Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides.

We have investigated the use of a cationic lipid preparation to enhance antisense oligonucleotide activity in human umbilical vein endothelial cells. A liposomal preparation containing the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) was found to increase by at least 1000-fold the potency of an antisense oligonucleotide (ISIS 1570) that hybridizes to the AUG translation initiation codon of human intercellular adhesion molecule-1. In the presence of 8 microM DOTMA, 6-15-fold more 35S-ISIS 1570 associated with cells, at oligonucleotide concentrations from 0.01 to 5 microM, than did in the absence of DOTMA. Both 35S-ISIS 1570 association with cells and antisense activity were increased as a function of DOTMA concentration and with increasing time of incubation with the cationic lipid. Fluorescein-labeled ISIS 1570 was used to assess the intracellular distribution of the oligonucleotide in the presence and absence of DOTMA. In the absence of DOTMA, the oligonucleotide localized to discrete structures in the cytoplasm of the cell, resulting in a punctate fluorescence pattern. In the presence of DOTMA, cellular fluorescence markedly increased and the oligonucleotide localized within the nucleus, as well as to discrete structures in the cytoplasm. Accumulation of the oligonucleotide in the nucleus in the presence of DOTMA was time and temperature dependent. Nuclear accumulation was inhibited by preincubation of the cells with monensin but not chloroquine, NH4Cl, nocodazole, colcemid, or brefeldin A. These data demonstrate that cationic lipids increase antisense activity by increasing the amount of oligonucleotide associated with cells and altering intracellular distribution of the oligonucleotide.     

Continuous arteriovenous hemodialysis: an alternative therapy for acute renal failure associated with critical illness.

Critically ill patients often cannot tolerate conventional hemodialysis because of hemodynamic instability. Continuous arteriovenous hemofiltration provides control of fluid and electrolyte balance but is inefficient in the management of azotemia. Continuous arteriovenous hemodialysis (CAVHD) combines dialysis with hemofiltration. We performed 15 CAVHD treatments of 2 or more days' duration in 12 critically ill patients aged 23 to 85 (mean 64.4) years who had acute oliguric renal failure as a component of multiple organ system failure and who were unsuitable for conventional hemodialysis. The total treatment time was 106 days. The serum creatinine and urea levels were controlled in all the patients during CAVHD. The ultrafiltrate losses were sufficient to allow appropriate nutrition and fluid administration and still maintain a negative fluid balance. Renal function returned in five patients (42%), of whom four survived to be discharged home. CAVHD is an effective means of managing acute oliguric renal failure in critically ill patients.     

How accurate was GMENAC?--A retrospective review of supply projections for diagnostic radiologists.

In 1982, the Graduate Medical Education National Advisory Committee (GMENAC), a prominent national panel, predicted there would be 25,650 full-time equivalent (FTE) diagnostic radiologists, a 34% oversupply, by 1990. The radiologists involved in GMENAC, however, using models developed by the American College of Radiology, projected 19,800 FTE diagnostic radiologists in 1990, which was similar to the GMENAC estimate of need. The disagreement arose principally from different assumptions about residents entering the specialty. Recent data show there actually were approximately 21,900 FTE diagnostic radiologists in 1990. The radiologists' projection was 10% below this figure; the GMENAC projection was 17% above it. GMENAC erred principally in assuming diagnostic radiology residencies would not replace general radiology residencies, but rather be an addition to them. The radiologists erred principally in their assumption about the effects of the financial problems of hospitals on the number of residency positions. Accurate long-term projection of physician supply in individual specialties may well not be feasible.     

Natural killer cells in children with malignant solid tumors. Effect of recombinant interferon-alpha and interleukin-2 on natural killer cell function against tumor cell lines

Natural killer (NK) cells and NK cell activity were determined in three groups (newly diagnosed [n = 21], on therapy [n = 21], and off therapy [n = 18]) of children with various types of malignant solid tumors and in a control group (n = 26) by means of Leu-7 and Leu-11b monoclonal antibodies and a 4-hour 51Cr-release assay, respectively. The erythroleukemia cell line K562 was used as a target cell. The newly diagnosed group included eight patients with localized disease (Stage I-II), ten with bulky but nonmetastatic disease (Stage III), and three with metastases (Stage IV). The mean percent of NK cell activity in the newly diagnosed group was significantly higher than that of the control group. Children with Stage III tumors at diagnosis had higher mean NK cell function than those with Stage I-II and Stage IV. On therapy patients had significantly fewer NK cells and lower NK cell cytotoxicity than those in the other groups studied. We also studied the following: (1) the in vitro effect of recombinant interferon-alpha (rIFN-alpha) and recombinant interleukin-2 (rIL-2) on NK cell function of peripheral blood lymphocytes (PBL) from children with solid malignancies; and (2) the susceptibility of neuroblastoma-derived (CHP-126 and SKNSH) and rhabdomyosarcoma-derived (A-204) cell lines to NK cell lysis. Both rIFN-alpha and rIL-2 enhanced NK cell activity of PBL from children with malignancies and healthy children against K562 and solid tumor cell lines. The enhancing effect or rIL-2 was greater than that of rIFN-alpha. CHP-126 and SKNSH cell lines were susceptible to NK cell lysis mediated by the PBL of children with neuroblastoma and the control group. The A-204 cell line was less sensitive than K562 to NK cell cytotoxicity. Our results suggest a potential therapeutic role for both cytokines in the treatment of malignant solid tumors of childhood.     

