Case report: thrombotic microangiopathy post-intravenous immunoglobulin in the context of BK nephropathy and renal transplantation.

Intravenous immunoglobulin (IVIg) is a blood product with immunomodulating properties that have been widely applied in the management of renal transplant recipients. In general, IVIg has been considered a relatively safe therapy, with most adverse events being mild and transient. Although rare, a serious and well-recognized complication of IVIg is large-vessel thrombotic events, which are thought to be related to hyperviscosity. We describe here two cases in which there was a temporal relationships between the administration of IVIg, an acute decline in allograft function, and the histologic finding of de novo thrombotic microangiopathy (TMA). In both cases, IVIg had been administered to facilitate immunosuppressive dose reduction in the context of BK nephropathy. We believe this is the first report of TMA associated with IVIg administration in renal allograft recipients.

Sirolimus: a single center experience in combination with calcineurin inhibitors.

A retrospective survey was performed to establish patient and graft outcome for all 41 patients at our centre receiving sirolimus (SRL) in combination with calcineurin inhibition (CNI) as primary therapy for the 6 years prior to March 2002. Patient mortality [12%; n = 5 (TTP, lymphoma, mucormycosis, and small bowel perforation] was significantly higher at 3 months compared with those not receiving SRL, but not thereafter. 12.8% had delayed graft function and 33% had one or more episodes of rejection in the first 6 months. Mean GFR at 12 months was significantly lower (47.3 mL/min) in the SRL group compared with those not receiving SRL (51.3 mL/min). Twenty-two patients had a 12-month protocol biopsy; CNI toxicity was present in 36%. SRL was associated with significant hyperlipidaemia (serum cholesterol, 5.2 +/- 1.4 at baseline vs 7.3 +/- 1.7 mmol/L at 3 months, P <.001; triglycerides, 2.3 +/- 1.4 at baseline vs 2.7 +/- 1.1 mmol/L at 3 months, P <.05). Mild thrombocytopenia occurred in 23% but was not associated with haemorrhagic events. LDH increased by 62%, remaining elevated out to 2 years post engraftment. Seven patients developed insulin requiring diabetes mellitus, similar to the rate observed in our general transplant population.Thus, in this early experience, SRL in combination with CNI was associated with significant mortality and morbidity including CNI toxicity, presumably a reflection of a heavy burden of immunosuppression. However, 1-year graft survival on SRL was similar to the mean Australia-wide graft survival regardless of immunosuppression. The future use of SRL may center around CNI sparing and avoidance type protocols.

Analysis of the involvement of ocs-like bZip-binding elements in the differential strength of the bidirectional mas1'2' promoter.

The ocs-like elements of the bidirectional mas1'2' promoter of Agrobacterium tumefaciens, mas1' and mas2', were analyzed to elucidate their role in the expression conferred by this promoter. Tetramers of the elements were cloned upstream of the beta-glucuronidase-coding region linked to the 35S promoter deleted at -54. Transient expression assays with tobacco (Nicotiana tabacum) and potato (Solanum tuberosum) protoplasts showed that tetramers of the mas1' element had 3- to 8-fold enhancing activity, respectively. Enhancement obtained by tetramers of the mas2' element was higher, suggesting that this element plays a role in the stronger promoter activity from the 2' side. Three cDNA clones with higher homology to the tobacco transcription factor TGA1a were isolated from a potato root expression library. Overexpression of the proteins encoded by these cDNA clones in Escherichia coli and analysis of DNA-binding activity in bacterial extracts showed that all three factors could bind strongly to the mas1' ocs-like element. In contrast, only two of the mas-binding factors exhibited significant binding to the mas2' element. Southern analysis revealed the presence of a small, multigene family encoding the mas-binding factors in potato.

Expression of a calmodulin methylation mutant affects the growth and development of transgenic tobacco plants.

