Long-Term Follow-Up of the Edmonton Protocol of Islet Transplantation in the United States.

We report the long-term follow-up of the efficacy and safety of islet transplantation in seven type 1 diabetic subjects from the United States enrolled in the multicenter international Edmonton Protocol who had persistent islet function after completion of the Edmonton Protocol. Subjects were followed up to 12 years with serial testing for sustained islet allograft function as measured by C-peptide. All seven subjects demonstrated continued islet function longer than a decade from the time of first islet transplantation. One subject remained insulin independent without the need for diabetic medications or supplemental transplants. One subject who was insulin-independent for over 8 years experienced graft failure 10.9 years after the first islet transplant. The remaining six subjects demonstrated continued islet function upon trial completion, although three had received a supplemental islet transplant each. At trial completion, five subjects were receiving insulin and two remained insulin independent, although one was treated with liraglutide. The median hemoglobin A1c was 6.3% (45 mmol/mol). All subjects experienced progressive decline in the C-peptide/glucose ratio. No patients experienced severe hypoglycemia, opportunistic infection, or lymphoma. Thus, although the rate and duration of insulin independence was low, the Edmonton Protocol was safe in the long term. Alternative approaches to islet transplantation are under investigation.

Enterobacter cloacae bloodstream infections traced to contaminated human albumin.

In August 1996, a patient in Kansas developed an Enterobacter cloacae bloodstream infection (BSI) shortly after receiving Albuminar, a brand of human albumin. Albuminar contamination was suspected. A case-control study of patients with primary gram-negative bacterial BSIs showed that patients with E. cloacae BSIs were significantly more likely than patients with non-E. cloacae gram-negative BSIs to have received Albuminar within 3 days of developing their BSIs (3 of 5 vs. 0 of 9; OR, undefined; P=.03). The E. cloacae isolate from the Kansas patient was found by pulsed-field gel electrophoresis to be identical to the isolate from the patient's Albuminar vial, to isolates from 2 previously unopened Albuminar vials, and to an isolate from a Wisconsin patient who had received Albuminar. A worldwide recall of approximately 116,000 Albuminar vials took place. This multistate outbreak was detected because of clinical astuteness and prompt reporting. Combined epidemiological and laboratory approaches are valuable when investigating potentially contaminated blood components and plasma derivatives.

Both adhesion to immobilized vitronectin and FcepsilonRI cross-linking cause enhanced focal adhesion kinase phosphorylation in murine mast cells.

Murine mast cells adhere spontaneously to plate-bound vitronectin (VNPB) via alphav-containing integrins, and this adhesive interaction results in an augmented interleukin-3 (IL-3)-dependent mast-cell proliferation. In this report we demonstrate that the activation of murine mast cells through alphav-integrin, as well as through the high affinity immunoglobulin E (IgE) receptor (FcepsilonRI), results in enhanced tyrosine phosphorylation of focal adhesion kinase (FAK), a cytoplasmic protein tyrosine kinase involved in mitogenic and oncogenic signal transduction. While mast cell adhesion to VNPB resulted in enhanced FAK phosphorylation, treatment with soluble vitronectin (VNSOL) failed to do so. Spontaneous mast cell adhesion to entactin (EN) did not induce tyrosine phosphorylation of FAK, demonstrating that not all adhesive interactions lead to the same sequence of biochemical events. Because FAK has intrinsic tyrosine kinase activity, we examined whether activating mast cells via alphav-integrins, or via FcepsilonRI-cross-linking stimulated the in vitro kinase activity of FAK. Both pathways were found independently to activate FAK in mast cells and together appeared additive. Protein kinase C depletion in mast cells and calcium depletion in the medium caused decreased tyrosine phosphorylation of FAK, indicating that optimal tyrosine phosphorylation of FAK is regulated by both pathways. These data are consistent with the conclusion that the tyrosine phosphorylation of FAK represents at least one example of a point of convergence in the intracellular tyrosine phosphorylation cascades induced by alphav integrin-and FcepsilonRI-mediated signal transduction pathways in mast cells.

Induction and enhancement of Fc(epsilon)RI-dependent mast cell degranulation following coculture with activated T cells: dependency on ICAM-1- and leukocyte function-associated antigen (LFA)-1-mediated heterotypic aggregation.

Activated mast cells are known to reside in close apposition to T cells in various inflammatory processes. In this regard, we have reported that activated mast cells form heterotypic aggregates with activated lymphocytes. To determine whether this interaction would result in mast cell degranulation, we examined the effect of EL-4, 2B4, or freshly isolated T cells, activated by PMA or immobilized anti-CD3 mAb, on histamine release from murine bone marrow-derived cultured mast cells (BMCMC). Coculturing BMCMC with activated but not with resting T cells resulted in significant histamine release. Also, Fc(epsilon)RI cross-linking-induced degranulation was augmented when BMCMC were cocultured with activated T cells. Supernatants of activated T cells failed to exert the stimulatory effect. Separation of the two cell populations with a porous membrane prevented degranulation, indicating that BMCMC activation was adhesion dependent. Indeed, the kinetics of histamine release paralleled the kinetics of the formation of heterotypic aggregates, which peaked after 12 h of coculture. Introduction of anti-LFA-1 and anti-intercellular adhesion molecule-1 mAb inhibited the adhesion-induced mast cell degranulation. These data suggest a heretofore unrecognized mast cell activation pathway induced by LFA-1/intercellular adhesion molecule-1-mediated heterotypic aggregation with activated T cells.

