected and typed by using PCR reverse line blot assays. Among 557 invasive oropharyngeal squamous cell carcinomas, 72% were positive for HPV and 62% for vaccine types HPV16 or 18. Prevalence of HPV-16/18 was lower in women (53%) than in men (66%), and lower in non-Hispanic Black patients (31%) than in other racial/ethnic groups (68%–80%). Results indicate that vaccines could prevent most oropharyngeal cancers in the United States, but their effect may vary by demographic variables.     

Prospective Associations between Bilingualism and Executive Function in Latino Children: Sustained Effects While Controlling for Biculturalism

The study purpose was to test 1-year prospective associations between English–Spanish bilingualism and executive function in 5th to 6th grade students while controlling for biculturalism. Participants included 182 US Latino students (50 % female). Self-report surveys assessed biculturalism, bilingualism, and executive function (i.e., working memory, organizational skills, inhibitory control, and emotional control, as well as a summary executive function score). General linear model regressions demonstrated that bilingualism significantly predicted the summary executive function score as well as working memory such that bilingual proficiency was positively related to executive function. Results are the first to demonstrate (a) prospective associations between bilingualism to executive function while controlling for the potential third variable of biculturalism, and (b) a principal role for working memory in this relationship. Since executive function is associated with a host of health outcomes, one implication of study findings is that bilingualism may have an indirect protective influence on youth development.     

Construct validity of individual and summary performance metrics associated with a computer-based laparoscopic simulator

Background

Computer-based surgical simulators capture a multitude of metrics based on different aspects of performance, such as speed, accuracy, and movement efficiency. However, without rigorous assessment, it may be unclear whether all, some, or none of these metrics actually reflect technical skill, which can compromise educational efforts on these simulators. We assessed the construct validity of individual performance metrics on the LapVR simulator (Immersion Medical, San Jose, CA, USA) and used these data to create task-specific summary metrics.

Methods

Medical students with no prior laparoscopic experience (novices, N = 12), junior surgical residents with some laparoscopic experience (intermediates, N = 12), and experienced surgeons (experts, N = 11) all completed three repetitions of four LapVR simulator tasks. The tasks included three basic skills (peg transfer, cutting, clipping) and one procedural skill (adhesiolysis).

Results

We selected 36 individual metrics on the four tasks that assessed six different aspects of performance, including speed, motion path length, respect for tissue, accuracy, task-specific errors, and successful task completion. Four of seven individual metrics assessed for peg transfer, six of ten metrics for cutting, four of nine metrics for clipping, and three of ten metrics for adhesiolysis discriminated between experience levels. Time and motion path length were significant on all four tasks. We used the validated individual metrics to create summary equations for each task, which successfully distinguished between the different experience levels.

Conclusion

Educators should maintain some skepticism when reviewing the plethora of metrics captured by computer-based simulators, as some but not all are valid. We showed the construct validity of a limited number of individual metrics and developed summary metrics for the LapVR. The summary metrics provide a succinct way of assessing skill with a single metric for each task, but require further validation.     

Human endothelial cell-based assay for endotoxin as sensitive as the conventional Limulus Amebocyte Lysate assay

Endotoxin, also known as lipopolysaccharide (LPS) produced by bacteria can be present in any liquid or on any biomaterial even if the material is sterile. Endotoxin in mammals can cause fever, inflammation, cell and tissue damage and irreversible septic shock and death. In the body, endothelial cells making up the blood vasculature and endothelial cells in vitro rapidly react to minute amounts of endotoxin resulting in a rapid induction of the cell adhesion molecule E-selectin. In this study we have used immunofluorescent staining to evaluate the expression of E-selectin on human microvascular endothelial cells from the skin (HDMEC) and human umbilical vein endothelial cells (HUVEC) exposed to various concentrations of LPS. In addition, the sensitivity of detection was compared with the most widely used assay for the presence of endotoxin, the Limulus Amebocyte Lysate assay (LAL). The detection of E-selectin on endothelial cells in the presence of LPS for 4 h was found to be at least as sensitive in detecting the same concentration using the LAL assay. A cell adhesion molecule-enzyme immunosorbent assay was also developed and used to quantify LPS using the endothelial cell model. A comparison of LAL and the immunofluorescent staining method was carried out with solutions, nanoparticles, biomaterial extracts and endothelial cells grown directly on biomaterials. Under all conditions, the endothelial/E-selectin model system was positive for the test samples that were positive by LAL. Thus, we propose the use of this highly sensitive, rapid, reproducible assay for the routine testing of endotoxin in all steps in the manufacturing process of materials destined for use in humans. This can give a rapid feedback and localization of bacterial contamination sources with the LAL being reserved for the testing of the final product.