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AKI has been observed in cases of Ebola virus disease. We describe the protocol for the first known successful delivery of RRT with subsequent renal recovery in a patient with Ebola virus disease treated at Emory University Hospital, in Atlanta, Georgia. Providing RRT in Ebola virus disease is complex and requires meticulous attention to safety for the patient, healthcare workers, and the community. We specifically describe measures to decrease the risk of transmission of Ebola virus disease and report pilot data demonstrating no detectable Ebola virus genetic material in the spent RRT effluent waste. This article also proposes clinical practice guidelines for acute RRT in Ebola virus disease.

Acute kidney injury (AKI) is a common complication in the postoperative period whose risk increases with the complexity and type of surgery and preoperative chronic kidney disease (CKD). As expected, renal dysfunction in a critically ill patient during the postoperative period is associated with a range of perturbations including volume regulation, electrolyte, and acid/base disorders. The majority of AKI patients do not require renal replacement therapy and need a stepwise approach in recognizing and managing these complications in the perioperative period. The following chapter discusses the key principles in maximizing renal perfusion during the postoperative period and managing complications of decreased renal function including volume manegement, electrolyte disorders, and acid/base derangements.

The article is based on a paper delivered to the AWCEBD KEYWORDS Conference at Cirencester in 1999, where the focus was upon staff emotional support. The article proposes that some of the causes of teacher and intelligence; worker stress are organizational factors and perceptual bias, and that staff support; stress is an inevitable consequence of working in an environment supervision where human relationships are critical for the successful management of young people with social, emotional and behavioural difficulties.The author suggests that one way of alleviating this stress is through the introduction of supervision to the educational field. Supervision in other helping professions has been successful in providing support, changing perceptions, managing emotions and coping with stressful situations, and in so doing has improved relationships with others and work performance.

Given the recent interest in attention difficulties or deficits, it is legitimate to enquire what would be considered “normal” in terms of the length of time on task that one would expect of an individual of a given age group. This paper explores the issues surrounding selective attention and attention “types”, and conclude by setting down tentative guidelines for making judgements.

Retinoids play fundamental roles in CNS development, but their distribution, metabolism, and function within the mature human CNS are unknown. In these studies, extracts of autopsy tissues recovered from histopathologically confirmed control and Alzheimer diseased brains were tested for their ability to synthesize retinoic acid. Retinaldehyde dehydrogenase (RLDH), the enzyme that forms retinoic acid from retinaldehyde, was present in hippocampus, frontal cortex, and parietal cortex. The RLDH activity of hippocampus and parietal cortex from Alzheimer diseased brains was 1.5- to 2-fold higher (p < 0.05) compared to the controls. In contrast, the RLDH activity of frontal cortex was the same for both Alzheimer diseased and control groups. A cultured human glioblastoma (U251) and neuroblastoma (LA-N-5) cell line synthesized retinoic acid from retinaldehyde or retinol, suggesting that a variety of neural cell types possess this activity. LA-N-5 cells grown in vitamin A-depleted medium had higher (p < 0.05) RLDH activity (0.35 +/- 0.04 nmol/mg/h) than LA-N-5 cells grown in vitamin A-replete media (0.15 +/- 0.02 nmol/mg/h). This difference was lost when retinol was added back to the medium, confirming that a reduction in vitamin A supply can induce RLDH activity in neural cells. However, this feedback mechanism does not appear to explain the higher RLDH activity of Alzheimer diseased hippocampus and parietal cortex, because the overall vitamin A status as indicated by serum retinol and carotenoid levels and by hippocampal retinoid content was similar for the Alzheimer diseased and control groups. These studies establish the presence of retinoids and RLDH activity in human brain tissues, and indicate that retinoic acid synthesis is modulated in some regions of Alzheimer diseased brain.

In this study, patients with Philadelphia (Ph) negative and positive chronic myelogenous leukemia (CML) were analyzed by unicolor- and dual color- (DC-) fluorescence in situ hybridization (FISH) using abl and bcr cosmid probes. Unicolor- and DC-FISH analysis revealed a BCR-ABL fusion in a Ph-negative patient. DC-FISH to interphase nuclei revealed the BCR-ABL fusion, even though no metaphases were available for metaphase-FISH and conventional cytogenetic analysis. In the remaining cases, the findings of FISH were in agreement with the cytogenetic results.

The following notes describe the working of a scheme in a Surrey comprehensive school (Glebelands School, Cranleigh) whereby students perceived as being at risk of underachieving or disaffection have been paired with volunteers among adults who work in local organisations or companies. The organisation of the scheme, the procedures involved, and the general outcomes are described. Some implications or suggestions for enhancing the scheme are set down. The general conclusion is that the initiative has been successful in bringing about benefits for targeted students, their peers and teachers, and for the adult volunteers themselves, and that the scheme would usefully continue.

