Theo A. Schuurs

Time-dependent changes in donor brain death related processes

Donor brain death (BD) affects kidney function and survival after transplantation. Studies on brain dead kidney donors indicate that, besides inflammation and coagulation, cytoprotective gene expression is activated as well. Here, we evaluated in a time‐course experiment progression of these renal BD‐related processes. Animals were sacrificed 0.5, 1, 2 or 4 h after BD and compared to sham‐operated controls. Proinflammatory genes (E‐selectin, MCP‐1, Il‐6) were massively up‐regulated (p < 0.05) already 0.5 h after BD. Inducers of proinflammatory gene expression were either activated (NF‐κB) or induced in expression (Egr‐1) after 0.5 h of BD. Increased numbers of infiltrating granulocytes were seen in the interstitium from 0.5 h on. Also, expression of protective genes HO‐1 and HSP70 were increased within 0.5 h. Remarkably, reactive oxygen species formation was detectable only in the later phase of BD. Among 14 measured serum cytokines, MCP‐1 and KC‐protein were significantly elevated from 0.5 h on. In conclusion, a fast induction of proinflammatory and stress‐induced protective processes in brain dead donor kidneys was demonstrated, probably triggered by changes occurring during BD induction. Importantly, hypoxia appeared not to be one of the initial triggers, and early increased systemic levels of chemokines MCP‐1 and KC may be regarded as the starting point for the inflammatory cascade in brain dead donor kidneys.

Response of encapsulated rat pancreatic islets to hypoxia

Hypoxia contributes to encapsulated pancreatic islet graft failure. To gain insight into the mechanisms that lead to hypoxia-induced graft failure, encapsulated islet function, vitality, and cell replication were assessed after 2 and 5 days of hypoxic (1% O2) and normoxic (20% O2) culture. The mRNA expression levels of Bcl-2, Bax, inducible nitric oxide synthase (iNOS), and monocyte chemoattractant protein 1 (MCP-1) were assessed, as well as the amount of nitrite and MCP-1 in the culture medium. Hypoxia was associated with loss of encapsulated islet function and vitality, but not with an increase in islet cell replication. Loss of vitality was due to necrosis, and only modestly due to apoptosis. Hypoxia was not associated with changes in the Bcl-2/Bax mRNA ratio, but it did increase the expression of iNOS and MCP-1 mRNA. The increased mRNA levels were, however, not associated with elevated concentrations of nitrite nor with elevated levels of MCP-1 protein. The increased iNOS mRNA levels imply a role for NO in the completion of cell death by hypoxia. The increased MCP-1 mRNA levels suggest that encapsulated islets in vivo contribute to their own graft failure by attracting cytokine-producing macrophages. The discrepancy between iNOS mRNA and nitrite is explained by the longer half-life of NO during hypoxia. MCP-1 protein levels are underestimated as a consequence of the lower number of vital cells in combination with a higher proteolytic activity due to necrosis. Thus, strategies to eliminate hypoxia may not only improve islet function and vitality, but may also reduce the attraction of macrophages by encapsulated islets.

Targeted mutation of the SC3 hydrophobin gene of affects formation of aerial hyphae

The SC3 hydrophobin gene of Schizophyllum commune was disrupted by homologous integration of an SC3 genomic fragment interrupted by a phleomycin resistance cassette. The phenotype of the mutant was particularly clear in sealed plates in which formation of aerial hyphae was blocked. In non-sealed plates aerial hyphae did form but these were hydrophilic and not hydrophobic as in wild-type strains. Complementation with a genomic SC3 clone restored formation of hydrophobic aerial hyphae in sealed plates. In a dikaryon homozygous for the SC3 mutation normal sporulating fruiting bodies were produced but aerial hyphae were hydrophilic.

Rat islet isolation yield and function are donor strain dependent

Effective rat islet isolation is pertinent for successful islet transplantation and islet studies in vitro. To determine which rat strain yields the highest number of pure and functional islets, four commonly used rat strains were compared with regard to islet yield, islet purity and islet function. Secretory responses were assessed by stimulation with glucose, and by stimulation with glucose plus 3-isobutyl-1-methylxanthine (IBMX). We show that rat islet function and isolation yield are donor strain dependent. Albino Oxford (AO) rats donated twice as many islets than Wistar, Lewis and Sprague Dawley (SD) rats. Stimulation with glucose plus IBMX resulted in an average five-fold increase of the stimulation index of AO, Lewis, Wistar and SD rats compared to stimulation with glucose only. AO islets had improved secretory responses after a one-week culture period, but required the addition of IBMX to glucose to elicit a distinguished stimulated insulin secretion after 2 days of culture. Islets from SD rats showed inferior results with regard to purity immediately after isolation and with regard to function after short- and after long-time culture. Because Lewis islets possessed the highest secretory response to glucose (without IBMX) immediately after isolation, Lewis rats may be preferred as islet donors for immediate use. The addition of IBMX to glucose for in vitro functional testing is recommended because it elicits high insulin secretory responses of islets regardless of the rat strain. AO rats are preferred for culture experiments since the number of experimental animals is reduced two-fold compared to Lewis, Wistar and SD rats.