Radiological Screening Methods in Deceased Organ Donation: An Overview of Guidelines Worldwide.

Organ transplantation is performed worldwide, but policies regarding donor imaging are not uniform. An overview of the policies in different regions is missing. This study aims to investigate the various protocols worldwide on imaging in deceased organ donation. An online survey was created to determine the current policies. Competent authorities were approached to fill out the survey based on their current protocols. In total 32 of the 48 countries approached filled out the questionnaire (response rate 67%). In 16% of the countries no abdominal imaging is required prior to procurement. In 50%, abdominal ultrasound (US) is performed to screen the abdomen and in 19% an enhanced abdominal Computed Tomography (CT). In 15% of the countries both an unenhanced abdominal CT scan and abdominal US are performed. In 38% of the countries a chest radiographic (CXR) is performed to screen the thorax, in 28% only a chest CT, and in 34% both are performed. Policies regarding radiologic screening in deceased organ donors show a great variation between different countries. Consensus on which imaging method should be applied is missing. A uniform approach will contribute to quality and safety, justifying (inter)national exchange of organs.

Subcutaneous diphtheria and tetanus vaccines in children with haemophilia: A pilot study and review of the literature.

INTRODUCTION: Subcutaneous (SQ) vaccination has emerged as standard of care in children with severe bleeding disorders to reduce unnecessary factor exposure and avoid provoking an intramuscular bleed, but little is known about comparative immunogenicity to intramuscular (IM) vaccination. AIM: To confirm immunogenicity of Diphtheria Tetanus acellular Pertussis (DTaP) vaccines administered SQ to individuals <6 years old with haemophilia. METHODS: We performed a retrospective and prospective pilot study of tetanus and diphtheria antibody titres among patients evaluated at our Haemophilia Treatment Centre between 2015-2016. Children with haemophilia who had received three to four doses of DTaP containing vaccine administered SQ were eligible. RESULTS: Eight children met inclusion criteria. The mean age at the time of diphtheria and tetanus antibody testing was 21.1±17.8 months. All children who received SQ diphtheria and tetanus developed a positive antibody titre to both antigens. There was no statistically significant difference in distribution of titre values. The average time between the last dose of vaccine and antibody testing was 6.6±3.9 months among SQ vaccinated subjects. Minor injection site reactions were common with SQ vaccines. CONCLUSION: SQ administration of diphtheria and tetanus vaccination appears to be immunogenic in a pilot study of Haemophilia patients and supports this practice as the standard of care for this population.

Meeting report: international workshop on endocrine disruptors: exposure and potential impact on consumers health.

The French Agency for Food, Environmental and Occupational Health and Safety (Anses) hosted a two-day workshop on Endocrine Disruptors: Exposure and Potential Impact on Consumers Health, bringing together participants from international organizations, academia, research institutes and from German, Swedish, Danish and French governmental agencies. The main objective of the workshop was to share knowledge and experiences on endocrine disruptors (ED) exposure and potential impact on consumers' health, to identify current risk assessment practices and knowledge gaps and issue recommendations on research needs and future collaboration. The following topics were reviewed: (1) Definition of ED, (2) endpoints to be considered for Risk assessment (RA) of ED, (3) non-monotonic dose response curves, (4) studies to be considered for RA (regulatory versus academic studies), (5) point of departure and uncertainty factors, (6) exposure assessment, (7) regulatory issues related to ED. The opinions expressed during this workshop reflect day-to-day experiences from scientists, regulators, researchers, and others from many different countries in the fields of risk assessment, and were regarded by the attendees as an important basis for further discussions. Accordingly, the participants underlined the need for more exchange in the future to share experiences and improve the methodology related to risk assessment for endocrine disrupters.

Mandatory COVID-19 vaccination for healthcare workers: The experience of chaplains evaluating religious accommodation requests from coworkers.