Transgenic plants were constructed that express two foreign calmodulins (VU-1 and VU-3 calmodulins) derived from a cloned synthetic calmodulin gene. VU-1 calmodulin, similar to endogenous plant calmodulin, possesses a lysine residue at position 115 and undergoes posttranslational methylation. VU-3 calmodulin is a site-directed mutant of VU-1 calmodulin that is identical in sequence except for the substitution of an arginine at position 115 and thus is incapable of methylation. Both calmodulin genes, under the control of the cauliflower mosaic virus 35S promoter, were expressed in transgenic tobacco. Foreign calmodulin protein accumulated in plant tissues to levels equivalent to that of the endogenous calmodulin. All transformed lines of VU-1 plants were indistinguishable from untransformed controls with respect to growth and development. However, all transformed lines of VU-3 plants were characterized by decreased stem internode growth, reduced seed production, and reduced seed and pollen viability. The data suggest that these phenotypes are the result of the expression of the calmodulin mutant rather than the position of transferred DNA insertion or the overall alteration of calmodulin levels. Analyses of the activity of the purified transgenic calmodulins suggest that calmodulin-dependent NAD kinase is among the potential targets that may have altered regulation in VU-3 transgenic plants.

Functional analysis of cis-elements, auxin response and early developmental profiles of the mannopine synthase bidirectional promoter.

The dual MAS1'-2' promoter regulating two divergently transcribed mannopine synthase genes has been widely employed in plant expression vectors. As part of an effort towards its rational design as a genetic engineering tool, we have undertaken a functional analysis of the promoter by deletion mutagenesis and by the use of hybrid promoter constructs. Our results indicate that the central region of the intergenic promoter is composed of at least four domains. Three of these contain complementary sequences, which can potentially hybridize to form alternative palindromic structures. These three domains can function cooperatively, and in an orientation-independent manner, in imparting a sevenfold higher expression level at the 2' end relative to the corresponding 1'. The remaining domain is characterized by tracts of repeated A/T-rich elements, and appears to confer the weak activity at the MAS1' promoter end. However, even though this A/T-rich DNA segment is functional, our deletion analysis provided strong evidence that it is completely dispensable for wild-type promoter activity. In addition, the relative distances between these enhancer domains and the 1'-2' TATA-proximal regions can have a pronounced influence on the level of expression in both directions. In young tobacco seedlings, the two promoter ends are expressed in similar, if not identical, tissues in the aerial parts of the plants, but major differences can be observed in roots. Transient expression assays using hybrid promoter constructs showed that cis-elements that can respond to auxin induction signals are redundant in nature, in that they are dispersed throughout the promoter and showed no obvious consensus sequence.

Perturbation of the ubiquitin system causes leaf curling, vascular tissue alterations and necrotic lesions in a higher plant.

A ubiquitin variant with Lys48 changed to Arg acts in vitro as an inhibitor of ubiquitin dependent protein degradation. To assess the role of this proteolytic pathway in the life cycle of plants, we expressed the ubiquitin variant in Nicotiana tabacum. Expression of variant mono- or polyubiquitin leads to marked abnormalities in vascular tissue. In addition, overexpression of variant polyubiquitin induces discrete lesions on leaves. This indicates that perturbations of the ubiquitin system can induce a programmed necrotic response in plants.

A technique for avoidance of hypothermia during prolonged mechanical circulatory support with centrifugal pumps.

Systemic hypothermia became a major problem in one of our patients undergoing postcardiotomy mechanical circulatory support with a centrifugal pump. We have developed a technique to prevent systemic hypothermia in this setting by applying an adapted topical cardiac cooling device to the centrifugal pump heads.

Myocardial protection: a comparison of cold blood and cold crystalloid cardioplegia.

Two hundred seven consecutive patients were randomized into four groups based on left ventricular end-diastolic pressure and subsequently into groups receiving crystalloid cardioplegia or blood cardioplegia. Hemodynamic data and enzymatic evidence of myocardial ischemia were examined postoperatively. We found slight but significant improvement in the blood cardioplegia group regarding left ventricular stroke work index. Similarly, the levels of creatine kinase and serum glutamic oxaloacetic transaminase were slightly but significantly better with blood cardioplegia. We believe that the technique of blood cardioplegia offers a slight but statistically significant advantage.

Prisms for inspection of a seesaw nystagmus.

Two eyes may be simultaneously viewed one above the other by using an arrangement of two prisms with their bases arranged to the right and to the left. When these prisms are placed in front of the glabella and tilted slightly clockwise it is possible to view both eyes simultaneously and appreciate the seesaw motion of a nystagmus.