Allelic Variation in the Toll-Like Receptor Adaptor Protein Ticam2 Contributes to SARS-Coronavirus Pathogenesis in Mice.

Host genetic variation is known to contribute to differential pathogenesis following infection. Mouse models allow direct assessment of host genetic factors responsible for susceptibility to Severe Acute Respiratory Syndrome coronavirus (SARS-CoV). Based on an assessment of early stage lines from the Collaborative Cross mouse multi-parent population, we identified two lines showing highly divergent susceptibilities to SARS-CoV: the resistant CC003/Unc and the susceptible CC053/Unc. We generated 264 F2 mice between these strains, and infected them with SARS-CoV. Weight loss, pulmonary hemorrhage, and viral load were all highly correlated disease phenotypes. We identified a quantitative trait locus of major effect on chromosome 18 (27.1-58.6 Mb) which affected weight loss, viral titer and hemorrhage. Additionally, each of these three phenotypes had distinct quantitative trait loci [Chr 9 (weight loss), Chrs 7 and 12 (virus titer), and Chr 15 (hemorrhage)]. We identified Ticam2, an adaptor protein in the TLR signaling pathways, as a candidate driving differential disease at the Chr 18 locus. Ticam2-/- mice were highly susceptible to SARS-CoV infection, exhibiting increased weight loss and more pulmonary hemorrhage than control mice. These results indicate a critical role for Ticam2 in SARS-CoV disease, and highlight the importance of host genetic variation in disease responses.

Novel mutant green fluorescent protein protease substrates reveal the activation of specific caspases during apoptosis.

BACKGROUND: The caspase-mediated proteolysis of many cellular proteins is a critical event during programmed cell death or apoptosis. It is important to determine which caspases are activated in mammalian cells, and where and when activation occurs, upon receipt of specific death stimuli. Such information would be useful in the design of strategies to regulate the activation of caspases during apoptosis. RESULTS: We developed two novel fluorescent substrates that were specifically cleaved by caspase-1 or caspase-3. For in vitro studies, four-amino-acid recognition sequences, YVAD for caspase-1 and DEVD for caspase-3, were introduced between blue fluorescent protein (BFP) and green fluorescent protein (GFP), expressed in bacteria and purified. For in vivo studies, YVAD and DEVD were introduced between cyan fluorescent protein and yellow fluorescent protein, and expression was monitored in live mammalian cells. The proximity between fluorophores was determined using fluorescence resonance energy transfer. Purified substrates were cleaved following exposure to purified caspase-1 and caspase-3. In Cos-7 cells, caspase-1 and caspase-3 substrates were cleaved upon induction of apoptosis with staurosporine, a protein-kinase inhibitor, whereas caspase-3 but not caspase-1 substrate was cleaved upon treatment of cells with the DNA-damaging agent mitomycin c. CONCLUSIONS: These substrates allow the spatial activation of specific members of the caspase family to be deciphered during the initiation and execution phase of programmed cell death, and allow activation of specific caspases to be monitored both in vivo and in vitro. This technology is also likely to be useful for high-throughput screening of reagents that modulate caspase activity.

Diseases of meaning, manifestations of health, and metaphor.