Mandatory COVID-19 vaccination requirements for healthcare workers in the United States, instituted at the height of the pandemic to protect vulnerable patients and preserve the infrastructure of healthcare, nonetheless met with resistance by some members of the work force. As unprecedented numbers of employees sought religious accommodations, chaplain leaders were recruited by institutional leadership to adjudicate these requests, either alone or as part of a committee. This study reports results of a survey conducted from 6/1/2022 to 7/15/2022 with U.S. healthcare chaplains (n = 76) who were involved in the evaluation of coworker requests for religious exemption to the COVID-19 vaccine anytime during the pandemic until they accessed the survey. Chaplains were recruited online through national chaplaincy and ethics organizations. A mixed methods design facilitates integration of statistically significant associations with chaplains' in-depth reflections on their experience. Surveying the religious experts on the review committee affords a rare look into how the tension between the free exercise of religion in the workplace and the obligation to protect the public played out during the pandemic. The study further addresses a gap in research literature on the experience of chaplains during the pandemic and identifies unique features of moral injury experienced by a subset of healthcare providers. Chaplains largely perceived their involvement as promoting an ethical, informed process of review. Although all chaplains found this role stressful, high levels of meaning were protective against distress. Sources of distress identified included: ethical concern that granting exemptions would lead to the spread of the virus; inconsistencies in the review process; and, repeated exposure to coworkers' misunderstanding and political use of religious teachings. Featuring prominently in comments from chaplains was the difficulty navigating requests in the context of anti-science, anti-vaccine, and politically charged public discourse.

Earth's anomalous middle-age magmatism driven by plate slowdown.

The mid-Proterozoic or "boring billion" exhibited extremely stable environmental conditions, with little change in atmospheric oxygen levels, and mildly oxygenated shallow oceans. A limited number of passive margins with extremely long lifespans are observed from this time, suggesting that subdued tectonic activity-a plate slowdown-was the underlying reason for the environmental stability. However, the Proterozoic also has a unique magmatic and metamorphic record; massif-type anorthosites and anorogenic Rapakivi granites are largely confined to this period and the temperature/pressure (thermobaric ratio) of granulite facies metamorphism peaked at over 1500 °C/GPa during the Mesoproterozoic. Here, we develop a method of calculating plate velocities from the passive margin record, benchmarked against Phanerozoic tectonic velocities. We then extend this approach to geological observations from the Proterozoic, and provide the first quantitative constraints on Proterozoic plate velocities that substantiate the postulated slowdown. Using mantle evolution models, we calculate the consequences of this slowdown for mantle temperatures, magmatic regimes and metamorphic conditions in the crust. We show that higher mantle temperatures in the Proterozoic would have resulted in a larger proportion of intrusive magmatism, with mantle-derived melts emplaced at the Moho or into the lower crust, enabling the production of anorthosites and Rapakivi granites, and giving rise to extreme thermobaric ratios of crustal metamorphism when plate velocities were slowest.

[Legionella in drinking water : outcomes of an exchange of ideas on the prevention of waterborne legionellosis]. / Legionellen im Trinkwasserbereich : Ergebnisse eines Fachgespräches zur Prävention trinkwasserbedingter Legionellosen.

Although much is known about Legionella and the illness they cause, the relationship between their concentration in water and the risk of infection remains unclear. A comprehensive body of experience shows that the growth of Legionella in heated water distribution systems can be effectively controlled by following the preventive measures described in the generally acknowledged codes of practice. As orientation to trigger action, a technical action level of 100 colony-forming units in 100 ml water has found wide acceptance as the maximally tolerable concentration of Legionella in drinking water hygiene. However, this value as well as the concept for controlling the growth of Legionella in installations has developed historically. In part, the focus differs between drinking water hygiene and hospital hygiene. Also, there is no harmonized European approach for controlling Legionella. In spite of such differences, there are important international parallels in the assessment of the occurrence of Legionella, in experience with controlling them, and in recommendations for prevention. There is a need particularly for adequate studies to clarify the risk of infection as well as for the publication of existing data and experience showing the efficacy of measures for prevention, disinfection and system upgrading. Such data are necessary to support evidence-based prevention of Legionella infections and to create a better epidemiological data base in Germany. One chance for reaching this target would be to improve practices in diagnosis, reporting and central data evaluation-not only of illness, but including also data on Legionella occurrence. More frequent Legionella testing of pneumonia patients is a prerequisite for this, but particularly also for the rapid identification and removal of the source of infection. Further requirements include better training of planers, plumbers, and operators of drinking water installations about the approach to preventing Legionella contamination of drinking water installations described in standards and guidelines. The further development of practicable concepts for effective Legionella prevention requires good collaboration between public authorities responsible for drinking water and hospital hygiene on the federal and state levels and experts for the prevention of infection, hygiene, and sanitary installations.