We studied two cases of chronic myeloid leukemia (CML) having variant complexes translocations detected by trypsin G-banding and fluorescence in situ hybridization (FISH). Application of dual color- (DC-) FISH using abl and bcr cosmid probes permitted us to detect the bcr-abl fusion event on both interphase nuclei and metaphase spread. Furthermore, FISH using combinatorial hybridization (centromeric-library and library-library probes) demonstrated the content and the position of the translocations in CML patients with variant (complex type) Ph-positive rearrangements. FISH analysis appears to be superior than conventional cytogenetic analysis.

Background Continuous renal replacement therapy (CRRT) is an important tool in the care of critically ill patients. However, the impact of a specific CRRT machine type on the successful delivery of CRRT is unclear. The purpose of this study was to evaluate the effectiveness of CRRT delivery with an intensive care unit (ICU) bedside nurse delivery model for CRRT while comparing circuit patency and circuit exchange rates in 2 Food and Drug Administration–approved CRRT devices. This article presents the data comparing circuit exchange rates for 2 different CRRT machines. Materials and Methods A group of ICU nurses were selected to undergo expanded training in CRRT operation and empowered to deliver all aspects of CRRT. The ICU nurses then provided all aspects of CRRT on 2 Food and Drug Administration–approved CRRT devices for 6 months. Each device was used exclusively in the designated ICU for a 2-week run-in period followed by 3-month data collection period. The primary end point for the study was the differences in average number of filter exchanges per day during each CRRT event. Results A total of 45 unique patients who underwent 64 separate CRRT treatment periods were included. Four CRRT events were excluded (see text for details). Twenty-eight CRRT events occurred in the NxStage System One arm (NxStage Medical, Lawrence, Mass) and 32 events in the Gambro Prismaflex arm (Gambro Renal Products, Boulder, Colo). Average (SD) filter exchanges per day was 0.443 (0.60) for the NxStage System One machine and 0.553 (0.65) for Gambro Prismaflex machine (P = .09). Conclusions There was no demonstrable difference in circuit patency as defined by the rate of filter exchanges per day of CRRT therapy.

OBJECTIVE To report the pharmacokinetic and pharmacodynamic properties of vancomycin in 4 patients undergoing high-volume continuous venovenous hemofiltration (CVVH). CASE SUMMARY Data from 4 patients prescribed high-volume CVVH for acute renal failure treated with vancomycin were analyzed. Vancomycin plasma concentrations were measured 4 and 24 hours after the end of a 1-hour vancomycin infusion. The mean therapy fluid rate on initiation of vancomycin was 56.2 mL/kg/h (range 48.0–65.5). The mean loading dose of vancomycin was 18.3 mg/kg (range 14.7–19.7). Median vancomycin concentration 4 hours after the dose was 18.1 mg/L (range 13.1–30.0). At 24 hours after the dose, only 1 patient had a detectable vancomycin concentration (5.2 mg/L). DISCUSSION There was a large variability in the clearance of vancomycin in this patient population. Current strategies for dosing vancomycin may lead to subtherapeutic trough concentrations. Vancomycin dosing in this patient population should be based on a detailed assessment of the CVVH prescription, vancomycin concentrations, and clinical needs and response. CONCLUSIONS An initial vancomycin dose of 20–25 mg/kg with frequent monitoring and adjustment is recommended for patients receiving high-volume CVVH.

We have evaluated the in vivo effects of 13-cis retinoic acid (13-cis RA) on human antibody responses to immunization with tetanus toxoid (TT) and keyhole limpet hemocyanin (KLH). Subjects with severe cystic acne were immunized with suboptimal doses (10 micrograms) of KLH 7 d and 3 months after starting retinoid therapy (13-cis RA, 1 mg/kg/day for 4 mo). A standard booster immunization with TT was given along with the initial KLH sensitization. A control group of acne patients received identical immunization regimens, but no 13-cis RA. Plasma retinoid levels were evaluated by reverse-phase HPLC and confirmed that blood-level concentrations of 13-cis RA and metabolites in these acne patients reached values previously demonstrated to be immunomodulatory in vitro. The retinoid had no effect on responses to TT as reflected by the characteristics of increased anti-TT IgG levels or the isotype distribution of the antibody. In contrast, the anti-KLH response was significantly enhanced in the 13-cis-RA-treated group. Whereas anti-KLH antibody was detected in only 4 of 13 control subjects after the secondary immunization, 10 of 13 retinoid-treated subjects had measurable levels of anti-KLH IgG (p less than 0.05). Among the responders, no differences were noted in the isotype distribution of anti-KLH antibody. These results showing enhanced anti-KLH responses induced by 13-cis RA therapy represent the first demonstration in humans that in vivo administration of a retinoid can modulate antigen-specific immune responses.

