Analysis of reporter gene expression in ovine dermis and afferent lymph dendritic cells in vitro and in vivo.

Plasmid DNA administration has revolutionised approaches to vaccination, and many studies have demonstrated the generation of both humoral and cytotoxic T cell responses which confer protection against live pathogen challenge. However, the mechanisms underlying DNA vaccination are poorly understood. Several studies have suggested the involvement of professional antigen presenting cells such as dendritic cells (DC), but direct evidence for this is lacking. We have used the pseudoafferent lymphatic cannulation model in sheep to study the expression of a plasmid encoding enhanced green fluorescent protein (EGFP) by afferent lymph DC following administration to skin. The cells were analysed by flow cytometry. Preliminary studies were carried out to determine if the pEGFP would function in sheep cells in vitro. The results showed that electroporation of sheep skin fibroblasts, primary macrophages, and afferent lymph DC with 30 microg pEGFP resulted in varying degrees of fluorescence in these cells e.g. 35% of skin cells examined at 48 h, and 7% of afferent lymph DC examined after 4 h. Following intradermal injection of 120 microg of pEGFP, small numbers of fluorescent DC (1-5%) were evident by flow cytometry after 1-4 h. The fluorescent DC continued to drain into the lymphatics over a period of 24 h. Analysis by PCR showed that free pEGFP appeared in the afferent lymph plasma within 1 h of injection, peaking at 2 h and becoming undetectable after 6 h. The results suggest that primary immune responses may be initiated by uptake of soluble protein antigen by afferent lymph DC and by free plasmid rapidly draining to the lymphatics where it may be taken up by DC in the lymph plasma and the local lymph node.

Differing effects of pancreas-kidney transplantation with systemic versus portal venous drainage on cholesteryl ester transfer in IDDM subjects.

OBJECTIVE: Cholesteryl ester transfer (CET) is accelerated in patients with IDDM treated with conventional (subcutaneous) insulin therapy (CIT) and a number of other disorders associated with premature cardiovascular disease. We have shown that in IDDM this disturbance is closely linked to iatrogenic hyperinsulinemia (HI), because it was reversed when insulin was administered by the intraportal (i.p.) route. In this study, we sought to determine whether HI after successful pancreas-kidney transplantation (PKT) has the same adverse effect on CET. RESEARCH DESIGN AND METHODS: CET was measured by both mass and isotopic assays and compared in two groups of euglycemic non-insulin-requiring IDDM PKT patients with either systemically draining allografts and persistent HI or grafts with portal vein anastomoses that were normoinsulinemic (PK-P). A third group of eight nondiabetic kidney transplant (KT) patients receiving the same immunosuppressive drugs served as control subjects. RESULTS: CET in pancreas-kidney transplantation subjects with systemic venous drainage (PK-S) was increased (P < 0.001) to the same level we have reported previously in IDDM patients receiving CIT and was significantly higher (P < 0.001) than in those subjects with PK-P. CET in the PK-P group did not differ from that of the KT control patients. CONCLUSIONS: CET is affected by variations in systemic insulin levels in pancreas transplant patients with allografts that have differing venous drainage. Because high systemic insulin levels are linked to the activation of (ET, euglycemic HI IDDM pancreas allograft recipients may continue to be at high risk for macrovascular complications.

Safety and feasibility of renal blood flow determination during kidney transplant surgery with perfusion ultrasonography.

Contrast-enhanced perfusion patterns of newly transplanted kidneys were determined in 10 patients. Albumin-stabilized sonicated microspheres were injected into the iliac-renal artery of the transplanted kidney while continuous two-dimensional ultrasound images were recorded. Doppler derived resistance index (RI) of the transplanted kidney's blood flow before injection of contrast (0.68 +/- 0.8) did not differ significantly from RI measured immediately after injection (0.72 +/- 0.13) or RI 24 h after surgery (0.69 +/- 0.11). Heart rate, mean arterial pressure, central venous pressure, and electrocardiogram (ECG) signs for ischemia did not change during contrast injections. Renal scintigraphy and renal biopsy revealed acute tubular necrosis and/or rejection in two patients at 24-48 h. Videodensitometry was used to assess the ratio of inner to outer peak pixel intensity from the recorded tomographic images in six patients. In both patients with acute rejection, the inner to outer cortex peak pixel intensity was greater than 1, whereas it was less than 1 in the remaining four patients with normal postoperative renal function. Visual scores (0-3) of contrast enhancement for three doses of Albunex were evaluated (0.5 mL, 1.0 mL, 2.0 mL). Two milliliters always enabled perfusion assessment. In seven patients the identical dose of Albunex was injected immediately before and 30 s after 2 mg of verapamil was injected directly into the renal artery at the time of surgery. The contrast enhancement score before verapamil (1.4 +/- 0.6) was significantly less than the enhancement score after (2.1 +/- 0.6), implying greater renal blood flow after verapamil.(ABSTRACT TRUNCATED AT 250 WORDS)