T cells compete for access to antigen-bearing antigen-presenting cells.

These studies tested whether antigenic competition between T cells occurs. We generated CD8(+) T cell responses in H-2(b) mice against the dominant ovalbumin epitope SIINFEKL (ova8) and subdominant epitope KRVVFDKL, using either vaccinia virus expressing ovalbumin (VV-ova) or peptide-pulsed dendritic cells. CD8(+) T cell responses were visualized by major histocompatibility complex class I-peptide tetrameric molecules. Transfer of transgenic T cells with high affinity for ova8 (OT1 T cells) completely inhibited the response of host antigen-specific T cells to either antigen, demonstrating that T cells can directly compete with each other for response to antigen. OT1 cells also inhibited CD8(+) T cell responses to an unrelated peptide, SIYRYGGL, providing it was presented on the same dendritic cells as ova8. These inhibitions were not due to a more rapid clearance of virus or antigen-presenting cells (APCs) by the OT1 cells. Rather, the inhibition was caused by competition for antigen and antigen-bearing cells, since it could be overcome by the injection of large numbers of antigen-pulsed dendritic cells. These results imply that common properties of T cell responses, such as epitope dominance and secondary response affinity maturation, are the result of competitive interactions between antigen-bearing APC and T cell subsets.

Activation-induced inhibition of interleukin 6-mediated T cell survival and signal transducer and activator of transcription 1 signaling.

The cytokines interleukin (IL)-2, IL-4, IL-6, IL-7, and IL-15 have all previously been shown to inhibit resting T cell death in vitro. We have found a difference in the response of T cells to IL-6, depending on the activation status of the cells. IL-6 inhibited the death of naive T cells, but had no effect on the death of either superantigen-activated T cells, or T cells bearing memory markers. This was true even when the resting and activated T cells were isolated from the same animal; thus, the determining factor for IL-6 insensitivity was the activation status or activation history of the cell, and not the milieu in the animal from which the cells were isolated. Activated T cells expressed lower levels of IL-6 receptors on their surfaces, yet there were sufficient levels of receptors for signaling, as we observed similar levels of signal transducer and activator of transcription (Stat)3 phosphorylation in resting and activated T cells treated with IL-6. However, there was profound inhibition of IL-6-induced Stat1 phosphorylation in activated T cells compared with resting T cells. These data suggest that there is activation-induced inhibition of IL-6 receptor signaling in T cells. This inhibition appears to be specific for some but not all of the IL-6-mediated signaling cascades in these cells.

Disparity in management of diabetes and coronary heart disease risk factors by sex in DCCT/EDIC.

AIMS: Coronary heart disease (CHD) is a major cause of morbidity and mortality in patients with diabetes. Sex disparity in the treatment of modifiable CHD risk factors in patients with Type 2 diabetes has been reported previously; however, there is little comparable information in Type 1 diabetes. METHODS: We performed a cross-sectional analysis of 1153 subjects with Type 1 diabetes in the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) cohort to compare achievement of metabolic and CHD risk factor goals and use of recommended risk factor interventions between the sexes. RESULTS: Women were less likely than men to achieve glycated haemoglobin (HbA1c)<7.0% [adjusted odds ratio (AOR) 0.76, 95% confidence interval (CI) 0.57-0.995] or<8.0% (AOR 0.74, 95% CI 0.58-0.95). Achievement of target lipid levels was not significantly different between the sexes. As in the non-diabetic population, men had higher blood pressure. Women were significantly less likely than men to report using aspirin (AOR 0.77, 0.60-0.99) and angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor blockers (ARBs) (AOR 0.62, 0.49-0.80) and statins (AOR 0.56, 0.43-0.73), even after adjusting for blood pressure and lipid levels, respectively. Reported use of statins was also lower in women than men in the subset that developed a low-density lipoprotein (LDL) cholesterol level>3.4 mmol/l (39% vs. 60%, P<0.05). CONCLUSIONS: In Type 1 diabetes, women report lower frequency than men in the use of interventions that decrease CHD risk. These findings are consistent with reports in the Type 2 diabetic population, showing that risk-reducing measures are underused in women with diabetes.