Disease and health are commonly thought of as distinct opposites. We propose a different view in which both may be seen to be facets of healthy functioning, each necessary for the other, each giving rise to the other. Thus, disease may be thought of as a manifestation of health. It is the healthy response of an organism striving to maintain physical, psychologic, and spiritual equilibrium. Disease is not necessarily to be avoided, blocked, or suppressed. Rather, it should be understood to be a process of transformation. The process should therefore be facilitated because it is an integral part of the dynamic equilibrium that we ordinarily think of as health. In many cases, perhaps all, people get ill because there is something going "wrong" in their lives. This could occur in a whole range of ways-relationships, environment, food, or job. Our view, however, is that disease is a meaningful state that can inform health workers how to help patients to heal themselves. In this way, instead of being meaningless, people's problems become diseases of meaning, enabling people to see that things are not necessarily "going wrong" but are, in fact, helping them become stronger, to live more fully and with more understanding. Seen from this perspective, depression; cancer; heart disease; neurodegenerative and autoimmune disease; dementia; and conditions such as community violence, genocide, and the problem of environmental devastation are "diseases of meaning." World Health Organization forecasts make it clear that diseases of meaning will continue well into the next millennium to be the major cause of suffering and death worldwide. To deal with them, the world needs to reformulate the biomolecular paradigm that has been exploited in the last two centuries. It does not address the reasons why these diseases arise, attending mainly to their molecular consequences. A paradigm that includes the importance of meaning must now be given top priority. The concept that diseases are a manifestation of health-a call to a different relationship with ourselves and our environment, both animate and inanimate- is in itself a different approach. Programs for care and education based upon it would have immediate application in medicine, industry, education and ecology. We believe that this model would have far-reaching consequences for the understanding, treatment, and prevention of diseases and behaviors that lead to violence and environmental destruction.

First International Pharmacoeconomic Conference on Alzheimer's Disease: report and summary.

The First International Pharmacoeconomic Conference on Alzheimer's Disease (AD) was held in Amsterdam in July 1998. The meeting was held under the auspices of the International Working Group for Harmonization of Dementia Drug Guidelines (http://dementia.ion.ucl.ac.uk/harmon), bringing together academics, clinicians, purchasers, and representatives from industry. Presentations were given on the methodology of pharmacoeconomic studies in AD, particularly focusing on caregiver burden, quality of life (QOL), and resource utilization. Three economic models of AD were presented based on data from the United States, Canada, and the United Kingdom. In two studies, these data were then used to model the cost-effectiveness and effect on cost of treatment with donepezil. Both studies suggested a possible cost advantage for the use of donepezil, when compared with no placebo or treatment, particularly when donepezil is used appropriately in mild-to-moderate AD. These data need to be interpreted with care, as none of the cost or utility information were collected during the clinical trials. Additional data from a 2-year clinical trial of selegiline and vitamin E suggest that cognitive measures may be poor predictors of economic outcome, which is better measured directly. Both economic models of donepezil rely on short-term cognitive data to predict long-term outcome, a methodf that may not be useful in predicting economic savings. The issues facing pharmacoeconomists, researchers, clinicians, and families in the future were addressed in a series of workshops using a method of strategic futuring. The workshops attempted to see 7 years into the future for a range of areas, including consumer and caregiver use of pharmacoeconomic data; early detection and prevention; Japanese perspectives; activities of daily life and what will be daily life activities; caregiver burden; QOL at the end of life; new uses for new information and communication technology in clinical research; and physicians' use of pharmacoeconomic data. A range of exciting futures were predicted, although common themes that arose when considering barriers to achieving these futures included cost, education, political will, confidentiality, privacy, and ethics. The first conference was deemed to have been a success, having attracted more than 160 delegates and many distinguished speaker. A second conference is planned for the year 2000. Over the next 2 years, research needs to be broadened particularly in the methodological areas of resource utilization, QOL, and caregiver burden. Data from clinical trials with relevant economic and QOL outcomes will be needed by purchasers if drug treatments for dementia are to gain widespread use. It is also hoped that the models described at the meeting may become more freely available to politicians, purchasers, clinicians, and caregivers to help them make better decisions about treatment.

Role of ICE-like proteases in endothelial cell hypoxic and reperfusion injury.

