Prolonged insulin independence after islet allotransplants in recipients with type 1 diabetes.

We sought to determine the long-term outcomes in type 1 diabetic recipients of intraportal alloislet transplants on a modified immunosuppressive protocol. Six recipients with hypoglycemia unawareness received one to two islet infusions. Induction therapy was with antithymocyte globulin (ATG) plus etanercept for tumor necrosis factor-alpha blockade. Recipients received cyclosporine and everolimus for maintenance immunosuppression for the first year posttransplant, with mycophenolic acid or mycophenolate mofetil subsequently substituted for everolimus. Recipients have been followed for 1173 +/- 270 days since their last infusion for islet graft function (insulin independence, hemoglobin A(1c) levels and C-peptide production) and for adverse events associated with the study protocol. Of the six recipients, five were insulin-independent at 1 year, and four continue to be insulin-independent at a mean of 3.4 +/- 0.4 years posttransplant. None of the six recipients experienced recurrence of severe hypoglycemia. Measured glomerular filtration rate decreased from 110.5 +/- 21.2 mL/min/1.73 m(2) pretransplant to 82.6 +/-19.1 mL/min/1.73 m(2) at 1 year posttransplant. In conclusion, islet transplants restored insulin independence for a mean of >3 years in four of six recipients treated with ATG and etanercept induction therapy and with cyclosporine and, initially, everolimus for maintenance. Our results suggest this immunosuppressive protocol may allow long-term graft survival.

Feasibility evaluation of a new pericardial bioprosthesis with dye mediated photo-oxidized bovine pericardial tissue.

BACKGROUND AND AIMS OF THE STUDY: In an attempt to overcome the problem of calcification of bioprostheses, a novel bovine pericardial tissue valve preserved with a non-aldehyde, dye mediated photo-oxidation process (PhotoFixTM) developed by CarboMedics, Inc. The device was evaluated by implantation in the mitral position of juvenile sheep with a mean age of 3.5 months. MATERIALS AND METHODS: Thirteen valves were evaluated; six experimental valves with photo-oxidized tissue, five identically designed valves with glutaraldehyde-fixed tissue, and two Carpentier-Edwards pericardial valves as controls. RESULTS: Four of the six animals in the photo-oxidized group were free of calcification when electively sacrificed at 152 days, 152 days, one year and 1.5 years respectively. One animal was electively sacrificed at 98 days. Pathologic findings indicated minimal calcification of the valve due to uneven stress distribution from two bent stent posts that most likely occurred during surgical implantation, but the device was still functional. The sixth animal with photo-oxidized tissue died at 131 days due to massive calcification of a single leaflet. There was no pathologic evidence of infection. Explants at five months from additional and still continuing sheep studies per FDA guidelines have not reproduced single leaflet calcification in over forty explanted valves with photo-oxidized tissue. The glutaraldehyde-fixed valves all exhibited some calcification at explant; two of these animals died early due to the mineralization. Both control animals with Carpentier-Edwards valves died early from mitral stenosis due to extensive device calcification. CONCLUSIONS: The fact that four of six valves with photo-oxidized tissue remained free of any signs of calcification for up to 1.5 years, while none of the other valves did, suggests that photo-oxidation is a promising method of preserving and fixing tissue for use in bioprostheses. These results suggest that photo-oxidized bioprosthetic valves may be clinically valuable and warrant further study.

Removal of baboon and human antiporcine IgG and IgM natural antibodies by immunoadsorption. Results of in vitro and in vivo studies.

The safe and effective removal of xenoreactive antibodies in the peritransplant period is likely to be critical for the clinical application of xenotransplantation involving disparate donor species, such as the pig. In an effort to develop an improved method for antibody removal in xenotransplantation, we have studied reusable antihuman antibody (Ig) columns in vitro and in vivo. Two types of columns were tested: (1) an antihuman Ig column containing polyclonal sheep antihuman IgG (heavy- and light-chain-specific) conjugated to sepharose CL-4B (Ig-Therasorb), and (2) an antihuman Ig column using polyclonal antihuman IgM (mu-chain-specific) conjugated to sepharose. Passage of human or baboon plasma through the Ig-Therasorb column resulted in 97.5% and 78.4% mean reductions in total IgG and IgM, respectively. Reductions in total IgG and IgM correlated with lowering of antipig IgG (54-486 fold) and IgM (9-54 fold) antibody titers as assessed by pig endothelial cell ELISA. The ability of the Ig-Therasorb to significantly reduce IgM may be attributed to the light chain specificity of this column. With the anti-IgM column, marked reductions in total (82.6-83.9%) and antipig (27-54 fold) IgM in human and baboon plasma occurred, while levels of total and xenoreactive IgG were slightly affected. Other than a dilutional effect, neither column resulted in significant reduction in albumin, fibrinogen, factor 5, and factor 8. Repeated in vivo use of either column in baboons achieved reductions in IgG and IgM that closely followed the results of our in vitro studies. No subject morbidity or mortality occurred. Use of the Ig-Therasorb column with immunosuppression in two baboons receiving pig renal xenografts achieved sustained reductions in antipig antibodies and prevented hyperacute rejection. Subjects were sacrificed at 11 and 13 days posttransplant with functioning xenografts and were found to have no evidence of vascular xenograft rejection. We conclude that anti-Ig columns represent a safe and effective method for antibody removal, without several of the limitations of other antibody removal techniques. Also, columns appear to be safe for repeated antibody removal in the posttransplant period.