Immediate impact of COVID-19 on transplant activity in the Netherlands.

The rapid emergence of the COVID-19 pandemic is unprecedented and poses an unparalleled obstacle in the sixty-five year history of organ transplantation. Worldwide, the delivery of transplant care is severely challenged by matters concerning - but not limited to - organ procurement, risk of SARS-CoV-2 transmission, screening strategies of donors and recipients, decisions to postpone or proceed with transplantation, the attributable risk of immunosuppression for COVID-19 and entrenched health care resources and capacity. The transplant community is faced with choosing a lesser of two evils: initiating immunosuppression and potentially accepting detrimental outcome when transplant recipients develop COVID-19 versus postponing transplantation and accepting associated waitlist mortality. Notably, prioritization of health care services for COVID-19 care raises concerns about allocation of resources to deliver care for transplant patients who might otherwise have excellent 1-year and 10-year survival rates. Children and young adults with end-stage organ disease in particular seem more disadvantaged by withholding transplantation because of capacity issues than from medical consequences of SARS-CoV-2. This report details the nationwide response of the Dutch transplant community to these issues and the immediate consequences for transplant activity. Worrisome, there was a significant decrease in organ donation numbers affecting all organ transplant services. In addition, there was a detrimental effect on transplantation numbers in children with end-organ failure. Ongoing efforts focus on mitigation of not only primary but also secondary harm of the pandemic and to find right definitions and momentum to restore the transplant programs.

Impact of patatin-like phospholipase domain containing 3 rs738409 G/G genotype on hepatic decompensation and mortality in patients with portal hypertension.

BACKGROUND: The rs738409 C>G p.I148M variant in the patatin-like phospholipase domain containing 3 (PNPLA3)-gene promotes triglyceride accumulation in hepatocytes and hepatic stellate cell activation and has previously been linked to hepatic steatosis/liver fibrosis. AIM: To investigate its impact on hepatic decompensation and (liver-related) mortality in patients who had already developed portal hypertension. Moreover, we assessed its link with hepatic steatosis as evaluated by controlled attenuation parameter. METHODS: We performed a retrospective analysis in prospectively characterised patients with viral hepatitis/fatty liver disease-induced portal hypertension (hepatic venous pressure gradient [HVPG] ≥ 6 mm Hg) diagnosed at the Medical University of Vienna who underwent HVPG measurement (until 2013; n = 372; longitudinal study) or simultaneous HVPG and controlled attenuation parameter measurement (2014-2017; n = 153; cross-sectional study). RESULTS: While survival was similar between PNPLA3-C/C and -C/G patients, we observed substantially increased mortality in PNPLA3-G/G patients. PNPLA3-G/G had no impact on mortality in the subgroup of patients with viral hepatitis; however, we observed a strong independent association between PNPLA3-G/G and hepatic decompensation (adjusted subdistribution hazard ratio [aSHR]: 2.1, 95% confidence interval [95% CI]: 1.1-4; P = 0.024) as well as mortality (overall: aSHR: 2.2, 95% CI: 1.22-3.98; P = 0.009; liver-related: aSHR: 2.2, 95% CI: 1.08-4.46; P = 0.029) in patients with fatty liver disease. Interestingly, even in the subgroup of patients who had already progressed to clinically significant portal hypertension (HVPG ≥ 10 mm Hg), PNPLA3-G/G substantially increased mortality (aSHR: 2.33, 95% CI: 1.27-4.29; P = 0.006). PNPLA3-genotype had no influence on controlled attenuation parameter or the prevalence of values ≥248 dB/m. CONCLUSION: PNPLA3-G/G-genotype seems to double the risks of hepatic decompensation and (liver-related) mortality in patients with portal hypertension due to fatty liver disease. Further studies are warranted to investigate potential underlying pathophysiological mechanisms unrelated to hepatic steatosis.

Genomic-scale analysis of gene expression in resting and activated T cells.

