P2X receptor characterization and IL-1/IL-1Ra release from human endothelial cells.

BACKGROUND AND PURPOSE: The pro-inflammatory cytokine, interleukin-1beta (IL-1beta), has been implicated in the pathogenesis of atherosclerosis, potentially via its release from vascular endothelium. Endothelial cells (EC) synthesize IL-1beta in response to inflammatory stimuli, but the demonstration and mechanism of release of IL-1 from ECs remains unclear. In activated monocytes, efficient release of bioactive IL-1beta occurred via activation of ATP-gated P2X(7) receptors (P2X(7)Rs). Activation of P2X(7)R in ECs from human umbilical vein (HUVECs) released IL-1 receptor antagonist (IL-1Ra). The purpose of this study was to provide a quantitative investigation of P2XR expression and function, in parallel with IL-1beta and IL-1Ra synthesis, processing and release, in HUVECs under pro-inflammatory conditions. EXPERIMENTAL APPROACH: Quantitative RT-PCR, immunoblotting, ELISA, flow cytometry, and whole-cell patch clamp recordings were used to determine protein expression and receptor function. IL-8-luciferase-reporter was used as an IL-1 sensitive bioassay. KEY RESULTS: HUVECs expressed P2X(4)R and P2X(7)R subtypes and both were significantly up-regulated under inflammatory conditions. P2X(7)R currents were increased 3-fold by inflammatory stimuli, whereas no P2X(4)R-mediated currents were detected. Caspase-1, but not IL-1beta, was present intracellularly under basal conditions; inflammatory stimuli activated the synthesis of intracellular pro-IL-1beta and increased caspase-1 levels. Activation of P2X(7)Rs resulted in low-level release of bioactive IL-1beta and simultaneous release of IL-1Ra. The net biological effect of release was anti-inflammatory. CONCLUSIONS AND IMPLICATIONS: Endothelial P2X(7)Rs induced secretion of both pro- and anti-inflammatory IL-1 receptor ligands, the balance of which may provide a means for altering the inflammatory state of the arterial vessel wall.

Peripubertal ontogeny and estrogen stimulation of cholecystokinin and preproenkephalin mRNA in the rat hypothalamus and limbic system.

The neuropeptide cholecystokinin (CCK) is expressed in limbic system and hypothalamic nuclei that form a circuit that regulates the display of the female rodent reproductive behavior, lordosis. CCK mRNA and peptide levels fluctuate across the estrous cycle and have been shown to be modulated by estrogen exposure. The objective of these experiments was to examine the expression of CCK mRNA during postnatal development of this limbic-hypothalamic, lordosis regulating circuit, and to determine the age at which CCK mRNA expression becomes responsive to estrogen stimulation, by using quantitative in situ hybridization histochemistry. CCK mRNA levels were below the level of detectability within the circuit during the postnatal period, but increased during the peripubertal period. Rats were injected with either estradiol benzoate (EB), EB and progesterone, progesterone, or oil, and were killed 48 hours later on postnatal day (PND) 15, 20, and 25. Alternate brain sections were processed for CCK and preproenkephalin (PPE) mRNA in situ hybridization histochemistry. EB treatment induced CCK mRNA expression in the central portion of the medial preoptic nucleus and posterodorsal medial amygdala at PND 20 and 25, respectively. However, EB treatment increased PPE mRNA levels within the nuclei of the circuit at all ages examined. Progesterone had neither an independent nor additive effect on the EB induction of these neuropeptide messages. The estrogenic induction of CCK mRNA appears to be dependent on estrogen sensitive pathways of neurotransmission, or components of second messenger pathways which regulate CCK mRNA expression in the adult limbic-hypothalamic lordosis regulating circuit, which are not functional before PND 18-25.

Infusion of CCK-A receptor mRNA antisense oligodeoxynucleotides inhibits lordosis behavior.

