Inhibition of lysosomal degradative functions in RPE cells by a retinoid component of lipofuscin

PURPOSE: To investigate the effect of the lipofuscin component N-retinylidene-N-retinylethanolamine (A2-E) on degradative functions of lysosomes in human retinal pigment epithelial (RPE) cells and to evaluate its mechanism of action. METHODS: A2-E was coupled to low-density lipoprotein (LDL). Human RPE cell cultures were loaded with the A2-E/LDL complex, and controls were run with medium containing LDL alone. To determine whether A2-E accumulated in lysosomes, cells were fractionated in a Percoll gradient, and protein degradation was determined by metabolic labeling and measurement of the release of low-molecular-weight radioactivity. Lysosomal degradation was distinguished from nonlysosomal degradation by inclusion of NH4Cl in the medium. The metabolism of sulfated glycosaminoglycans was studied by radiosulfate incorporation in pulse-chase experiments. Intralysosomal pH was determined using a fluorescent lysosomotropic pH indicator. RESULTS: A2-E accumulated almost exclusively in the lysosomal compartment. Lysosomal protein degradation was reduced in a dose-dependent fashion in A2-E-treated cells. The selectivity of A2-E on lysosomal function was demonstrated by its lack of effect on degradation of extralysosomal protein. Lysosomal glycosaminoglycan catabolism of RPE cells was also strongly inhibited by A2-E. Lysosomal pH was increased by A2-E. CONCLUSIONS: The findings indicate that accumulation of A2-E in RPE cells interferes with lysosomal functions as exemplified by its inhibitory effect on protein and glycosaminoglycan catabolic pathways. The quaternary amine character of the A2-E apparently causes a perturbation of the acidic intralysosomal milieu, resulting in diminished hydrolase action and consequent accumulation of undegraded material. Such mechanism could be operative in retinal diseases associated with excessive lipofuscin accumulation including age-related macular degeneration.     

In vivo significance of ICAM-1--dependent leukocyte adhesion in early corneal angiogenesis

PURPOSE: Numerous investigations have stressed the significance of leukocytes in early angiogenesis. Leukocytes invade the cornea, and the location of their extravasation corresponds to the site of vessel ingrowth. The interactions between leukocytes and vascular endothelium are mediated by various proteins, including adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1). In this study, the role of ICAM-1 during early corneal angiogenesis was evaluated in vivo. METHODS: Corneal neovascularization was induced in New Zealand White rabbits by use of intrastromal pellets containing 750 ng vascular endothelial growth factor (VEGF). The fluorescent dye rhodamine 6G was used to stain leukocytes in vivo. Leukocyte adhesion and vessel growth were quantified in vivo by high-resolution fluorescence angiography. To inhibit ICAM-1 interactions a microemulsion containing anti-ICAM-1 antibody was applied topically. RESULTS: Limbal vessels showed increased leukocyte adhesion 24 hours after pellet implantation: The number of rolling and sticking leukocytes was significantly increased compared with the number in control animals (P < 0.01). Treatment with anti-ICAM-1 antibody resulted in reduced leukocyte sticking and increased leukocyte rolling. The area covered by new blood vessels was significantly diminished in eyes treated with anti-ICAM-1 (P < 0.05). CONCLUSIONS: The results support the hypothesis that ICAM-1-mediated leukocyte adhesion is a key event in early angiogenesis. This model may serve for investigation of the significance of adhesion molecules by in vivo observation and quantification.     

Financing the World Health Organisation: global importance of extrabudgetary funds

From 1948, when WHO was established, the Organisation has relied on the assessed contributions of its member states for its regular budget. However, since the early 1980s the WHO World Health Assembly has had a policy of zero real growth for the regular budget and has had to rely increasingly, therefore, on attracting additional voluntary contributions, called extrabudgetary funds (EBFs). Between 1984-85 and 1992-93 the real value of the EBFs apparently increased by more than 60% and in the 1990-91 biennium expenditure of extrabudgetary funds exceeded the regular budget for the first time. All WHO programmes, except the Assembly and the Executive Board, receive some EBFs. However, three cosponsored and six large regular programmes account for about 70% of these EBFs, mainly for vertically managed programmes in the areas of disease control, health promotion and human reproduction. Eighty percent of all EBFs received by WHO for assisted activities have been contributed by donor governments, with the top 10 countries (in Europe, North America and Japan) contributing about 90% of this total, whereas the UN funds and the World Bank have donated only about 6% of the total to date. By contrast, about 70% of the regular budget expenditure has been for organisational expenses and for the support of programmes in the area of health systems. Despite the fact that the more successful programmes are heavily reliant on EBFs, there are strong indications that donors, particularly donor governments, are reluctant to maintain the current level of funding without major reforms in the leadership and management of the Organisation. This has major implications for WHO's international role as the leading UN specialised agency for health.     