Murine APC activation in the xenogeneic MLC.

Purified human T cells proliferate in response to direct and indirect presentation of human alloantigens. However, until recently it was believed that human T cells could respond only indirectly to murine xenoantigens. We recently used the mixed leucocyte culture (MLC) to demonstrate that purified human T cells proliferate in response to direct presentation of murine xenoantigens by murine antigen-presenting cells (APC) in the presence of human cytokines. We suggested that cytokines might function poorly across species barriers. In this study, we demonstrate that although proliferation occurs in the presence of exogenously added cytokines, the precursor frequency of responding human T cells is much lower in a xenogeneic than in an allogeneic MLC. We demonstrate that human T cells also proliferate in response to murine APC in the presence of murine cytokines, and murine cytokines augment the proliferative response seen in a human anti-human MLC, ruling out the possibility that an absolute cytokine incompatibility exists between these species. We show that exogenously added human IL-1 causes maximal proliferation of human T cells in response to murine xenoantigens only when added early in the culture. We further demonstrate that murine APC preincubated in human rIL-1, and washed extensively, prior to use as stimulating cells, cause human T-cell proliferation without the need for exogenously added cytokines. Finally, we noted that during interactions of human T cells and murine APC, little to no IL-1 is produced, whereas after the addition of exogenous IL-1, a marked increase in the production of IL-1 is seen. These data suggest that during interactions between human T cells and murine APC, the murine cells do not receive adequate stimulation to produce sufficient costimulatory signals to allow proliferation of the human T cells.

CD3 negative "small agranular lymphocytes" are natural killer cells.

We describe here that CD3-, CD16+ and/or CD56+ small lymphocytes, in a highly reproducible fashion, mediate a significant level of K562 killing that is, on a "per cell" basis, comparable to the cytolytic activity of CD3- LGL. The CD3- small lymphocytes appeared to have no granules based on light and electron microscopy and lack of right-angle scatter on the FACS; we thus refer to them as small "agranular" lymphocytes (SAL). The lytic activity against K562 is inhibited by treatment with either L-leucine methyl ester or EGTA, which are reported to effect granule-dependent killing. We suggest that the SAL have lytic molecules in their cytoplasm (which are sensitive to these treatments) but that these molecules are not organized into discrete granules as found in LGL. The CD3- SAL are phenotypically very similar to LGL and both SAL and LGL mediated equal and reproducible antibody-dependent cell-mediated cytotoxicity. These observations force redefinition of the concept of NK cells to include both CD3- LGL and CD3- SAL.

Comparison of 6 and 8 hourly tobramycin dosing intervals in treatment of pulmonary exacerbations in cystic fibrosis patients.

The efficacy and toxicity of a shortened tobramycin dosing interval in the treatment of exacerbations of Pseudomonas aeruginosa pulmonary infection in cystic fibrosis patients were evaluated prospectively. Patients ages 13 to 30 years received 34 treatment courses and were randomized by pairs to receive tobramycin administered either every 6 or 8 hours. Peak serum concentrations were adjusted to 8 to 10 micrograms/ml; thus a larger total daily dosage was administered to patients receiving tobramycin every 6 hours. The shorter dosing interval was associated with better pulmonary function at follow-up and significantly longer time before next hospital admission for a pulmonary exacerbation. During the study hospitalization there were no differences in pulmonary function tests, clinical score, sputum carriage of P. aeruginosa, toxicity or necessary length of hospitalization. A 6-hour tobramycin dosing interval was more efficacious than an 8-hour dosing interval in the treatment of cystic fibrosis patients.