The purpose of this study was to establish the prevalence of Barrett's esophagus and erosive esophagitis in a group of patients undergoing upper endoscopy for dyspepsia. Computerized endoscopy records were retrospectively evaluated to identify patients who underwent upper endoscopy for dyspepsia. Objective findings were recorded, including the presence of Barrett's esophagus, erosive esophagitis, and peptic ulcer disease. Among 264 patients, Barrett's esophagus was found in 16 (6.1%). The mean length of Barrett's was 2.0 cm, and the majority (81.3%) was short segment. Erosive esophagitis was found in 62 patients (23.%), and peptic ulcer disease was found in 25 patients (9.5%). Approximately 30% of patients undergoing endoscopy for dyspepsia had esophageal lesions. The prevalence of Barrett's in this population was 6%. Based on these results, a trial of acid suppression may benefit a third of patients with dyspepsia. Current screening practices for Barrett's in reflux patients alone may need to be reevaluated.

Chromoendoscopy and magnification endoscopy appear to be a valuable adjuncts for the detection and classification of BE. These techniques may also prove to be useful aids in surveillance protocols for identifying dysplastic epithelium or early cancer within a segment of BE. Ideally, the use of these techniques would enable the endoscopist to rule in or out the presence of IM and of dysplastic or cancerous epithelium by obtaining only a minimal number of targeted biopsy specimens, or potentially performing no biopsies at all. This could transform upper endoscopy into a much more effective screening and surveillance tool for BE. Several problems currently exist for the use of chromoendoscopy for BE. Results of studies reporting the accuracy of chromoendoscopy remain mixed,and are likely explained by the wide range of techniques and materials used in the investigations. Staining adds several steps, and likely several minutes, to an upper endoscopy. Staining within the esophagus is often patchy and uneven. In addition, poor spraying technique exaggerates the irregular uptake by the mucosa. There is a high false-positive rate when staining gastric-type epithelium and denuded epithelium. Areas of dysplasia or cancer may take up stain in an irregular manner, or may not stain at all. Chromoendoscopy is a relatively new technique in the management of BE and depends on the skill and experience of the endoscopist. Magnification, however, only allows the endoscopist to observe small areas of mucosa at a time, increasing the overall complexity and length of the procedure. The learning curve for this procedure is relatively short, however, and endoscopists can usually become proficient in the technique quickly. Currently, the greatest body of literature exists concerning the use of methylene blue for diagnosing BE. At the present time, chromoendoscopy and magnification endoscopy appear to be most beneficial in detecting IM in short segments of esophageal columnar-appearing mucosa. If used consistently by practicing physicians, the accuracy of biopsies for IM could be improved. If endoscopic ablative therapy for HGD and early adenocarcinoma becomes accepted, sensitive methods of detecting residual BE after ablation will be needed to help guide additional endoscopic therapy. Chromoendoscopy and magnification endoscopy could prove helpful in this setting. Further research in this field remains to be performed. As a first step, a uniform classification system for staining and magnification patterns should be devised. If investigators can reach a consensus, and validate classification, terminology, and pattern-types, future studies could be performed using "common and similar language." More controlled investigations with larger numbers of patients must be performed before tissue staining and magnification endoscopy become a part of the practicing endoscopist's armamentarium. The ultimate aims of chromoendoscopy and magnification endoscopy in the setting of BE are to show improved outcomes--namely, early detection of cancer and improved survival rates. These goals have not yet been realized and meeting them will require well-designed studies in the future.

In acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) there are frequently complex karyotypes with multiple structurally altered chromosomes, many of which are marker chromosomes of unknown origin. The aim of this study was to apply comparative genomic hybridization (CGH) to cases of AML or MDS in transformation submitted for routine cytogenetic analysis to investigate whether this approach would yield any further information and, if possible, to predict which cases would benefit from CGH analysis. Nineteen cases with AML or MDS in transformation were analyzed. CGH revealed nine cases with gains or losses of chromosomal material. In six of these cases the chromosomal location of this material was not apparent from cytogenetic analysis especially when multiple markers were present. By using fluorescence in situ hybridization (FISH) with specific libraries for the chromosome regions that showed discordance between CGH and conventional cytogenetics, we were able to identify the chromosome location of material within the karyotype. In this group of six patients, four cases of an unbalanced translocation involving regions of chromosomes 5 and 17 were characterized. Three of these cases had additional abnormalities, including two cases with regions of amplification in which oncogenes are located (MYC, MLL) and one case with a dic(7;21)(p10;p10). In all six cases it was possible to characterize complex chromosomal aberrations such as derivative chromosomes, marker chromosomes, and ring chromosomes. This study demonstrates that CGH can detect true gain and loss of critical chromosome regions more accurately than conventional karyotyping in cases with very complex karyotypes, and can thus prove useful in predicting prognosis and pinpointing areas of the genome that require further study. Also, CGH can be a useful technique to identify the origin of marker chromosomes, and it can assist in choice of probes for confirmatory FISH, when there is no clue provided from the analysis of G-banded chromosomes.

