Quantifying the radiant exposure and effective dose in patients treated for actinic keratoses with topical photodynamic therapy using daylight and LED white light.

Daylight photodynamic therapy (dl-PDT) is as effective as conventional PDT (c-PDT) for treating actinic keratoses but has the advantage of reducing patient discomfort significantly. Topical dl-PDT and white light-PDT (wl-PDT) differ from c-PDT by way of light sources and methodology. We measured the variables associated with light dose delivery to skin surface and influence of geometry using a radiometer, a spectral radiometer and an illuminance meter. The associated errors of the measurement methods were assessed. The spectral and spatial distribution of the radiant energy from the LED white light source was evaluated in order to define the maximum treatment area, setup and treatment protocol for wl-PDT. We compared the data with two red LED light sources we use for c-PDT. The calculated effective light dose is the product of the normalised absorption spectrum of the photosensitizer, protoporphyrin IX (PpIX), the irradiance spectrum and the treatment time. The effective light dose from daylight ranged from 3 ± 0.4 to 44 ± 6 J cm-2due to varying weather conditions. The effective light dose for wl-PDT was reproducible for treatments but it varied across the treatment area between 4 ± 0.1 J cm-2 at the edge and 9 ± 0.1 J cm-2 centrally. The effective light dose for the red waveband (615-645 nm) was 0.42 ± 0.05 J cm-2 on a clear day, 0.05 ± 0.01 J cm-2 on an overcast day and 0.9 ± 0.01 J cm-2 using the white light. This compares with 0.95 ± 0.01 and 0.84 ± 0.01 J cm-2 for c-PDT devices. Estimated errors associated with indirect determination of daylight effective light dose were very significant, particularly for effective light doses less than 5 J cm-2 (up to 83% for irradiance calculations). The primary source of error is in establishment of the relationship between irradiance or illuminance and effective dose. Use of the O'Mahoney model is recommended using a calibrated logging illuminance meter with the detector in the plane of the treatment area.

Requirement for Pbx1 in skeletal patterning and programming chondrocyte proliferation and differentiation.

Pbx1 and a subset of homeodomain proteins collaboratively bind DNA as higher-order molecular complexes with unknown consequences for mammalian development. Pbx1 contributions were investigated through characterization of Pbx1-deficient mice. Pbx1 mutants died at embryonic day 15/16 with severe hypoplasia or aplasia of multiple organs and widespread patterning defects of the axial and appendicular skeleton. An obligatory role for Pbx1 in limb axis patterning was apparent from malformations of proximal skeletal elements, but distal structures were unaffected. In addition to multiple rib and vertebral malformations, neural crest cell-derived skeletal structures of the second branchial arch were morphologically transformed into elements reminiscent of first arch-derived cartilages. Although the skeletal malformations did not phenocopy single or compound Hox gene defects, they were restricted to domains specified by Hox proteins bearing Pbx dimerization motifs and unaccompanied by alterations in Hox gene expression. In affected domains of limbs and ribs, chondrocyte proliferation was markedly diminished and there was a notable increase of hypertrophic chondrocytes, accompanied by premature ossification of bone. The pattern of expression of genes known to regulate chondrocyte differentiation was not perturbed in Pbx1-deficient cartilage at early days of embryonic skeletogenesis, however precocious expression of Col1a1, a marker of bone formation, was found. These studies demonstrate a role for Pbx1 in multiple developmental programs and reveal a novel function in co-ordinating the extent and/or timing of proliferation with terminal differentiation. This impacts on the rate of endochondral ossification and bone formation and suggests a mechanistic basis for most of the observed skeletal malformations.

The Hox cofactor and proto-oncogene Pbx1 is required for maintenance of definitive hematopoiesis in the fetal liver.

Pbx1 is the product of a proto-oncogene originally discovered at the site of chromosomal translocations in acute leukemias. It binds DNA as a complex with a broad subset of homeodomain proteins, but its contributions to hematopoiesis have not been established. This paper reports that Pbx1 is expressed in hematopoietic progenitors during murine embryonic development and that its absence results in severe anemia and embryonic lethality at embryonic day 15 (E15) or E16. Definitive myeloerythroid lineages are present in Pbx1(-/-) fetal livers, but the total numbers of colony-forming cells are substantially reduced. Fetal liver hypoplasia reflects quantitative as well as qualitative defects in the most primitive multilineage progenitors and their lineage-restricted progeny. Hematopoietic stem cells from Pbx1(-/-) embryos have reduced colony-forming activity and are unable to establish multilineage hematopoiesis in competitive reconstitution experiments. Common myeloid progenitors (CMPs), the earliest known myeloerythroid-restricted progenitors, are markedly depleted in Pbx1(-/-) embryos at E14 and display clonogenic defects in erythroid colony formation. Comparative cell-cycle indexes suggest that these defects result largely from insufficient proliferation. Megakaryocyte- and erythrocyte-committed progenitors are also reduced in number and show decreased erythroid colony-forming potential. Taken together, these data indicate that Pbx1 is essential for the function of hematopoietic progenitors with erythropoietic potential and that its loss creates a proliferative constriction at the level of the CMP. Thus, Pbx1 is required for the maintenance, but not the initiation, of definitive hematopoiesis and contributes to the mitotic amplifications of progenitor subsets through which mature erythrocytes are generated. (Blood. 2001;98:618-626)