Because of its location between blood and tissue, the endothelium is particularly vulnerable to hypoxic/reperfusion injury, but the mechanisms responsible for this injury are not known. A number of recent findings suggest that hypoxia and reperfusion injures neuronal cells via apoptosis. Apoptosis has recently been shown to depend on the activation of a class of proteases with homology to Interleukin-1 beta converting enzyme (ICE) protease. Therefore, we examined the effect of specific inhibitors of ICE-like proteases on hypoxic and reperfusion injury in cultured EAhy926 endothelial cells. Pretreatment of cells with ICE inhibitor II (Ac-YVAD-CMK), ICE inhibitor III (Z-Asp-2,6-dichlorobenzoyloxy-methylketone-Z-Asp-CH2-DCB+ ++), or ICE inhibitor IV (Ac-YVKD-CHO) (all at 10-100 microM) did not protect cells from hypoxic injury. However, pretreatment of cells with ICE inhibitor III and to a lesser extent with ICE inhibitor II, but not with ICE inhibitor IV, protected cells from reperfusion injury. The protective effect of ICE inhibitor III was not dependent upon pH, but was associated with decreased release of arachidonic acid from cells. These findings suggest that reperfusion injury to EAhy926 endothelial cells involves ICE-like proteases. The identity of the protease(s) is not known but it does not appear to be a YAMA-type protease based upon ICE inhibitor specificity. Our data also indicate that a potential target of this protease is phospholipase A2 (PLA2).

Contribution of pH-dependent group II phospholipase A2 to chemical hypoxic injury in rat hepatocytes.

Numerous studies have suggested that enhanced membrane phospholipid degradation contributes to hypoxic and ischemic injury. Recently, acidosis has been found to potently protect against hypoxic and ischemic injury. To investigate the interrelationships of these two events in hypoxic injury, we studied the role of a pH-dependent group II phospholipase A2 (PLA2, E.C. 3.1.1.4) in chemical hypoxic injury in rat hepatocytes. Northern blot analysis of RNA extracted from normoxic and hypoxic rat hepatocytes with group II rat liver PLA2-specific oligonucleotide cDNA probes revealed a 0.9 kb transcript whose abundance was significantly increased in rat hepatocytes within 15 min after initiation of chemical hypoxia and remained high until cells lost viability. Immunofluorescence staining of hepatocytes with polyclonal antibodies that recognize group II PLA2 demonstrated a substantial increase in the level of PLA2 protein in hypoxic rat hepatocytes within 30 min of initiation of hypoxic injury. Treatment of hepatocytes with group II PLA2-specific anti-sense DNA oligonucleotides: 1) abolished accumulation of PLA2 protein in hypoxic rat hepatocytes as assessed by immunofluorescence staining with anti-PLA2 antibodies; 2) decreased the enzymatic activity of PLA2 manifested as decreased arachidonic acid release in hypoxic hepatocytes; and 3) significantly delayed cell death evoked by chemical hypoxia as indicated by a decrease in propidium iodide uptake. These findings suggest that pH-dependent group II PLA2 plays an important role in chemical hypoxic injury of rat hepatocytes.

Down-regulation of HCW9 mRNA in rat hepatocytes during chemical hypoxia involves both transcriptional and posttranscriptional mechanisms.

HCW9 cDNA encodes a rat protein with 95% homology to mouse phospholipase A2 activating protein (PLAP). Its mRNA, which is substantially decreased in rat hepatocytes during chemical hypoxic injury, was found to be expressed in all rat tissues examined, including liver, heart, brain, spleen, lung, skeletal muscle, kidney, and testis. To elucidate the mechanisms responsible for this hypoxia-induced down-regulation of HCW9 mRNA levels, the transcription rate and half-life of HCW9 mRNA were measured. Nuclear run-off assays revealed a 54-57% inhibition in the transcription rate of HCW9 gene during chemical hypoxic injury. The half-life of HCW9 mRNA decreased from approximately 15 min under normoxic conditions to approximately 7 min during chemical hypoxic injury. These findings suggest that HCW9 expression in rat hepatocytes is regulated at both the transcriptional and posttranscriptional levels during chemical hypoxia.

Cloning of a rat cDNA encoding a novel LIM domain protein with high homology to rat RIL.