All-trans-retinoic acid (ATRA) has been shown to be one of the most potent chemical inducers of human neuroblastoma differentiation. The recent discovery that the stereoisomer of ATRA, 9-cis-retinoic acid (9-cis-RA), binds to both the retinoic acid and retinoid X series of receptors prompted us to evaluate the ability of this compound to promote differentiation of this cell type. Using the LA-N-5 cell line, we have now determined that 9-cis-RA can induce the differentiation of human neuroblastoma cells as evidenced by dose-dependent inhibition of cell proliferation, neurite outgrowth, increased acetylcholinesterase activity, and reduction of N-myc mRNA expression. In comparing the effects of 9-cis-RA to ATRA, we found that while both compounds induced qualitatively similar cholinergic (versus adrenergic) features in LA-N-5 cells, 9-cis-RA was 5-to-10-fold more potent than ATRA in its antiproliferative and differentiation activity. These results were supported by transient transfection experiments utilizing chloramphenicol acetyltransferase (CAT) plasmid constructs containing a retinoic acid responsive regulatory element which showed a 2-to-3-fold increase in reporter gene activity induced with 9-cis-RA over that seen with ATRA at pharmacologically relevant retinoid concentrations (> 10(-8) M). Furthermore, we have determined that 9-cis-RA can significantly enhance mRNA levels of the nuclear retinoic acid receptors alpha and beta in LA-N-5 cells. Taken together, these findings have established the ability of 9-cis-RA to induce neuroblastoma differentiation and suggest that this retinoic acid isomer may have better therapeutic characteristics than ATRA.

THE POLYETHERSULFONE MEMBRANE “EXPRESS” HAS A HIGHER PIPERACILLIN/TAZOBACTAM CLEARANCE INDEPENDANT OF THE DIALYSIS DOSE. Milen Amde, Charbel Salem, Joseph Groszek, Michael Connor, Seth Bauer, William Fissell; Cleveland Clinic Foundation, Cleveland, Ohio. In the intensive care setting, infection remains the leading cause of death in AKI. The goal of our study is to assess the impact of membrane types and continuous veno-venous hemodialysis (CVVHD) dose on the clearance and serum levels of piperacillin-tazobactam (piptazo). Using high-performance liquid chromatography (HPLC), we measured the serum and dialysate concentrations of pip-tazo. We then calculated the total clearance and area under the curve (AUC) of the drug in 28 anuric ICU patients who were on CVVHD for uninterrupted 24 hours and received 3.375 g Pip-Tazo q 8 hours (n=21) or q 12 hours (n=7). The 15 patients on the polyacrylnitrile M100 had a total pip clearance of 50.3±17.56 ml/min compared to 64.28±25.77 ml/min in the 13 patients on the polyethersulfone membrane Express. We used regression analysis to examine the association between total clearance, trough level, AUC and the variables; filter type, CVVHD dose and dosing interval (the latter was included in the trough level and AUC analysis only). We showed a trend towards lower total clearance with the M100 membrane compared to the Express membrane (coefficient of -11.62, p=0.0203). There was a trend towards lower AUC with higher CVVHD dose (coefficient of -83.2, p=0.0281) and less frequent dosing (-3.8 coefficient for q12 vs q8, p=0.0322) independent of membrane type. In conclusion, the membrane type and the CVVHD dose should be considered when deciding on antibiotics dosage in the critically ill patient. Larger studies are needed to study the impact of CRRT on antibiotics target pharmacokinetic levels and whether more frequent dosing of antibiotics would affect outcomes.