Cholecystokinin (CCK) acting on discrete receptors in the limbic-hypothalamic circuit modulates lordosis behavior. Neurons in the medial preoptic nucleus (MPN) express CCK-A subtype receptor mRNA, and site-specific infusions of CCK facilitate lordosis, suggesting that CCK-A receptor activation positively modulates lordosis. In the present study, we demonstrated CCK binding in the central portion of the MPN (MPNc) and the disruption of lordosis behavior by reducing the expression of CCK-A receptors in this nucleus. Antisense oligodeoxynucleotides (ODN) specific for CCK-A receptor mRNA were infused into the MPN of ovariectomized female rats. The expression of estrogen-induced lordosis behavior was blocked in animals receiving infusions of antisense ODN into the MPN (LQ = 10.0 +/- 1.0) compared to animals receiving infusions of nonsense ODN (containing the same nucleotide bases in a random order; LQ = 92.5 +/- 7.5). In vitro, AR42J pancreatic acinar carcinoma cells treated with antisense ODN had lower levels of CCK-A and CCK-B subtype receptor binding than nonsense ODN treated cells. In vivo, however, infusions of CCK-A mRNA antisense ODN did not alter CCK-B receptor binding levels. These results suggest that CCK, acting via CCK-A receptors in the MPNc, is critical for the display of lordosis behavior.

Estrogen modulation of opioid and cholecystokinin systems in the limbic-hypothalamic circuit.

The display of lordosis behavior has been correlated with the estrogen-induced expression of cholecystokinin (CCK) and enkephalin within the limbic-hypothalamic circuit. These neuropeptides have opposing effects on lordosis; for example, in the medial preoptic nucleus, CCK facilitates and opiates inhibit lordosis. Antisense oligodeoxynucleotide blockade of receptor expression indicated that CCK modulates lordosis in the medial preoptic nucleus through the CCK(A)-receptor. Sequence-specific antibodies directed against delta- and mu-opiate receptor proteins labeled fibers in the medial preoptic nucleus. Estrogen treatment of ovariectomized rats or etorphine (a nonselective opiate agonist) treatment altered the appearance of the immunoreactivity from a diffuse pattern to one of distinctly stained mu-opiate receptor immunoreactive cells and varicose fibers in the medial preoptic nucleus. Such a pattern of staining reflects an internalization of mu-opiate receptors following agonist stimulation. This type of internalization has been used as an indication of synaptic activity. The distribution of receptor internalization surrounds the distribution of CCK cells in the medial preoptic nucleus, suggesting that endogenous opioid peptides may modulate estrogen-induced CCK mRNA expression. Interestingly, nonselective and delta-opiate receptor selective antagonists potentiated the estrogen-induced CCK mRNA expression in the medial preoptic nucleus. Together, these results suggest that endogenous opioid peptides may modulate the estrogenic upregulation of CCK mRNA expression and demonstrate an important level of regulation of gene expression in which synaptic activity modifies hormonal input.

Streptococcus pneumoniae colonization in the young child: association with otitis media and resistance to penicillin.

OBJECTIVE: To determine the nasopharyngeal colonization rate of penicillin-susceptible and penicillin-resistant strains of Streptococcus pneumoniae in young children, and to assess its relationship with the incidence of otitis media. DESIGN: Observational study in 215 children younger than 6 years of age who received care in the Vanderbilt Vaccine Clinic from September 1, 1992, to August 31, 1993. RESULTS: Of 842 nasopharyngeal cultures obtained, results for 44% of the cultures were positive for S. pneumoniae; 73% of the isolates were serotypes 6, 14, 19, or 23. Younger children had significantly higher rates of pneumococcal colonization than older children, with a peak at 1 year of age. By microdilution susceptibility testing, 37% of the cultures with positive results were intermediately or highly resistant to penicillin. Significantly more serotype 19 and 23 isolates were penicillin resistant than organisms of other serotypes. Children younger than 2 years of age had a twofold higher percentage of resistant isolates than those older than 2 years of age. A significant association was noted between nasopharyngeal carriage of S. pneumoniae and acute otitis media (p = 0.0002); however, the incidence of acute otitis media did not differ significantly between children colonized with penicillin-susceptible or penicillin-resistant strains. Unresolved otitis media was diagnosed more often in children who were colonized with resistant organisms than in children colonized with susceptible strains (p = 0.04). CONCLUSIONS: There was a high rate of nasopharyngeal carriage of penicillin-resistant S. pneumoniae in this population of young children. Nasopharyngeal colonization was associated with an increased incidence of acute otitis media, and penicillin resistance was associated with an increased incidence of unresolved otitis media.

Comparative trial of large-particle aerosol and nose drop administration of live attenuated influenza vaccines.