A complementary DNA (1392 bp) encoding a protein with high homology to rat reversion-induced LIM (RIL) protein was cloned from rat hepatocytes by differential screening of a subtractive (normoxic minus hypoxic) lambda GEM-2 cDNA library. This cDNA clone, denoted CLP-36, encodes a 327-amino-acid (aa) protein that contained a restrictively conserved LIM (a Cys-rich domain with consensus aa sequence C-X2-C-X17-19-H-X2-C-X2-C-X2-C-X16-20-C-X2-C/H/D that was initially identified in homeodomain proteins, Lin-11 [Freyd et al., Nature 344 (1990) 876-879], Isl-1 [Karlsson et al., Nature 344 (1990) 879-882] and Mec-3 [Way et al., Cell 54 (1988) 5-16] in the C-terminal portion (aa 192-327). It shared an overall 45.1% identity to a rat LIM domain RIL protein [Kiess et al., Oncogene 10 (1995) 61-68], with 62.0% identity in the N terminus (aa 1-89) and 50.0% identity in the C terminus (aa 188-327). The rat CLP-36 mRNA is expressed most abundantly in heart, lung and liver, moderately in spleen and skeletal muscle, and at extremely low levels (if at all) in testis and brain tissues. Northern blotting analysis of total RNA extracted from normoxic or hypoxic rat hepatocytes, with a fragment of clone CLP-36 as probe, demonstrated a single band with a mobility corresponding to a size of 1.4 kb, whose level was significantly decreased during chemical hypoxia.

Liquid chromatographic determination of flucytosine in capsules: collaborative study.

A liquid chromatographic method for the determination of flucytosine in capsules was collaboratively studied by 7 laboratories. The method uses a C18 reverse phase column, water-methanol-acetic acid mobile phase containing 1-octanesulfonic acid sodium salt, p-aminobenzoic acid as internal standard, and photometric detection at 285 nm. The mean recovery value (+/- SD) of flucytosine from a synthetic formulation representing capsules was 99.2 +/- 1.72% (CV = 1.73%). Composited samples of 250 and 500 mg commercial capsules gave assay values of (mean +/- SD) 103.17 +/- 2.21 and 99.29 +/- 1.29% of declared, respectively. CV values were 2.15 and 1.30%. Reproducibility and repeatability CVs were 2.19 and 1.50%, respectively, for the 250 mg capsules, and 1.34 and 0.63%, respectively, for the 500 mg capsules. The method has been adopted official first action.

Liquid chromatographic determination of flucytosine in capsules.

A liquid chromatographic method has been developed for determination of flucytosine in capsules. Flucytosine and p-aminobenzoic acid, the internal standard, are separated on a C18 reverse phase column using water-methanol-acetic acid mobile phase containing 1-octane-sulfonic acid sodium salt. Compounds are detected photometrically at 285 nm. Mean assay results for 250 and 500 mg commercial capsules were 101.5% (n = 5) of declared, respectively. Mean recovery of flucytosine added to commercial capsules was 99.3%.

Liquid chromatographic determination of allopurinol in tablets: collaborative study.

A liquid chromatographic method for the determination of allopurinol in tablets was collaboratively studied by 7 laboratories. The method uses a C18 reverse phase column, a 0.05M ammonium phosphate mobile phase, hypoxanthine as the internal standard, and photometric detection at 254 nm. Collaborators were supplied with samples of 2 commercial tablets and 1 synthetic tablet powder. The mean recovery value of allopurinol from the synthetic tablet powder was 100.0%. The combined mean coefficient of variation for all 3 types of sample analyzed was less than 2%. The method has been adopted official first action.

Differential pulse polarographic analysis of thyroid hormone: determination of iodine, thyroxine, and liothyronine.

A differential pulse polarographic method for the analysis of thyroid and thyroid tablets for total iodine, thyroxine, and liothyronine is described. The procedure for iodine, which is also applicable to individual tablet assay, consists of ashing the sample, coverting iodide to iodate, and analyzing by differential pulse polarography. The procedure for thyroxine and liothyronine involves hydrolysis of the sample with barium hydroxide and isolation and separation of the iodoamino acids using ion exchangers, followed by differential pulse polarographic determination in a supporting electrolyte composed of 0.5 N Na2CO3 in 20% 2-propanol containing 1% tetrabutylammonium bromide. The differential pulse polarographic results for iodine agree with values obtained using the USP XIX procedure, and the quantities of thyroxine and liothyronine found agree with literature values.