Cutaneous protection against ultraviolet B (UVB) radiation damage by endogenous glutathione (GSH) was evaluated in the epidermis of the hairless mouse by measuring the influence of GSH depletion on sunburn cell (SBC) formation. Cellular GSH exerts antioxidant effects and recent studies have suggested a role for oxygen radicals in the production of SBC. Hairless mice (Skh/h 1) received oral treatment with buthionine S,R-sulfoximine (BSO), an irreversible inhibitor of gamma-glutamylcysteine synthetase, to deplete cutaneous GSH; 4 d later their ears were exposed to UVB radiation. BSO treatment significantly reduced GSH levels in the epidermis to 10-15% of control levels. Twenty-four hours after UVB exposure, SBC counts in the ears of animals with and without BSO treatment were measured, and those exposed to UVB were found to have increased. Greater numbers of SBC were found in the ears of BSO-treated mice exposed to 15 or 20 mJ/cm2 UVB, than in non-BSO-treated mice exposed to the same UVB doses. At higher UVB doses, there were no statistically significant differences between the groups. The results show that endogenous GSH provides the epidermis with measurable protection against injury by low or moderate UVB doses.

The induction of ornithine decarboxylase (ODC) activity may be an essential component of skin tumor promotion. ODC requires pyridoxal 5′-phosphate (PLP) as a cofactor. We have measured the epidermal PLP concentration and investigated its relationship to DNA synthesis and ODC activity in the hairless mouse. The epidermal PLP concentration was approximately 1.0 μg/g. When tape-stripping was used to induce ODC activity in the epidermis the concentration of PLP was significantly elevated 4.5 h later at the time of peak ODC activity and when DNA synthesis was reduced. Systemic treatment with the vitamin B-6 antagonist 4′-deoxypyridoxine (4-DOP) significantly reduced the epidermal PLP concentration and DNA synthesis. The ODC activity induced in the epidermis 4.5 h after tape-strip- ping in 4-DOP-treated mice was only 17% of that induced in untreated tape-stripped controls. In in vitro experiments it was shown that while 4-DOP does not inhibit ODC activity, a major metabolite of 4-DOP, 4-DOP-phosphate ( K i .06 mM), does. In mixing experiments it was shown that the epidermal extracts from 4-DOP-treated mice did not contain significant amounts of ODC inhibitors. 4-DOP may inhibit ODC induction in the epidermis by depleting the PIP content.

One hundred and one subjects (52 men, 49 women) with low probability coronary disease and normal resting left ventricular ejection fraction performed upright bicycle exercise to target without angina, ischemic ECG change, or segmental wall motion abnormality. Fifteen millicuries of Tc-99m pertechnetate were injected at rest and during peak exercise, and images were processed with first pass technique (RNA). The responses were not uniform. Group 1 was characterized by the resting end diastolic volume index (EDVI) usually greater than 80 ml/M2, a peripheral resistance drop (decreases R) of 0.20-0.50, an increase in cardiac index (increases CI) of 2.8-5.5 1/minute/M2, accompanied by decreasing EDVI and ESVI with exercise, stroke volume index (SVI) remaining essentially unchanged, and left ventricular ejection fraction (EF) usually increasing. Group 2 was characterized by the resting EDVI less than 80 ml/M2, and with the same decreases R and increases CI; EDVI, ESVI, SVI all increased and EF tended to decrease. Group 3 was characterized by decreases R greater than 0.50, increases CI greater than 5.5 1/minute M2, the exercise EDVI usually increasing, ESVI usually decreasing, SVI usually increasing, and EF usually increasing. There were no significant differences (alpha = 0.01) in exercise-related changes between men and women within each group.

Induction of mouse epidermal ornithine decarboxylase activity by u.v. radiation has been studied with a monochromator. Dose-response studies were carried out with 10 nm increments between 260 and 320 nm. Time-course studies were carried out at 280, 290 and 300 nm. Ornithine decarboxylase activity, assessed at 24 h post-irradiation, was induced in a dose-dependent fashion between 260 and 310 nm with peak activity at 280-290 nm. These data suggest that the chromophore is a protein.

The ratio of peak systolic pressure to end-systolic volume (PSP/ESV) is a measure of contractility that is relatively independent of loading conditions. To define the relation of this index to the natural history of chronic mitral insufficiency, follow-up studies were performed in 76 patients. All had isolated mitral insufficiency and were followed up for an average of 48 months. None underwent surgery. Cardiac volumes, ejection fraction and PSP/ESV ratio were calculated and Cox multiple regression analyses were performed to determine the relation of functional status, ejection fraction and PSP/ESV ratio to morbidity and mortality. Twenty-three patients died during follow-up; in 70% of those who died, the PSP/ESV ratio was reduced below the 20th percentile. However, as an independent predictor of mortality, this ratio was less sensitive (p greater than 0.05) than ejection fraction (p less than 0.01). Similarly, functional status change was predicted more accurately by ejection fraction (p less than 0.01) than by the PSP/ESV ratio (p greater than 0.05). Thus, although a decreased PSP/ESV ratio was associated with a higher mortality rate, other clinical and laboratory variables were superior to this index for determining morbidity and mortality in patients with isolated mitral insufficiency.