A transactivation-deficient mouse model provides insights into Trp53 regulation and function.

The gene Trp53 is among the most frequently mutated and studied genes in human cancer, but the mechanisms by which it suppresses tumour formation remain unclear. We generated mice with an allele encoding changes at Leu25 and Trp26, known to be essential for transcriptional transactivation and Mdm2 binding, to enable analyses of Trp53 structure and function in vivo. The mutant Trp53 was abundant, its level was not affected by DNA damage and it bound DNA constitutively; however, it showed defects in cell-cycle regulation and apoptosis. Both mutant and Trp53-null mouse embryonic fibroblasts (MEFs) were readily transformed by oncogenes, and the corresponding mice were prone to tumours. We conclude that the determining pathway for Trp53 tumour-suppressor function in mice requires the transactivation domain.

Kit/stem cell factor receptor-induced activation of phosphatidylinositol 3'-kinase is essential for male fertility.

The c-kit-encoded transmembrane tyrosine kinase receptor for stem cell factor (Kit/SCF-R) is required for normal haematopoiesis, melanogenesis and gametogenesis. However, the roles of individual Kit/SCF-R-induced signalling pathways in the control of developmental processes in the intact animal are completely unknown. To examine the function of SCF-induced phosphatidylinositol (PI) 3'-kinase activation in vivo, we employed the Cre-loxP system to mutate the codon for Tyr719, the PI 3'-kinase binding site in Kit/SCF-R, to Phe in the genome of mice by homologous recombination. Homozygous (Y719F/Y719F) mutant mice are viable. The mutation completely disrupted PI 3'-kinase binding to Kit/SCF-R and reduced SCF-induced PI 3'-kinase-dependent activation of Akt by 90%. The mutation induced a gender- and tissue-specific defect. Although there are no haematopoietic or pigmentation defects in homozygous mutant mice, males are sterile due to a block in spermatogenesis, with initially decreased proliferation and subsequent extensive apoptosis occurring at the spermatogonial stem-cell level. In contrast, female homozygotes are fully fertile. This is the first report so far demonstrating the role of an individual signalling pathway downstream of Kit/SCF-R in the intact animal. It provides the first in vivo model for male sterility caused by a discrete signalling pathway defect affecting early germ cells.

A safe new procedure for high-risk patients with symptomatic gallstones.

Cholecystectomy is associated with an appreciable mortality rate in elderly high-risk patients. Patients aged over 60 years with symptomatic gallstones, at high operative risk, underwent cholecystotomy under local anaesthesia through a 3-cm incision. Stones were removed and clearance was demonstrated endoscopically and by tube cholecystography. Catheter drainage was continued for 7 days until a further cholecystogram confirmed clearance. The procedure was attempted in 26 patients with concomitant cardiovascular, respiratory or malignant disease. Successful removal of all gallbladder stones was possible in 24 patients. Four patients had common bile duct stones demonstrated on cholecystography, all of which were successfully treated by endoscopic sphincterotomy. All patients are symptom-free at a mean follow-up of 36 weeks with no recurrent stones on ultrasonography.

Cystic duct obliteration and gallbladder mucosal destruction: a feasible alternative to cholecystectomy.

'Chemical cholecystectomy' has been proposed as an alternative to removal of the gallbladder. This study assessed cystic duct obliteration using bipolar electrocoagulation (with sham cannulation controls) and gallbladder mucosal treatment with tetracycline (or saline controls) in 29 mongrel dogs. Cystic duct obstruction was assessed by tube cholecystography at day 14, and epithelial damage by histology at day 42. Electrocoagulation by duct diathermy effectively occluded the cystic duct in 14 of 19 animals; however, this was associated with mucocele formation unless mucosal treatment with tetracycline was also performed. Immediate instillation of tetracycline after duct electrocoagulation produced only partial epithelial damage. The combination of duct electrocoagulation and delayed tetracycline instillation at 14 days produced complete destruction of all gallbladder epithelium and an effective chemical cholecystectomy.