Recent advances in gene array technology and isolation of lymphocytes now allow comprehensive analysis of gene expression in many different types of T cells. So far only a few sets of results have been published. However it is already clear that these analyses provide accurate measurements of gene expression in T cells. This technology offers the first opportunity to examine global and subtle changes in gene expression in response to specific stimuli.

Analysis of tumor-associated stromal cells using SCID GFP transgenic mice: contribution of local and bone marrow-derived host cells.

The green fluorescence protein (GFP) from the UBI-GFP/BL6 transgenic line was bred into C57BL/6J-scid and C.B-17-scid mice for investigating host-tumor cell interactions. These mice express high levels of GFP under the control of the ubiquitin promoter in virtually all cells examined. In tumor tissue generated by implanting tumor cells in the GFP transgenic SCID mice, the tumor cells and tumor-associated murine host cells were clearly distinguished by GFP expression. A population of cells expressing the endothelial cell marker VEGFR-2/Flk-1, and the progenitor markers c-Kit and Sca-1, were incorporated into tumor tissue. The majority of the Flk-1-positive cells were hematopoietic-derived cells that coexpressed CD45. To investigate the contribution of bone marrow-derived cells to the formation of tumor vessels and stroma, tumor cells were implanted in nontransgenic SCID mice that received a bone marrow transplant from GFP-expressing SCID mice. Although GFP-positive cells were readily detected by histology in tumors taken from bone marrow transplanted animals, they were spatially isolated and lacked organization. In contrast, if tumors were implanted in nontransgenic SCID mice adjacent to a patch of transplanted GFP-expressing skin, these tumors recruited GFP-positive cells that organized into tumor vessels. The results demonstrate that hematopoietic-derived cells, including Flk-1+/CD45+ cells, readily colonized the tumor stroma but were minimally incorporated in the tumor vasculature. The majority of the tumor vessels were instead recruited from tissue adjacent to the tumor. The expression of Flk-1 on nonendothelial, tumor-associated host cells raises the possibility that VEGF antagonists, such as Avastin, could inhibit tumor growth by a mechanism involving hematopoietic-derived CD45+/Flk-1+ cells, in addition to direct suppression of endothelial cell function.

Host-cell-determined methylation of specific Epstein-Barr virus promoters regulates the choice between distinct viral latency programs.

Epstein-Barr virus (EBV) is capable of adopting three distinct forms of latency: the type III latency program, in which six EBV-encoded nuclear antigens (EBNAs) are expressed, and the type I and type II latency programs, in which only a single viral nuclear protein, EBNA1, is produced. Several groups have reported heavy CpG methylation of the EBV genome in Burkitt's lymphoma cell lines which maintain type I latency, and loss of viral genome methylation in tumor cell lines has been correlated with a switch to type III latency. Here, evidence that the type III latency program must be inactivated by methylation to allow EBV to enter the type I or type II restricted latency program is provided. The data demonstrates that the EBNA1 gene promoter, Qp, active in types I and II latency, is encompassed by a CpG island which is protected from methylation. CpG methylation inactivates the type III latency program and consequently allows the type I or II latency program to operate by alleviating EBNA1-mediated repression of Qp. Methylation of the type III latency EBNA gene promoter, Cp, appears to be essential to prevent type III latency, since EBNA1 is expressed in all latently infected cells and, as shown here, is the only viral antigen required for activation of Cp. EBV is thus a pathogen which subverts host-cell-determined methylation to regulate distinct genetic programs.

Constitutive activation of Epstein-Barr virus (EBV) nuclear antigen 1 gene transcription by IRF1 and IRF2 during restricted EBV latency.