After the subcutaneous injection of retinoyl β-glucuronide (RAG), both RAG and retinoic acid (RA), formed by the hydrolysis of RAG in vivo, achieved peak plasma concentrations within 1–2 h. Thereafter, RA was rapidly cleared from the plasma whereas RAG was eliminated much more slowly. No significant changes were noted in the peak (2 h) plasma levels of RAG for treatment periods up to 56 days (one injection of RAG/day), in the clearance rate of RAG from plasma, or in plasma retinol concentrations. Similarly, no consistent decrease in plasma levels of the RA hydrolysis product was observed. Mice undergoing these long-term chronic treatments with RAG did not show any clinical manifestations of retinoid toxicity. Taken together, our findings that chronic dosing with RAG produces sustained levels of both the parent compound and the RA hydrolysis product, combined with the apparent low toxicity of RAG, suggest that RAG could be a safe and useful alternative to some retinoids which are presently being utilized in the clinic.

The purpose of this study was to examine the dose response and time course relationships between PUVA (psoralen + UVA) depletion of skin glutathione (GSH) and the induction of inflammation. Dorsal skin fold thickness (DSFT), an index of cutaneous edema, was used as a noninvasive measure of inflammation. Ornithine decarboxylase (ODC) was used as a measure of epidermal damage. Female hairless mice were given 8-methoxypsoralen (8-MOP) (dissolved in corn oil) by gavage at different doses, and 2h later the mice were irradiated with 5 J/cm2 UVA. At 24 h, DSFT measurements were taken, the mice were killed, and re-measurements were taken, the mice were killed, and reduced GSH, glutathione disulfide (GSSG), and glutathione-S-transferase were measured in the epidermis and dermis. Epidermal GSH was depleted 0, 11, 45, 87, and 98% from vehicle and/or UVA-treated levels (0.7 mM) after 0.1, 0.5, 5, 25, and 50 mg/kg, respectively. In the dermis GSH decreased from 0.3mM by 47, 87, and 91% after 5, 25, and 50 mg/kg 8-MOP, respectively. Increases in DSFT of 20, 141, and 242% were observed after 5, 25, and 50 mg/kg doses, respectively. GSSG accounted for a small portion of total GSH in the skin after PUVA treatment. The maximal decreases in GSH were not observed until 24–48h after PUVA treatment. PUVA treatment leads to dose-related increases in dermal edema, epidermal ODC, and depletion of GSH levels from both compartments in the skin. The time course of glutathione loss suggests that PUVA may interfere with its resynthesis or utilization from the circulation.

The retention of tritium-labeled all- trans -retinoic acid (RA), 13- cis -retinoic acid (13- cis -RA), aromatic retinoid, and arotinoid ethyl ester in the epidermis and dermis of hairless mice was measured up to 24 h following a single topical application in an acetone vehicle. The radioactivity in the tissues was extractable with chloroform:methanol, and the identities of the radioactive species extracted were confirmed as retinoids using thin-layer chromatography. All the retinoids were absorbed into the skin rapidly. After an initial period of distribution and penetration (lasting for about 1 h) the amount of the applied retinoid remaining in the epidermis and dermis decreased more slowly, obeying first order (exponential) decay kinetics. Both the arotinoid and the aromatic retinoid persisted for longer in the epidermis than equivalent doses of RA or 13- cis -RA. The half-lives of the retinoids in the dermis tended to be longer than in the epidermis, except for the arotinoid. Aromatic retinoid persisted for longest in the dermis with a half-life of 11 h.

The cytotoxicity, and ability to induce the expression of prophage in Salmonella typhimurium LT2 strains has been examined for 4 furocoumarins, 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP), 3-carbethoxypsoralen (3-CP), and 5-methylisopsoralen (5-MI) given with ultraviolet-A radiation (u.v.A). 8-MOP and 5-MOP have a linear tricyclic structure and possess two photoreactive sites, the 3,4 and 4',5' double bonds, enabling them to form both monoadducts and interstrand cross-links with DNA. The angular structure of 5-MI imposes steric constraints preventing it from forming interstrand cross-links with DNA although it possesses both of the photoreactive double bonds. The substituent group at position C-3 in 3-CP blocks the 3,4 reactive site and 3-CP is thus incapable of forming interstrand cross-links; 3-CP is reportedly, unlike the other three furocoumarins, non-carcinogenic in mice. 8-MOP, 5-MOP, and 5-MI were very cytotoxic to both the base-pair (TA1535) and frame-shift (TA1538) tester strains when given with u.v.A. Comparable amounts of 3-CP given with u.v.A were much less toxic. 8-MOP, 5-MOP, and 5-MI were potent inducers of prophage expression in both TA1535 and TA1538. 3-CP was a very poor inducer of prophage. The cytotoxicity and prophage inducing ability of 5-MI + u.v.A indicate that these actions are not necessarily restricted to the DNA crosslinking psoralens. The lower toxicity of 3-CP + u.v.A is not a simple function of the ability of 3-CP to form only monoadducts with DNA. The ability or inability to induce the expression of prophage in S. typhimurium may be a rapid and useful screen for the potential phototoxicity and carcinogenicity of novel psoralens.

