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.

Shiitake Flagellate Dermatitis: the First Case Reported in Ireland.

Shiitake (Lentinula edodes) is the second most commonly consumed mushroom worldwide. The first case of shiitake mushroom flagellate dermatitis was described in Japan in 1977 and it is now being reported in the western world. We describe the first reported case in Ireland.

Alpha-1-antitrypsin deficiency-related panniculitis: two cases with diverse clinical courses.

Alpha-1-antitrypsin deficiency (AATD)-related panniculitis is an extremely rare and underdiagnosed entity, and there is a paucity of data on its treatment. We report two cases of AATD-related panniculitis. The first was a 24-year-old woman with known AATD who presented with painful leg ulcers refractory to treatment with corticosteroids and colchicine. She had a good response to α1-antitrypsin infusions but required dose adjustment due to flares in disease activity. The second case was a 38-year-old woman who presented with painful nodules on the legs refractory to corticosteroid therapy. Laboratory investigations revealed severe AATD. She had an excellent response to colchicine therapy. In both these cases of AATD, panniculitis was the first clinical manifestation of the disease. AATD-related panniculitis may have none of the typical clinical clues for AATD, such as a family history, cirrhosis or emphysema. Early identification may help prevent these complications from developing.

An uncommon dermatophyte infection: two cases of cutaneous infection with Trichophyton verrucosum.

Zoophilic dermatophytes can cause highly inflammatory cutaneous infections. Cattle represent the largest reservoir for the zoophilic dermatophyte Trichophyton verrucosum. Effective vaccination programmes have contributed to a low rate of livestock infection in central and northern Europe, and T. verrucosum infection is relatively more common in southern Europe and in Arabic countries. Transmission to humans typically results from direct contact with infected livestock. It may also be transmitted from person to person. We report two cases of T. verrucosum skin infections in Irish farmers. In both cases, effective treatment was delayed due to misdiagnosis of the condition as a bacterial infection in the primary care setting. Both cases responded rapidly to treatment with oral terbinafine. Culture of T. verrucosum can take 3 weeks or longer to grow, therefore a high index of clinical suspicion is necessary, and skin scrapings for potassium hydroxide microscopy and culture are essential for accurate diagnosis.

A rare case of arterial thrombosis in a 37-year-old male with Factor V Leiden mutation.

BACKGROUND: The classic triad of symptoms seen in chronic mesenteric ischaemia is post-prandial pain, sitophobia (fear of food) and progressive weight loss. Patients with mesenteric ischaemia secondary to a prothrombotic state such as that rendered by the Factor V Leiden mutation, are substantially younger than the typical elderly patient in whom embolic disease triggered by atrial fibrillation is the main underlying cause. METHOD: This is one such case report documenting arterial thrombosis in a 37-year-old male with a subsequently identified heterozygous Factor V Leiden mutation. CONCLUSION: Factor V Leiden mutation is a contributing risk factor in cases of small bowel infarction.

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)

The role of RNA editing of kainate receptors in synaptic plasticity and seizures.

The ionotropic glutamate receptor subunit GluR6 undergoes developmentally and regionally regulated Q/R site RNA editing that reduces the calcium permeability of GluR6-containing kainate receptors. To investigate the functional significance of this editing in vivo, we engineered mice deficient in GluR6 Q/R site editing. In these mutant mice but not in wild types, NMDA receptor-independent long-term potentiation (LTP) could be induced at the medial perforant path-dentate gyrus synapse. This indicates that kainate receptors with unedited GluR6 subunits can mediate LTP. Behavioral analyses revealed no differences from wild types, but mutant mice were more vulnerable to kainate-induced seizures. Together, these results suggest that GluR6 Q/R site RNA editing may modulate synaptic plasticity and seizure vulnerability.

Identification of the kainate receptor subunits underlying modulation of excitatory synaptic transmission in the CA3 region of the hippocampus.

To understand the physiological role of kainate receptors and their participation in seizure induction in animal models of epilepsy, it will be necessary to develop a comprehensive description of their action in the CA3 region of the hippocampus. Activation of presynaptic kainate receptors depresses excitatory synaptic transmission at mossy fiber and associational-commissural inputs to CA3 pyramidal neurons (Vignes et al., 1998; Bortolotto et al., 1999; Kamiya and Ozawa, 2000). In this study, we use gene-targeted mice lacking glutamate receptor 5 (GluR5) or GluR6 kainate receptor subunits to identify the receptor subunits that comprise the kainate receptors responsible for presynaptic modulation of CA3 transmission. We found that bath application of kainate (3 microm) profoundly reduced EPSCs at mossy fiber and collateral synapses in neurons from wild-type and GluR5(-/-) mice but had no effect on EPSCs in neurons from GluR6(-/-) mice. These results therefore contrast with previous studies that supported a role for GluR5-containing receptors at mossy fiber and associational-commissural synapses (Vignes et al., 1998; Bortolotto et al., 1999). Surprisingly, at perforant path synapses kainate receptor activation enhanced transmission; this potentiation was abolished in both GluR5 and GluR6 knock-out mice. Kainate receptors thus play multiple and complex roles to modulate excitatory synaptic transmission in the CA3 region of the hippocampus.

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.