The Epstein-Barr virus (EBV) EBNA1 gene promoter active in the type I program of restricted viral latency was recently identified and shown to reside in the viral BamHI Q fragment. This promoter, Qp, is active in a wide variety of cell lines and has an architecture reminiscent of eukaryotic housekeeping gene promoters (B. C. Schaefer, J. L. Strominger, and S. H. Speck, Proc. Natl. Acad. Sci. USA 92:10565-10569, 1995; B. C. Schaefer, J. L. Strominger, and S. H. Speck, Mol. Cell. Biol. 17:364-377, 1997). Here we demonstrate by deletion analysis that the important cis-acting elements regulating Qp are clustered in a relatively small region (ca. 80 bp) surrounding the site of transcription initiation. Immediately upstream of the site of initiation is a region which is protected from DNase I digestion by crude nuclear extracts. Electrophoretic mobility shift analyses (EMSA) employing probes spanning this region demonstrated the presence of two major protein complexes. Deletion analysis of Qp demonstrated that at least one of these complexes plays an important role in Qp activity. Evidence that interferon response factor 2 (IRF2) is a major constituent of the most prominent EMSA complex and that IRF1 may be a minor component of this complex is presented. Transfections into IRF1-/-, IRF2-/-, and IRF1,2-/- fibroblasts demonstrated that absence of both IRF1 and IRF2 reduced Qp activity to approximately the same extent as mutation of the IRF-binding site in Qp, strongly implicating IRF2, and perhaps IRF1, in the regulation of Qp activity. Notably, transcription from Qp was not inducible by either alpha or gamma interferon in EBV-negative B cells but rather was shown to be constitutively activated by IRF1 and IRF2. This observation suggests that IRF1 and IRF2 have a previously unrecognized role as constitutive activators of specific genes. Additionally, data presented indicate that a protein complex containing the nonhistone architectural protein HMG-I(Y) binds to the region identified as the major transcription initiation site for Qp. This observation raises the possibility that HMG-I(Y)-induced DNA bending plays a role in the initiation of transcription from Qp.

Topotecan, a new active drug in the second-line treatment of small-cell lung cancer: a phase II study in patients with refractory and sensitive disease. The European Organization for Research and Treatment of Cancer Early Clinical Studies Group and New Drug Development Office, and the Lung Cancer Cooperative Group.

PURPOSE: To assess activity and toxicity of topotecan in previously treated small-cell lung cancer (SCLC) patients. PATIENTS AND METHODS: Patients with measurable SCLC, progressive after one first-line regimen, were eligible for the study. Two groups of patients were selected: (1) patients who failed first-line treatment < or = 3 months from chemotherapy discontinuation (refractory group); and (2) patients who responded to first-line treatment and progressed greater than 3 months after chemotherapy discontinuation (sensitive group). Topotecan was administered as a 30-minute daily infusion at a dose of 1.5 mg/m2 for 5 consecutive days, every 3 weeks. RESULTS: One hundred one patients were entered onto the study and 403 courses were administered. Ninety-two patients (47 refractory and 45 sensitive) were eligible and assessable for response. Among refractory patients, there were two partial responses (PRs) and one complete response (CR), for an overall response rate of 6.4% (95% confidence interval [CI], 1.3% to 17.6%), whereas in the sensitive group, there were 11 PRs and six CRs, for an overall response rate of 37.8% (95% CI, 23.8% to 53.5%). Overall median duration of response was 7.6 months. Median survival was 5.4 months; median survival of refractory patients was 4.7 months, whereas that of sensitive patients was 6.9 months (P = .002). Median survival of responding patients was 12.5 months. Toxicity was mainly hematologic. Leukopenia, although short-lived, was universal, with grade III and IV neutropenia occurring in 28% and 46.8% of cycles, respectively. Nonhematological toxicity was mild. Fatigue/malaise was reported in 39.3% of cycles and transient elevation of liver enzymes in 17%. CONCLUSION: Topotecan has significant activity in SCLC, particularly in patients sensitive to prior chemotherapy, with predictable and manageable toxicity. The incorporation of topotecan in combination chemotherapy regimens for future treatment of SCLC is warranted.

Characterization of the solubility of a poorly soluble hydroxylated metabolite in human urine and its implications for potential renal toxicity.