In experimental studies of u.v.-skin-carcinogenesis u.v.-radiation is usually given in discrete amounts over a protracted period of time. Epidermal polyamine profiles were investigated in hairless mice after single and multiple exposures to ultraviolet-B (u.v.B). Hairless mice were irradiated with u.v.B from FS40 sunlamps and sacrificed after 1, 5, 10 or 20 days of daily irradiation with 0.9 kJ/sq m u.v.B at 6, 24 or 48 h after the final irradiation. Epidermis was analyzed for ornithine decarboxylase (ODC) activity, and for its putrescine, spermidine and spermine content. Skin biopsies were examined for histological changes. As previously reported epidermal ODC activity was induced 6 h after one irradiation with u.v.B and reached a maximum activity at approximately 24 h. In contrast after 5, 10 or 20 daily irradiations with u.v.B the epidermal ODC activity was maximal at approximately 6 h after the final irradiation and by 24 h had returned towards control levels. The magnitude of the ODC activity measured 6 h after irradiation increased with the number of irradiations. A similar pattern was seen with epidermal putrescine levels where a marked shift from a peak at approximately 24 h after one irradiation with u.v.B to a peak at approximately 6 h after 20 days of irradiation with u.v.B occurred. Spermidine levels increased as the number of u.v.B exposures was increased and spermine levels tended to decrease. The spermidine/spermine ratio increased most rapidly during the first 5 exposures, and remained elevated through to 20 days of daily irradiation. Chronic irradiation with u.v.B results in rapid induction of ODC activity and putrescine accumulation in the epidermis, events also elevated by chemical or viral transformation.

The metabolism of [2-14C] + [3′,5′,9(n)-3H]pteroylglutamate, [2-14C] + [3′,5′,9(n)-3H]10-formylpteroylglutamate, and [2-14C] + [3′,5′,9(n)-3H]10-formylpteroylpentaglutamate in rats is described. All compounds were reduced and incorporated into the folate pool. Radiolabelled folates and a number of cleavage products labelled with 3H or 14C only were excreted in the urine. An excess of 14C over 3H was present in the faeces. Bile was found to contain only intact folate derivatives. The livers of the rats were examined and the retained folates shown to be polyglutamates in all cases. Some of the urinary catabolites identified were derived from p-aminobenzoyl- l -glutamate. The metabolism of this compound in the rat was examined and found to vary according to the route of administration. The metabolic fate of the folate molecule in vivo is discussed.

Supplemental antioxidants may have a role in ameliorating or preventing the actinic damage that can lead to cutaneous disorders such as skin cancer, hyperpigmentation, and premature aging. Glutathione is an important endogenous antioxidant and fulfills various protective functions in the skin. Irradiation of hairless mice with short (UVB) or long (UVA) wavelength ultraviolet radiation or with UVA combined with a photosensitizing psoralen (PUVA) can deplete skin glutathione levels. Ultraviolet B irradiation causes rapid transient fluctuations in the epidermal glutathione level and the relative amount present as the oxidized form. Ultraviolet A irradiation can deplete epidermal and dermal glutathione for several hours but requires much higher doses than UVB. PUVA treatments may lead to extensive and prolonged depletions of epidermal and dermal glutathione, the severity of which is dependent on the psoralen dose and may last for several days. These transient depletions, oxidations, and sometimes rapid recoveries of cutaneous glutathione levels are compatible with a role for glutathione as an endogenous photoprotective agent in the skin. Experimental evidence supports such a role: for example severe skin edema develops in mice only after about 50% of the glutathione has been depleted by PUVA treatment. Although different mechanisms are involved in each case, glutathione depletion may contribute to the production of phototoxicity by UVB, UVA, and by PUVA. Understanding the depletion mechanisms may allow the development of strategies aimed at preventing loss of cutaneous glutathione, and at reinforcing the natural protective functions of this critical cell component.