Techniques for vascular access when venous entry is impossible. Route depends on urgency and the agent to be administered

When a patient requires parenteral fluid or drug administration and venous cannulation cannot be performed, consider less typical routes. Intraosseus infusions are usually more effective in children than adults, but intraosseus cannulation failure may occur in as many as 20% of patients. Intra-arterial infusions are possible if pump pressures are kept high. Hypodermoclysis (infusion into the subcutaneous tissues) can correct moderate dehydration. Administering resuscitative drugs endobronchially is usually safe and effective, although pulmonary function may be somewhat compromised. A number of drugs may be given sublingually, either by injection or topical application. Finally, the corpora cavernosa of the penis may be used for short-term, large-volume fluid administration.     

Visual cortex activation recorded by dynamic emission computed tomography of inhaled xenon 133

Regional cerebral blood flow (CBF) was studied tomographically with ¹³³Xe administered by inhalation over a 1-min period at a concentration of 10 mCi/l. A fast rotating (dynamic) single-photon emission computed tomograph with four detector heads was used, an instrument that has been found to be well suited for detecting focal ischemia. In the present study its ability to detect focal hyperemia was investigated in 13 normal subjects studied during rest and during visual stimulation. A flickering light seen with eyes open and closed, increased blood flow in the visual cortex by 35% and 22% respectively. Looking at different pictures displayed on a screen raised regional CBF by 26%. The most complex task, reading and copying a text, increased blood flow by 45%. Averaging the different tasks resulted in a mean regional CBF increase in the visual cortex of 35%. The result is comparable with that obtained by positron emission tomography. Both forms of isotope tomography offer unique possibilities for the study of brain function in health and disease, possibilities not matched by X-ray tomography. The low cost and ready availability of appropriate single-photon radionucleides (¹³³Xe and ¹²⁷Xe) are mentioned.Supported by the Danish Medical Research Council, the Danish Sclerose Association, and the Johann and Hanne Weimann Foundation.     

Steady-state kinetics of imipramine in patients

Steady-state plasma level kinetics were studied in 76 patients given imipramine (IP) 150 to 225 mg/day for 2–5 weeks. IP was given in three divided doses at 8.00 a.m., 1.00 p.m. and 5.00 p.m. Plasma concentrations of IP and its active metabolite desipramine (DMI) were determined by quantitative in situ thin-layer chromatography. The plasma levels of IP and DMI showed pronounced flucutations throughout the day with a ratio of about 2 between highest and lowest level. Patients with steady-state levels of IP and/or DMI below 50 g/l reached this within 1 week of treatment. Patients with higher steady-state levels reached steady-state concentrations within 2–3 weeks. There were some intraindividual fluctuations in plasma levels from week to week after steady state had been reached (coefficient of variation: 10–20%). Interindividually, the steady-state levels corrected to a dose of 3.5 mg/kg per day varied considerably: IP: 6–356 g/l, DMI: 24–659 g/l and IP+DMI: 58–809 g/l. The steady-state plasma levels showed a skew distribution that became normal by logarithmic transformation. The IP/DMI ratio ranged from 0.07 to 5.5 with a median value of 0.47. Compared to data from amitriptyline treated patients the IP/DMI ratios had significantly lower median value and larger variation than the corresponding plasma level ratios of amitriptyline/nortriptyline. Several statistically significant differences in steady-state levels between age groups were found. For IP: Women aged 30–39 had lower levels than women aged 20–29, 40–49, and 50–59, and men aged 50–59 and 60–65; men aged 30–39 had lower levels than men aged 60–65. For DMI: Women aged 30–39 had lower levels than women aged 50–59.     

Bladder Cancer and Arsenic Exposure： Differences in the Two Populations Enrolled in A Study in Southwest Taiwan

Objective Analyses of bladder cancer mortality in the Black Foot Disease (BFD) endemic area of southwest Taiwan conducted by Morales et al. showed a discontinuity in risk at 400 μg/L arsenic in the drinking water in a stratified analysis and no discontinuity in a continuous analysis.     

