Synergistic effect of low dose Cyclosporine A and human interleukin 10 overexpression on acute rejection in rat lung allotransplantation

Objective: Electroporation mediated transfer of plasmid DNA into peripheral muscle results in high transfection efficiency. The aim of this study was to investigate the effect of gene transfer of human IL-10 (hIL-10) into the tibialis anterior muscle (MTA) in combination with low dose Cyclosporine A (CsA) on acute rejection of lung allografts in the rat. Methods: Lung allotransplantation was performed from male BN donor to male Fisher F344 rats. Gene transfer was achieved by intramuscular injection into the MTA of the recipient followed by electroporation (4×20 ms impulses at 200 V/cm) 24 h prior to the transplantation. Group A (n=5) received CsA (2.5 mg/kg bw ip) for 5 days post-transplant and group B (n=5) 2.5 μg of PCIK hIL-10 (plasmid expression vector containing human CMV immediate early gene promoter and enhancer) and a low dose CsA (2.5 mg/kg bw i.p.). Graft function was assessed by blood gas at day 5 after exclusion of the native lung. Animals were sacrificed and blood was drawn to measure serum hIL-10 levels (ELISA) and tissue was sampled for histological grading of rejection. Results: Local expression of hIL-10 was confirmed at the mRNA level by in situ hybridization. All group A control animals showed severe signs of rejection. At day 5 all grafts in group B showed good gas exchange mean PaO2 233±123 mmHg, vs 44±8 mmHg in group A. Histological examination revealed moderate to severe rejection in all animals in group A (IIIB, ISHLT) in contrast to low moderate rejection in group B (II–IIIA). hIL-10 serum levels on day 5 were 14±7 pg/ml in group B vs. 0 in group A. Conclusions: Electroporation mediated hIL-10 overexpression in a peripheral muscle of the recipient in combination with low dose CsA reduces acute rejection in this model of rat lung allotransplantation.     

Effects of posture on stimulated ventilation in quadriplegia

Quadriplegics are able to compensate for alterations of operational length of the diaphragm by reflexly increasing neural drive to the diaphragm. This increase in neural drive is adequate to maintain required tidal volume and minute ventilation during quiet breathing in these patients with limited inspiratory muscle function. It is not known, however, if this neural compensation is sufficient to preserve ventilation when the diaphragm is stressed by simultaneously changing its operational length and increasing ventilatory demands. This issue was explored in 7 quadriplegics whose vital capacity was reduced to 15 to 53% of predicted. The diaphragm was stressed by shortening its length from the supine to a 60 degree tilted position, and also by inducing hyperventilation by having the subjects rebreathe 7% CO2. Response to this stress was recorded by monitoring the ventilatory response to rebreathing CO2 (delta VE/delta PCO2), and also by measuring mouth pressure 0.1 s after occluding the airway at the start of inspiration during CO2 rebreathing (delta P0.1/delta PCO2). A change from the supine to the tilted position caused an increase in resting end-expiratory volume of 0.8 +/- 0.2 L (SD) and therefore shortened the diaphragm. Despite this shortening of diaphragm length and the stress of CO2 rebreathing, there was no significant change in delta VE/delta PCO2 and delta P0.1/delta PCO2 with changes in posture. The delta VE/delta PCO2 was 0.82 +/- 0.42 L/min/mm Hg supine versus 0.95 +/- 0.65 L/min/mm Hg when tilted. The delta P0.1/delta PCO2 was 0.18 +/- 0.08 cm H2O/mm Hg supine versus 0.20 +/- 0.10 cm H2O/mm Hg tilted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antigenic differences between human platelets and megakaryocytes.

To investigate the apparent paradox in the observation that most patients with immune thrombocytopenias have normal or increased numbers of megakaryocytes (MKs), the extent of antigenic cross-reactivity between normal platelets and MK was examined. Indirect immunofluorescence and ultrastructural studies were carried out by means of four antisera specific for platelets: anti-GpIb, anti-GpIIb/IIIa, anti-PLA1, and an antiserum from a patient with quinidine-induced thrombocytopenia. Following incubation of freshly collected marrow with these antisera, MK were first identified by phase-contrast microscopy and then inspected for fluorescence. Almost all MKs were found reactive with the last three antisera, albeit to a variable extent. In contrast, only 24% reacted with anti-GpIb. The pattern of fluorescence, ie, rim, partial or cytoplasmic, appeared to be related to the extent of MK fragmentation. Only rim fluorescence of living MKs could be interpreted to indicate that the platelet epitope was exposed on the surface of the precursor cell. The observations suggest that platelet antigens are variably expressed on the plasma membranes of MKs. In a clinical setting, the heterogeneity among platelet target antigens and the extent to which these are exposed on MKs at various stages of maturation may dictate the severity of the thrombocytopenia and degree of ineffective thrombocytopoiesis.     

A proton relaxation enhancement investigation of the binding of fatty acid spin labels to human serum albumin

Proton relaxation enhancement (PRE) values for fatty acid spin labels bound to human serum albumin have been investigated using the inversion-recovery method at 24 MHz. At 0.1 mM protein concentration and a label-to-protein ratio of one-to-one, the PRE value for 12-Doxylsterate-albumin complex is 7.8 +/- 2.3, whereas the PRE values for 5-Doxylstearate and 16-Doxylstearate-albumin complexes are 1.5 +/- 0.6 and 1.7 +/- 0.7, respectively. Addition of 10-fold excess of stearic acid reduced the PRE values nearly to 1, indicating that the strong enhancements arise from direct binding of fatty acid spin labels to human serum albumin. PRE values for all three labels exhibit maxima as a function of the label-to-protein ratio, suggesting multiple binding sites for fatty acid spin labels with labels in the tightest binding sites not resulting in the most effective relaxation. Based on the rates of reduction of ESR signal amplitudes by sodium ascorbate, the difference in PRE values for the three fatty acid spin labels bound to albumin is attributed to the difference in water accessibility of the nitroxide moieties at various positions along the acyl chain, being greater at the C-12 position than at C-5 or C-16 position. The PRE value of 8 for 12-Doxylstearate bound to human serum albumin indicates that this complex may be a suitable paramagnetic contrast agent for in vivo NMR imaging.