α-Bungarotoxin blocks the nicotinic receptor mediated increase in cell number in a neuroendocrine cell line

Exposure of H69 small cell lung carcinoma cells to nicotinic agonists resulted in a significant increase (up to 100%) in cell number after 6 to 12 days. The effect of nicotine (10⁻⁸ M to 10⁻⁴ M) was both dose and time dependent as was that of another nicotinic agonist cytisine (10⁻⁶ M to 10⁻⁴ M). Interstingly, both the nicotine and cytisine induced increases in H69 cell number were blocked by α-bungarotoxin, as well as d-tubocurarine a nicotinic blocker which appears to interact with most nicotinic receptors. These results suggest that the nicotine induced increase in cell number is mediated through an interaction at the nicotinic α-bungarotoxin receptor. This idea is further supported by experiments which show (1) that H69 cells possess high affinity α-bungarotoxin sites (K_d = 25 nM, B_max = 10.4 fmol/10⁶ cells) with the characteristics of a nicotinic α-bungarotoxin receptor and (2) that the potencies of nicotinic receptor ligands in the α-bungarotoxin binding assay were similar to those observed in the functional studies. Northern analysis showed that mRNA for α7, a putative nicotinic α-bungarotoxin binding subunit, and for α5 were present in H69 cells. The present data provide further evidence that nicotine increases cell number in small cell lung carcinoma and are the first to show that this effect is mediated through an interaction at the nicotinic α-bungarotoxin receptor population. These results suggest that the α-bungarotoxin site may be involved in modulating proliferative responses in neuroendocrine derived SCLC cells.     

Systemic effects of inflammation in bronchiectasis

A group of bronchiectatic subjects in the clinically stable state were studied for systemic evidence of inflammation. The following parameters were evaluated: body weight, serum albumin, serum globulin, serum alpha 1-antitrypsin (alpha 1 AT) and peripheral white cell count. For serum albumin and globulin, comparison was made between subjects with bronchiectasis and control subjects with no known pulmonary disease matched for sex and age, and for serum alpha 1 AT and peripheral white cell count, matched for smoking habit as well. The bronchiectatic subjects showed systemic effects of inflammation as indicated by lower body weight and serum albumin (P less than 0.01), higher serum globulin (P less than 0.001), serum alpha 1 AT (P less than 0.05) and total leucocyte count (P less than 0.05). Differential white cell count showed that the elevation was distributed in most cell types. Correlation matrix was done for the above systemic parameters and indices of airway inflammation including sputum volume, purulence, and polymorph count and FEV1. There was an inverse correlation between total peripheral WBC count and FEV1 in percentage of predicted (P less than 0.01), and a positive correlation between sputum purulence and sputum polymorph score (P less than 0.05). This suggests that host peripheral leucocyte response may be a factor in the determination of lung function.     

Plasma Histamine After Methacholine, Allergen, and Aspirin Challenges

Plasma histamine levels were measured by radio-enzymatic technique in seven patients following 10 challenges: five methacholine challenge tests, four antigen inhalation challenge tests, and one oral aspirin challenge test. Baseline plasma histamine was the same in all patients except in the aspirin-challenged patient, who had a higher baseline histamine level. There was no statistical change in the level of histamine throughout the test in either the methacholine-challenged or the antigen-challenged patients, whereas there was a marked increase in histamine levels in the aspirin challenged patient. A possible explanation is that methacholine and antigen are inhaled and therefore have primarily local effects on the lung, whereas oral aspirin has a systemic effect with consequently systemic changes in histamine which are detectable as changes in plasma level.     

Hepatitis B infection in prostitutes

The prevalence of HBV markers in prostitutes is known to be higher than that of the normal population of similar age range in previous studies. This study showed a significant correlation between the duration of prostitution and the prevalence of HBV antibodies amongst the prostitutes. The prevalence of HBV antibodies doubled after the first year of prostitution. A two-year follow-up study of these prostitutes showed that 37% of HBsAg positive prostitutes lost the HBsAg. The seroconversion rate of HBsAg negative prostitutes was 10% during this two-year period. Early immunization against HBV infection should be considered in this high risk group in view of these findings.     

Electrophysiological properties of neurons in the rostral ventrolateral medulla of normotensive and spontaneously hypertensive rats

Single unit activities were recorded from the rostral ventrolateral medulla (RVL) of pentobarbital-anesthetized normotensive Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Throughout the recording period, arterial blood pressures of WKY (mean arterial pressure, MAP = 103.1 mm Hg) and SHR (MAP = 159.2 mm Hg) remained stable at the respective basal levels. The units recorded in this study were all spontaneously active and cardiac-locked. Two types of discharge patterns, namely single and double discharges, were identified. These single and double discharge units were found to distribute randomly in RVL. In WKY, 92.6% of RVL neurons exhibited single discharges whereas in SHR, the majority (57%) of RVL neurons exhibited double discharges. The mean firing rate of single discharge units in RVL of SHR was significantly higher than that of WKY, whereas the mean firing rate of double discharge units in WKY was similar to that of SHR. About half of the units studied were also tested for antidromic collision; all units tested could be antidromically activated from the intermediolateral column (IML) of the thoracic spinal cord and the lowest threshold sites were consistently localized within IML. In both groups of rats, the axonal conduction velocity of RVL neurons showed a bimodal distribution viz. the fast and slow conducting axons. The mean conduction velocities of each of these two groups of neurons in WKY and SHR were similar. Most of the double discharge units in WKY and SHR belonged to the fast conducting type.(ABSTRACT TRUNCATED AT 250 WORDS)