The solubility, in human urine, of the major hydroxylated metabolite (M1) of an experimental cognition enhancer was characterized through a series of in vitro experiments in an effort to estimate the probability of crystalluria occurring following oral administration of the parent compound. The aim of these experiments was to determine if a safety margin existed between clinically observed urine concentrations and the solubility of M1. The mean urine concentrations of M1 in young and elderly subjects following oral administration of the parent compound at the highest doses tested, were 4865 +/- 2368 ng/mL and 2764 +/- 791 ng/mL, respectively. In vitro solubility experiments with M1 were conducted in drug-free human urine (37 degrees C) from four male and four female healthy subjects under conditions of high and low urine osmolality. Mean concentrations (n = 16) of M1 in human urine to which solid M1 was added, were 3656 +/- 621 ng/mL, 4678 +/- 1169 ng/mL and 5378 +/- 2474 ng/mL after stirring for 24, 48 and 72 h, respectively, indicating that the ex vivo mean solubility of M1 in human urine is no greater then approximately 5 microg/mL. Addition of solid M1 to urine from human subjects dosed with the parent compound resulted in mean urine M1 concentrations 23.5% greater than those observed in vivo. The results from both experiments indicated a significant overlap between urine concentrations of M1 in vivo following the highest oral administration of the parent drug and M1 solubility measured in vitro, suggesting a high potential for in vivo saturation of urine with M1 with subsequent precipitation, crystalluria, and nephrotoxicity. Consequently, the results of these studies have placed restrictions on the dose that could be administered during clinical development of this compound.

The membrane-associated protein pKe#192/MAP17 in human keratinocytes.

UNLABELLED: In order to isolate genes that are upregulated in human keratinocytes upon loss of cell/matrix contact, a subtractive cDNA library was constructed from dispase-treated versus untreated keratinocytes. Among the cloned cDNAs one was pKe#192 having an open reading frame of 411 bp. By database analysis pKe#192 was found to be identical with the gene "MAP17" previously isolated from human kidney. Kyte-Doolittle hydrophobicity analyzes showed a hydrophobic amino terminus of 13 amino acids, a transmembrane region and a 61 amino acid hydrophilic carboxy-terminus and two potential phosphorylation sites. In order to study regulation of pKe#192/MAP17 expression, RNA was extracted from resting human keratinocytes and from keratinocytes stimulated by dispase-induced detachment from the growth substratum. Reverse transcription polymerase chain reaction did not reveal specific mRNA in resting keratinocytes, whereas mRNA was detectable after detachment. For further characterization poly- and monoclonal antibodies were generated against a recombinant fusion protein. Immunohistologic studies using the mono- and polyclonal antibodies showed staining of the upper layers of the stratum granulosum in normal human epidermis. The staining was colocalized with involucrin. Immunhistologic staining of frozen sections derived from lesional skin of bullous pemphigoid und pemphigus vulgaris indicated that pKe#192/MAP17 was upregulated in the epidermis adjacent to the blister. Taken together, the data demonstrate that pKe#192/MAP17 is expressed in keratinocytes and may be involved in epidermal physiology and pathology. KEYWORDS: bullous diseases/differentiation.

Rapid normalization of epidermal integrin expression after allografting of human keratinocytes.

Allogeneic keratinocyte grafts have beneficial effects on skin wounds, but the underlying interactions between graft and woundbed remain to be explored in detail. The epidermal integrins play a pivotal role in mediating cell-to-cell and cell-to-matrix interactions. In unwounded epidermis, alpha 2 beta 1-, alpha 3 beta 1-, alpha 6 beta 4-, alpha 5 beta 1-, and alpha v beta 5-integrins are confined to basal cells. During healing of incisional wounds, these integrins are also expressed in suprabasal cells, where they remain detectable even after epidermal integrity is fully reestablished. We examined the integrin subunits alpha 2, alpha 3, alpha 6, alpha 5, and alpha v in partial thickness burn wounds grafted with allogeneic keratinocytes and asked whether the effect of allogeneic keratinocyte grafts, i.e., fast reepithelialization, is reflected by an accelerated reversion to a normal integrin pattern. Biopsies were taken after wound debridement before grafting and 10 d after transplantation. After 10 d, a stratified epidermis had developed in all cases and integrins were mainly restricted to the basal cell layer of the neo-epidermis. alpha 2-, alpha 3-, alpha 6-, and alpha v-subunits were present at basal and/or lateral cell borders, duplicating the integrin pattern in normal epidermis. The findings indicate that grafting accelerates the shift of the epidermis from an inflammatory to a regenerative state, as reflected by the reversion of the integrin pattern from a "spread-and-migrate" to the "steady-state" phenotype.