Laryngeal transplantation in minipigs: early immunological outcomes.

Despite recent tissue-engineering advances, there is no effective way of replacing all the functions of the larynx in those requiring laryngectomy. A recent clinical transplant was a success. Using quantitative immunofluorescence targeted at immunologically relevant molecules, we have studied the early (48 h and 1 week) immunological responses within larynxes transplantated between seven pairs of National Institutes of Health (NIH) minipigs fully homozygous at the major histocompatibility complex (MHC) locus. There were only small changes in expression of some molecules (relative to interindividual variation) and these were clearest in samples from the subglottic region, where the areas of co-expression of CD25(+) CD45RC(-) CD8(-) and of CD163(+) CD172(+) MHC-II(-) increased at 1 week after transplant. In one case, infiltration by recipient T cells was analysed by T cell receptor (TCR) Vß spectratype analysis; this suggested that changes in the T cell repertoire occur in the donor subglottis mucosal tissues from day 0 to day 7, but that the donor and recipient mucosal Vß repertoires remain distinct. The observed lack of strong immunological responses to the trauma of surgery and ischaemia provides encouraging evidence to support clinical trials of laryngeal transplantation, and a basis on which to interpret future studies involving mismatches.

Laryngeal transplantation in minipigs: vascular, myologic and functional outcomes.

There is no effective way of replacing all the functions of the larynx in those requiring laryngectomy. Regenerative medicine offers promise, but cannot presently deliver implants with functioning neuromuscular units. A single well-documented laryngeal transplant in man was a qualified success, but more information is required before clinical trials may be proposed. We studied the early response of the larynx to laryngeal transplantation between 17 pairs of NIH minipigs full matched at the MHC2 locus. Following iterative technical improvements, pigs had good swallowing and a patent airway at 1 week. No significant changes in mucosal blood flux were observed compared with pre-operative measurements. Changes in muscle morphology and fibre phenotype were observed in transplant muscles retrieved after 7 days: the levels of fast and slow myosin heavy chain (MyHC) protein were reduced and embryonic MyHC was up regulated consistent with denervation induced atrophy. At 1 week laryngeal transplantation can result in good swallowing, and is not associated with clinical evidence of ischemia-reperfusion injury in MHC-matched pigs.

Growth and phosphorus nutrition of a Paris-type arbuscular mycorrhizal symbiosis.

• Paris -type arbuscular mycorrhizas (AM) are reportedly the most common morphological type of AM; however, most research has focused on the Arum -type. Asphodelus fistulosus , a common weed in southern Australia, forms Paris -type AM when colonised by Glomus coronatum . It is often found in sites with low nutrient levels, and may therefore be dependent on its AM associations for growth and phosphorus (P) nutrition. • A. fistulosus was inoculated with G. coronatum and grown in pots containing a soil/sand mixture with P added to give five soil P concentrations. The plants were grown in a glasshouse and harvested 6 and 9 wk after planting, at which times growth, P nutrition and colonisation were measured. • At low soil P, A. fistulosus showed very marked positive responses to colonisation both in P uptake and growth; both responses decreased with increasing P supply. Colonisation was not greatly reduced by increasing P supply. • This study appears to be one of the first detailed investigations of P responses in a Paris -type AM, providing insight into what is reportedly the more common but less well studied morphological type of AM.

Quantitative development of Paris-type arbuscular mycorrhizas formed between Asphodelus fistulosus and Glomus coronatum.

• Arum- and Paris-type symbioses are the two main morphological types of arbuscular mycorrhiza. Here, the developmental time-course of the Paris-type association formed from colonization of Asphodelus fistulosus (onion weed) by Glomus coronatum is presented. • Development was monitored over 27 d. Root colonization was assessed using a modification of the magnified intersects technique (MIT), for investigating the interdependence (thus IMIT) of structures. • Hyphal and arbusculate coils were found predominantly in the outer and inner cortex of the root, respectively. The interdependence of external hyphae, hyphal coils and arbusculate coils was determined during the relatively slow development of the symbiosis. • The time required for development of Paris-type arbuscular mycorrhizas is slower than for the Arum type, and both time and space influence the formation of hyphal coils. Use of IMIT for scoring colonization allows determination of the interdependence of different fungal structures, and thus the technique has potentially wide applications, such as in relating the presence of different structures to signals from molecular probes.

Transmembrane electric potential difference of germ tubes of arbuscular mycorrhizal fungi responds to external stimuli.

Measurements of the electric potential difference across the hyphal wall and the cell membrane were made on external hyphae of three species of arbuscular mycorrhizal fungus Gigaspora margarita, Scutellospora calospora and Glomus coronatum and on germ tubes of Gi. margarita. The values of transmembrane electric potential difference recorded (∼-40 mV) are less negative than those previously reported from hyphae of arbuscular mycorrhizal fungi closely associated with roots and from filamentous fungi. The external hyphae of arbuscular mycorrhizal fungi grown in soil had similar values of electric potential difference to those grown in soil-less culture, and to germ tubes. Thermodynamic calculations showed that despite these low values of electric potential difference, efficient high-affinity uptake of phosphate is possible. The transmembrane electric potential difference of germ tubes of Gi. margarita became more negative when plant root extract was added to the medium, showing for the first time that the early stages of interaction between plant and fungus occur via direct effects on the plasma membrane rather than via effects on gene expression. Addition of K+ reversibly depolarized the transmembrane electric potential difference of germ tubes of Gi. margarita, indicating that despite the low electric potential difference the fungus has control over the permeability of the plasmamembrane to K+ .