Characterization of a mast cell line that lacks the extracellular domain of membrane c-kit.

Expression of the c-kit proto-oncogene receptor on mast cells is essential for their normal proliferation and maturation as well as for several biological responses such as chemotaxis and attachment. In the present study we report that the interleukin-3 (IL-3)-dependent mast cell line CFTL-15 lacks the extracellular domain of the c-kit receptor. This observation was made after noting that the c-kit ligand stem cell factor (SCF) could not prevent IL-3 deprivation-induced mast cell apoptosis and that CFTL-15 cells did not proliferate in response to SCF. Flow cytometric analysis employing monoclonal anti-c-kit antibodies, and immunogold labelling with analysis by electron microscopy, subsequently showed a diminished expression of c-kit on CFTL-15 cells. There was no identifiable message for the extracellular domain of c-kit in these cells, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). These previously unrecognized properties of the CFTL-15 mast cell line allowed the examination of other biological consequences of the lack of c-kit on mast cells. Analysing the ability of these cells to adhere to surface-bound fibronectin, it was found that addition of SCF did not increase their adhesion to this substrate, in opposition to what is reported with other mast cells. Similarly, CFTL-15 mast cells did not adhere to fibroblasts, which is known to require c-kit expression. Also, there was no protein tyrosine phosphorylation in these cells in response to SCF. CFTL-15 cells underwent apoptosis on removal of IL-3 coincident with a decrease in endogenous Bcl-2 mRNA. Overexpression of Bcl-2 cDNA prolonged survival of Bcl-2-transfected CFTL-15 cells upon withdrawal of IL-3. Thus, the CFTL-15 cell line that lacks surface c-kit is not able to proliferate in response to SCF, undergoes apoptosis in the presence of SCF, and does not adhere to fibroblasts. These results confirm earlier studies on the functional consequences of c-kit and provide a novel experimental model for further investigation.

IL-3-dependent mast cells attach to plate-bound vitronectin. Demonstration of augmented proliferation in response to signals transduced via cell surface vitronectin receptors.

The adhesive interactions of activated mast cells with the extracellular matrix play an important role in anchorage and cellular motility. In this report we demonstrate that IL-3-dependent bone marrow-derived mast cells adhere to plate-bound vitronectin with high affinity in a saturable and dose-dependent manner. This adhesion interaction is unique in that it does not require prior mast cell activation through Fc epsilon RI or after treatment with PMA. It is inhibited by divalent cation chelation and by competitive inhibition with a synthetic Arginine-Glycine-Aspartate-Serine tetrapeptide. Polyclonal antisera for alpha v beta 3, an integrin known to bind vitronectin, inhibits attachment to plate-bound vitronectin in a dose-dependent manner. Comparison of the adhesion interactions for vitronectin, fibronectin, and laminin indicate that adhesion to vitronectin is greater than that seen with either fibronectin or laminin, either in the presence or absence of PMA. FACS analysis using a monoclonal hamster anti-murine vitronectin receptor (alpha v) antibody followed by a fluorescein-conjugated rabbit anti-hamster IgG revealed no change in surface vitronectin receptor expression after Fc epsilon RI-mediated cell activation. Proliferation assays with correction for cell viability revealed a 25% increase in cell number above the maximal IL-3 response over a 24-h period of adhesion to a vitronectin-coated surface and a 41% increase over 96 h of adhesion to vitronectin. Binding to plate-bound vitronectin was not able to sustain cell viability in the absence of IL-3. Thus, IL-3-dependent bone marrow-derived mast cells adhere to vitronectin, an extracellular matrix protein present throughout connective tissues. This interaction generates a signal that results in the augmentation of the maximal IL-3-dependent mast cell proliferative response, thus demonstrating at least one way in which the interaction between mast cells and extracellular matrix alter the biologic responsiveness of the mast cell.

The role of epidemic infectious diseases in the discovery of America.

As the world prepares to celebrate the quincentennial events surrounding the discovery of the New World by Christopher Columbus in 1492, a particular interest regarding the influence of epidemic infectious diseases on the history of the conquest of America has emerged. Contrary to popular belief, it was not the European guns or fierce soldiers that conquered the native Americans, but instead it was the common childhood illnesses brought from the Old World by the European conquistadors. Diseases such as smallpox, measles, and typhus annihilated most of the American native populations. Devastating epidemics resulted throughout the New World. We will review the consequences of introducing new infectious agents into a nonimmune population, discuss the major pathogens that were imported from the Old World, and focus on how these diseases may have affected the aboriginal depopulation of the Americas.