Healthy adult subjects (n = 198) were randomized to receive bivalent cold-adapted (ca) influenza A vaccine containing 10(7.4) TCID50 each of A/Kawasaki/9/86 (H1N1) and A/Los Angeles/2/87 (H3N2) by either nose drops (ND) or large-particle aerosol (LPA). All subjects had received monovalent inactivated influenza B vaccine intramuscularly in the previous year. Ninety percent of LPA recipients and 82% of ND recipients preferred intranasal administration to their previous experience with intramuscular vaccine. Twenty-six (27%) of 98 LPA recipients and 47 (49%) of 97 ND recipients reported swallowing vaccine (P = .008). Fever was observed uncommonly (< or = 5%), and incidence of respiratory symptoms was comparable between groups. Fourfold or greater hemagglutination antibody response to at least one of the influenza A vaccine viruses was significantly more frequent after LPA (64%) than ND vaccination (43%; P = .005). LPA administration of ca influenza to the nasopharynx was well tolerated, safe, and more immunogenic than ND delivery.

Fate of incorrectly addressed mailed questionnaires.

Mail surveys are a widely used and often inexpensive method for conducting epidemiologic studies, but may suffer from poor response rates. Some of this nonresponse may be due to letters that never reach study subjects rather than nonparticipation. We mailed letters to invalid addresses and to fictitious occupants at valid addresses in order to determine the proportion of letters that were not returned to us by the postal service as undeliverable. All letters mailed to invalid addresses were returned, whereas 13.3% (95% Cl 2.0-24.7%) of letters sent to fictitious occupants were not. Because some undeliverable mail is not returned, investigators may erroneously assume nonresponse for some subjects who never have the opportunity to participate.

Participation of high-risk subjects in colon cancer screening.

Siblings of colorectal cancer patients are estimated to be three times more likely to develop colorectal cancer. Although these high-risk siblings are appropriate candidates for colon cancer screening, the factors that affect their participation in screening programs are not known. A study was conducted to examine the factors that might influence participation in fecal occult blood test (FOBT) screening by high-risk siblings of recently diagnosed colon cancer patients and nonhigh-risk control siblings. Siblings were mailed a sample FOBT card, a letter informing them of their risk status, and an invitation to participate in the FOBT screening program. They were later interviewed by telephone about their intention to participate in FOBT screening. FOBT cards were returned by 52.2% of high-risk siblings compared with 37.7% of control siblings (P less than 0.005). Only 24.8% of the cancer siblings thought they were more likely to get colon cancer compared with others their own age, and 27.8% thought they were less likely. Perceived risk of cancer and demographic and health-related factors did not predict compliance beyond membership in the high-risk group. Siblings of colon cancer patients are more likely to participate in screening, and efforts to screen them could have a substantial impact on colorectal cancer. More work is needed to identify the factors responsible for compliance so that effective interventions can be developed.

Adenomas of the large bowel after cholecystectomy. A case-control study.

Cholecystectomy has been reported to be more common in patients with colon cancer. Since adenomas are believed to be precursors of colon cancer, we evaluated the association of cholecystectomy and adenomas in a case-control study. Cases were 244 patients with one or more adenomas who underwent colonoscopy at the North Carolina Memorial Hospital between May 1983 and August 1985. Controls were 281 patients without adenomas. Overall, cholecystectomy was found in 11.9% of cases and 10.3% of controls. White women with adenomas, however, were more likely to have had previous cholecystectomy than controls [odds ratio (OR) 2.5, 95% confidence interval (CI) 1.1-5.6]. Although women with adenomas were older and heavier than controls, the elevated odds ratio persisted after adjustment for these factors (OR 1.8; 95% CI 0.8-4.2). No association was observed for men or for nonwhite women. Cholecystectomy may predispose to colon adenomas and to colon cancer in some patients. Alternatively, common risk factors for gallstones, cholecystectomy, and colon cancer may explain the apparent risk of colon cancer or adenomas after cholecystectomy.

Colon adenomas in patients with hyperplastic polyps.

Although hyperplastic polyps are generally believed to have no malignant potential, recent work has suggested that they might be more common in patients with adenomas. We evaluated whether hyperplastic polyps could serve as a marker for patients who might benefit from colonoscopy. We retrospectively reviewed 1,588 consecutive colonoscopy reports and hospital charts on 1,407 different patients examined between May 1983 and August 1985: 242 patients had adenomas, and 94 had hyperplastic polyps. Of patients with hyperplastic polyps 93.6% had concomitant adenomas, as compared with 35.7% of those without, p less than 0.001. Adenomas proximal to the rectosigmoid were found in 61.7% of patients with hyperplastic polyps and in 25.3% of those without, p less than 0.001. Patients with hyperplastic polyps in the rectosigmoid had proximal adenomas more frequently (64.7%) than did those without rectosigmoid hyperplastic polyps (29.4%), p less than 0.001. We conclude that patients with hyperplastic polyps are more likely to have adenomas, and patients with rectosigmoid hyperplastic polyps are more likely to have proximal adenomas. Based on these preliminary data, we believe that the finding of hyperplastic polyps in the rectosigmoid might justify full colonoscopy and that this should be studied further.