Subunit composition of kainate receptors in hippocampal interneurons.

Kainate receptor activation affects GABAergic inhibition in the hippocampus by mechanisms that are thought to involve the GluR5 subunit. We report that disruption of the GluR5 subunit gene does not cause the loss of functional KARs in CA1 interneurons, nor does it prevent kainate-induced inhibition of evoked GABAergic synaptic transmission onto CA1 pyramidal cells. However, KAR function is abolished in mice lacking both GluR5 and GluR6 subunits, indicating that KARs in CA1 stratum radiatum interneurons are heteromeric receptors composed of both subunits. In addition, we show the presence of presynaptic KARs comprising the GluR6 but not the GluR5 subunit that modulate synaptic transmission between inhibitory interneurons. The existence of two separate populations of KARs in hippocampal interneurons adds to the complexity of KAR localization and function.

Generation and analysis of GluR5(Q636R) kainate receptor mutant mice.

The physiological significance of RNA editing of transcripts that code for kainate-preferring glutamate receptor subunits is unknown, despite the fact that the functional consequences of this molecular modification have been well characterized in cloned receptor subunits. RNA editing of the codon that encodes the glutamine/arginine (Q/R) site in the second membrane domain (MD2) of glutamate receptor 5 (GluR5) and GluR6 kainate receptor subunits produces receptors with reduced calcium permeabilities and single-channel conductances. Approximately 50% of the GluR5 subunit transcripts from adult rat brain are edited at the Q/R site in MD2. To address the role of glutamate receptor mRNA editing in the brain, we have made two strains of mice with mutations at amino acid 636, the Q/R-editing site in GluR5, using embryonic stem cell-mediated transgenesis. GluR5(RloxP/RloxP) mice encode an arginine at the Q/R site of the GluR5 subunit, whereas GluR5(wt(loxP)/wt(loxP)) mice encode a glutamine at this site, similar to wild-type mice. Mutant animals do not exhibit developmental abnormalities, nor do they show deficits in the behavioral paradigms tested in this study. Kainate receptor current densities were reduced by a factor of six in acutely isolated sensory neurons of dorsal root ganglia from GluR5(RloxP/RloxP) mice compared with neurons from wild-type mice. However, the editing mutant mice did not exhibit altered responses to thermal and chemical pain stimuli. Our investigations with the GluR5-editing mutant mice have therefore defined a set of physiological processes in which editing of the GluR5 subunit is unlikely to play an important role.

Selection of primary cell cultures with cre recombinase induced somatic mutations from transgenic mice.

Deletion of genes in defined cell types has been achieved using a combination of gene targeting techniques and the Cre- lox P recombination system. Here we present a method to selectively isolate genetically altered primary cell cultures based on the permanent activation of a drug-resistance gene by the Cre recombinase. Transgenic mice were generated harboring a dormant form of the hygromycin resistance gene. This mouse line was crossed with mice carrying a constitutive Cre gene and an endogenous floxed allele. Primary fibroblasts established from triple transgenic embryos displayed not only hygromycin resistance but also recombination of the endogenous floxed allele. These results prove the potential of this approach.

Death and dying in contemporary society: an evaluation of current attitudes and the rituals associated with death and dying and their relevance to recent understandings of health and healing.

This paper develops a model which is intended to help nurses and other health professionals in the understanding of contemporary views regarding death and dying and the associated issues of health and healing. The author contends that in the first half of the 20th century, society lost sight of the importance of rituals associated with death and dying and of the need for appropriate death education. Consequently patients and professionals alike found themselves unable to cope with the inevitability of death. Fear supplanted hope, and the health and well-being of society was deleteriously influenced. During the second half of the century, there has been a proliferation of thanatology research and literature. Health professionals are realizing the inadequacy of their knowledge of an issue which fundamentally and unavoidably affects everyone including themselves. The holistic approach to health care has been recognized by many researchers as being essential to health and healing, and therefore death and dying have to be addressed. Often nurses are the professionals left to deal with the patients' grief and anger, and it is therefore critical that they are conversant with the contemporary parallel issues of death and dying and health and healing. The author also firmly believes that before nurses can help people to overcome the fear of death and to optimize their lives, it is essential to examine the traditions of other cultures as well as personal experiences and coping mechanisms, before an understanding of other people's fears and beliefs concerning death and dying can be reached.

Positive selection of FLP-mediated unequal sister chromatid exchange products in mammalian cells.

Site-specific recombination provides a powerful tool for studying gene function at predetermined chromosomal sites. Here we describe the use of a blasticidin resistance system to select for recombination in mammalian cells using the yeast enzyme FLP. The vector is designed so that site-specific recombination reconstructs the antibiotic resistance marker within the sequences flanked by the FLP target sites. This approach allows the detection of DNA excised by FLP-mediated recombination and facilitates the recovery of recombination products that would not be detected by available screening strategies. We used this system to show that the molecules excised by intrachromosomal recombination between tandem FLP recombinase target sites do not reintegrate into the host genome at detectable frequencies. We further applied the direct selection approach to recover a rare FLP-mediated recombination event displaying the characteristics of an unequal sister chromatid exchange between FLP target sites. Implications of this approach for the generation of duplications to assess their effect on gene dosage and chromosome stability are discussed.