— The cutaneous toxicities of the furocoumarins 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP), 3-carbethoxypsoralen (3-CP), and 5-methylisopsoralen (5-MI) administered topically (2 or 4 mg) or orally (25 mg/kg) followed by irradiation with longwave ultraviolet light (UVA) (50 kJ/ m) were compared in hairless mice. Parameters measured were the induction of ornithine decarboxylase (ODC) activity, a potential marker for skin-carcinogens, S-adenosyl-L-methionine decarboxylase, (SAMDC) activity, and the induction of skin edema (increase in dorsal skin fold thickness) and erythema, both indicators of acute phototoxicity. Edema and erythema, and epidermal ODC activity were induced by topical and oral 5-MOP, 8-MOP and 5-MI when given with UVA. The erythema found after topical 5-MI + UVA was less than after 8-MOP + UVA or 5-MOP + UVA. and first occurred at 48 h. Edema and epidermal ODC activity were also induced at later times, being maximal at 48 h or more. 3-Carbethoxypsoralen given orally (25 mg/kg) or topically (4 mg) with UVA did not have a consistent effect on the parameters measured, and appeared to be non-toxic under these conditions. These results of these studies suggest that the low toxicity of 3-CP + UVA is not necessarily related to its ability to form only monoadducts with DNA. The ability to crosslink with DNA is not a prerequisite for ODC induction and the induction of skin edema since 5-MI, which can only form monoadducts with DNA, gave a similar response to 8-MOP and 5-MOP.

All-trans-retinoic acid formation from topically applied retinol has been demonstrated in the skin of skh/hr1 (hairless) mice. The all-trans-retinoic acid was identified on the basis of its chromatographic properties on HPLC at various pH values, its photoisomerization to reaction products identical to those formed from authentic all-trans-retinoic acid, and its co-chromatography with methyl retinoate after methylation with diazomethane. Topically applied retinol is about 2-fold less potent at inducing epidermal hyperplasia and 7-fold less potent at inhibiting the induction of epidermal ornithine decarboxylase by phorbol esters than all-trans-retinoic acid in this strain of mice. To elucidate the possible role all-trans-retinoic acid formation from retinol may have in these pharmacological activities, the epidermal and dermal all-trans-retinoic acid levels were compared in mice treated topically with retinol or [11-3H]-all-trans-retinoic acid. The levels of all-trans-retinoic acid found after retinol treatment were several orders of magnitude lower than those found after [11-3H]-all-trans-retinoic acid treatment, and they were insufficient to account for the difference in potencies between all-trans-retinoic acid and retinol. Retinol was eliminated from the epidermis at a rate similar to that of all-trans-retinoic acid after topical administration, but the initial tissue levels achieved were lower. These results suggest that the lower potencies of retinol may simply reflect lower tissue uptake.

Retinoids are potent inducers of epidermal hyperplasia in vivo. Epidermal transglutaminase activity and stratum corneum turnover were examined in all-trans-retinoic acid and arotinoid ethyl ester treated hairless mice, to evaluate the possible contribution of decreased epidermal cell loss to the induction of hyperplasia by retinoids. Stratum corneum turnover was enhanced, and the absolute amounts (activity/cm2), but not the specific activities, of soluble and particulate transglutaminase increased following retinoid treatment. Since epidermal differentiation was enhanced after retinoid treatment, the hyperplastic response is due to increased cell formation and not decreased cell loss.

The use of isotopically labelled compounds as metabolic tracers in in vivo and in vitro studies is particularly important in folate biochemistry, where the natural coenzyme forms occur in transient and trace amounts. However, during our studies on the whole body metabolism of radioactive folate tracers, we have now observed that some forms of isotopically labelled folate are biologically distinguishable from the unlabelled carrier molecules by an as yet unidentified secondary isotope effect. Folates excreted in the urine of rats dosed previously with mixtures of 14C- and 3H-labelled folate derivatives are apparently 3H enriched, that is contain more 3H than 14C relative to the dosed compound. This apparent 3H enrichment was largely due to the enhanced absorption of 3H-folate from the intestine, confirmed by studies with everted sac preparations of rat jejunum in which it was found that the 3H-labelled folate in the mixture was transported from the mucosal into the serosal compartment at a faster rate than both the 14C-labelled folate and the unlabelled carrier. Secondary isotope effects were also observed on ion-exchange chromatography, 3H-labelled folates with 3H at the C-9 position eluting fractionally earlier than the corresponding unlabelled or [2-14C]folate from DEAE-cellulose, a behaviour similar to that reported earlier for isotopically labelled 2-aminopurine1 and several amino acids2. Such isotope effects may be more widespread and more important than is generally accepted.