E5 murine monoclonal antiendotoxin antibody in gram-negative sepsis: a randomized controlled trial. E5 Study Investigators

Context  Knowledge and understanding of gram-negative sepsis have grown over the past 20 years, but the ability to treat severe sepsis successfully has not. Objective  To assess the efficacy and safety of E5 in the treatment of patients with severe gram-negative sepsis. Design  A multicenter, double-blind, randomized, placebo-controlled trial conducted at 136 US medical centers from April 1993 to April 1997, designed with 90% power to detect a 25% relative risk reduction, incorporating 2 planned interim analyses. Setting  Intensive care units at university medical centers, Veterans Affairs medical centers, and community hospitals. Patients  Adults aged 18 years or older, with signs and symptoms consistent with severe sepsis and documented or probable gram-negative infection. Intervention  Patients were assigned to receive 2 doses of either E5, a murine monoclonal antibody directed against endotoxin (n = 550; 2 mg/kg per day by intravenous infusion 24 hours apart) or placebo (n = 552). Main Outcome Measures  The primary end point was mortality at day 14; secondary end points were mortality at day 28, adverse event rates, and 14-day and 28-day mortality in the subgroup without shock at presentation. Results  The trial was stopped after the second interim analysis. A total of 1090 patients received study medication and 915 had gram-negative infection confirmed by culture. There were no statistically significant differences in mortality between the E5 and placebo groups at either day 14 (29.7% vs 31.1%; P = .67) or day 28 (38.5% vs 40.3%; P = .56). Patients presenting without shock had a slightly lower mortality when treated with E5 but the difference was not significant (28.9% vs 33.0% for the E5 and placebo groups, respectively, at day 28; P = .32). There was a similar profile of adverse event rates between E5 and placebo. Conclusions  Despite adequate sample size and high enrollment of patients with confirmed gram-negative sepsis, E5 did not improve short-term survival. Current study rationale and designs should be carefully reviewed before further large-scale studies of patients with sepsis are conducted.      

Foix-Chavany-Marie (anterior operculum) syndrome in childhood: a reappraisal of Worster-Drought syndrome

Foix-Chavany-Marie syndrome (FCMS) is a distinct clinical picture of suprabulbar (pseudobulbar) palsy due to bilateral anterior opercular lesions. Symptoms include anarthria/severe dysarthria and loss of voluntary muscular functions of the face and tongue, and problems with mastication and swallowing with preservation of reflex and autonomic functions. FCMS may be congenital or acquired as well as persistent or intermittent. The aetiology is heterogeneous; vascular events in adulthood, nearly exclusively affecting adults who experience multiple subsequent strokes; CNS infections; bilateral dysgenesis of the perisylvian region; and epileptic disorders. Of the six cases reported here, three children had FCMS as the result of meningoencephalitis, two children had FCMS due to a congenital bilateral perisylvian syndrome, and one child had intermittent FCMS due to an atypical benign partial epilepsy with partial status epilepticus. The congenital dysgenetic type of FCMS and its functional epileptogenic variant share clinical and EEG features suggesting a common pathogenesis. Consequently, an increased vulnerability of the perisylvian region to adverse events in utero is discussed. In honour of Worster-Drought, who described the clinical entity in children 40 years ago, the term Worster-Drought syndrome is proposed for this unique disorder in children.     

Cyclooxygenase-2 is a target of KRASD12, which facilitates the outgrowth of murine C26 colorectal liver metastases.

PURPOSE: Mutational activation of the KRAS oncogene and overexpression of cyclooxygenase-2 (COX-2) contribute to colorectal carcinoma (CRC) development, but the relationship between these two events is unclear. This study was designed to clarify that relationship and to assess the contribution of KRAS-dependent COX-2 to the seeding of CRC cells in the liver and to their outgrowth as liver metastases in an experimental mouse model. EXPERIMENTAL DESIGN: The effect of RNA interference-mediated KRAS knockdown on COX-2 expression and activity was tested in murine C26 CRC cells. The contribution of KRAS-dependent COX-2 to early metastatic tumor cell seeding (by intravital microscopy) and outgrowth of metastases in the liver (by bioluminescence imaging) was studied by using parecoxib, a novel and highly selective liver-activated COX-2 inhibitor. Intratumoral cell proliferation, apoptosis, and tumor-associated angiogenesis were assessed by immunohistochemistry on liver tissue sections. RESULTS: Stable knockdown of mutant KRAS(D12) in murine C26 CRC cells by RNA interference lead to a dramatic reduction of COX-2 synthesis and prostaglandin E2 production. Inhibition of host or tumor cell COX-2 activity had no effect on early metastatic cell seeding in the liver but greatly reduced intrahepatic tumor cell proliferation and the rate of liver metastasis outgrowth. COX-2 inhibition had no effect on early tumor vascularization or on tumor cell apoptosis. CONCLUSIONS: The high levels of COX-2 enzyme and prostaglandin production in C26 CRC cells are primarily caused by the presence of endogenous mutant KRAS(D12). Furthermore, COX-2 inhibition affects the tumoral rather than the vascular compartment during the early stages of C26 liver metastasis outgrowth.