Biliary tract surgery in the elderly.

Cholecystectomy is the most common surgical procedure in the elderly. Published reports on this operation suffer from small sample sizes, crude analyses, erroneous conclusions, and unsupported recommendations. The authors reviewed all first operations for cholelithiasis at the North Carolina Memorial Hospital from 1973 to 1982 to assess factors associated with postoperative morbidity in patients aged 70 years or older after biliary tract surgery. These elderly patients (n = 142) had more frequent postoperative complications (26.1% vs. 12.0%, p less than 0.0001), and greater mortality (9.2% vs. 2.7%, p less than 0.005) than did 839 younger patients. Patients with preoperative cardiac, renal or neurologic problems had more frequent postoperative complications whether they were old or young. Postoperative complications were more frequent in the elderly for non-emergency surgery (21.2% vs. 9.7%) but not for emergency surgery (44.8% vs. 42.4%). Logistic regression analysis was used to determine independent predictors of postoperative complications. Age greater than or equal to 70 years was associated with an increase in risk that was not statistically significant.

Trends in gastric cancer sex ratio in the United States.

Recent reports have suggested that the male/female ratio for gastric cancer has decreased over time and that the mean age of cases has increased. The authors calculated age- and sex-specific mortality rates for gastric cancer from 1950 through 1979 for whites using data from US Vital Statistics and population estimates from the US Census Bureau. Whereas sex ratios based on crude mortality rates showed a decrease over time from 1.7 in 1950 to 1.5 in 1979, similar ratios based on age-adjusted mortality rates showed an increase from 1.8 to 2.1. An increase in mean age at death over time was consistent with the increase in age of the population. The authors conclude that factors responsible for the decrease in gastric cancer mortality in the United States appear to have affected males and females equally. The overall data do not support the emergence of a new form of gastric cancer with a sex ratio or age pattern different from that found in the past.

Risk of stomach cancer after gastric surgery for benign conditions. A case-control study.

Patients with previous gastric surgery for benign conditions are thought to be at increased risk for gastric cancer based on studies from Europe. Recommendations have been made to screen these patients repeatedly in order to detect early gastric cancers. We conducted a case-control study to estimate the risk in the United States where the rates of gastric cancer are lower. There were 521 gastric cancer cases who were matched by age, race, sex, and date of admission to an equal number of hospital controls. The odds ratio for cancer after gastric surgery was 0.7 (P = 0.4) which indicates no increased risk. The sample size was sufficient to detect risks in the range reported in previous studies. The odds ratios rose with longer postoperative intervals but were based on small numbers and were not statistically significant. This case-control study failed to demonstrate an overall increased risk for gastric cancer after surgery. Further study is needed before general screening can be endorsed in the United States.

Effect of toxic thioureas on resistance of rats to growth in the lungs of intravenously and intratracheally seeded tumour cells.

Clonogenic growth (colony-forming efficiency, CFE) of i.v. injected allogeneic W256 tumour cells in the lungs was markedly enhanced by treatment of rats with alpha-naphthyl thiourea (ANTU) injected i.p. from 2 h before to 2 h after the tumour cells. ANTU specifically increases pulmonary vascular permeability in adult rats and causes acute pulmonary oedema and pleural effusion. Inhibition of drug toxicity to the lungs by tachyphylaxis, specific antimetabolites or iodides did not abolish the effect of ANTU on CFE. CFE was not increased when cells were seeded by i.v. injection the lungs affected by advanced pulmonary oedema at 6 to 24 h after treatment with drug. ANTU did not enhance growth of intratracheally injected cells. Although ANTU has no cytotoxic or immunosuppressive action, treatment of tumour-immunized rats with ANTU caused apparent "breakdown" of tumour immunity in 50% of rats, by causing growth of tumour colonies in the lungs. Possible mechanisms for the ANTU-induced decrease in innate resistance to growth of tumour in the lungs are discussed.