Helicobacter pylori induces apoptosis in human epithelial gastric cells by stress activated protein kinase pathway.

BACKGROUND: The pathway by which Helicobacter pylori induces apoptosis in gastric epithelial cells is not known. The aim of this study was to determine whether H. pylori-induced apoptosis is associated with SAPK/JNK activity in human gastric cancer KATO III cells. MATERIALS AND METHODS: H. pylori VacA toxin positive strain was incubated with KATO III cells for 0.5, 1, 2 or 24 hours. The SAPK/JNK protein was harvested from the KATO III cell lysate by precipitation with a C-jun fusion protein and its activity was measured by C-jun phosphorylation utilizing transblotting and phosphoserine antibody. Cellular apoptosis was demonstrated by DNA fragmentation. In addition, cell growth in coculture with H. pylori was determined over 72 hours. RESULTS: H. pylori significantly stimulated SAPK/JNK activity in KATO III cells with a peak at the 0.5 hour time point (3.6-fold vs. control, p <.05), but a return to basal levels by 2 hours. In addition, significant DNA fragmentation was observed after 24 hours in these cells but not in the control KATO III cells. Cell growth was inhibited in a dose dependent fashion in coculture with H. pylori. CONCLUSION: These results show that H. pylori triggers an increase in apoptosis in KATO III cells as reflected by DNA fragmentation. This effect was preceded and correlated with an increase in SAPK/JNK activity suggesting that the H. pylori-induced apoptosis in human gastric epithelial cells may be mediated by the SAPK/JNK pathway.

The antacid Talcid adsorbs and neutralizes all proteins secreted by H. pylori including VacA cytotoxin: a new mechanism for its ulcer-healing action?

BACKGROUND AND AIM: Helicobacter pylori culture supernatant containing VacA cytotoxin significantly inhibits gastric cell proliferation and delays healing of experimental gastric ulcers. Since cell proliferation is crucial for ulcer healing, the removal of inhibitory effects of H. pylori secreted cytotoxin would have a beneficial effect on the healing process. In this study, we determined whether the antacid Talcid can adsorb, remove, or neutralize H. pylori derived VacA cytotoxin responsible for the above deleterious actions. METHODS: Supernatants of viable H. pylori isogenic strains producing VacA cytotoxin [VacA(+)] and with disrupted cytotoxin gene not producing cytotoxin [VacA(-)] were incubated with either placebo, Talcid 10 mg/ml, omeprazole 10 mg/ml (positive control) for 1-24 h. Treated supernatants were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to evaluate proteins. We also studied the effect of supernatants on epidermal growth factor stimulated Kato III cell proliferation using BrdU labeling. RESULTS: Talcid very effectively removed from the H. pylori culture supernatant the approximately 90 kD VacA(+) cytotoxin at 3 and 24 h (99.5% removal vs. placebo-treated control; p<0.001). It also removed all other proteins, including 66-kD urease and 58-kD heat shock protein, secreted by both VacA(+) and VacA(-) H. pylori strains. Omeprazole was completely ineffective in this regard. Preincubation with Talcid completely abolished the inhibitory effect of VacA(+) H. pylori culture supernatant on epidermal growth factor stimulated Kato III cell proliferation. CONCLUSION: Adsorption and neutralization by Talcid of all H. pylori secreted proteins may explain, at least in part, the ulcer-healing action of this drug.

Helicobacter pylori culture supernatant interferes with epidermal growth factor-activated signal transduction in human gastric KATO III cells.

The mechanisms by which Helicobacter pylori infection leads to gastroduodenal ulceration remain poorly understood. Previous studies have shown that H. pylori vacuolating cytotoxin (VacA) inhibits proliferation of gastric epithelial cells, which suggests that H pylori may interfere with gastric mucosal repair mechanisms. In this study, we investigated the effects of H. pylori broth culture supernatants on epidermal growth factor (EGF)-mediated signal transduction pathways in a gastric carcinoma cell line (KATO III). Exposure of these cells to EGF resulted in increased expression and phosphorylation of the EGF receptor (EGF-R), increased ERK2 activity and phosphorylation, and increased c-fos protein levels. Preincubation of cells with broth culture supernatant from VacA (+) H. pylori strain 60190 inhibited the capacity of EGF to induce each of these effects. In contrast, preincubation of cells with broth culture supernatant from an isogenic VacA-mutant strain (H. pylori 60190-v1) failed to inhibit the effects of EGF. These results suggest that the H. pylori vacuolating cytotoxin interferes with EGF-activated signal transduction pathways, which are known to be essential for cell proliferation and ulcer healing.

Increased incidence of Clostridium difficile-associated diarrhea following decreased restriction of antibiotic use.

Removal of antimicrobial agents from formulary restriction status at our center was followed by an increase in the incidence of Clostridium difficile-associated diarrhea. The mean monthly incidence of C. difficile diarrhea for the 12-month period before institution of decreased restriction of antibiotic use was 3.4 infections per 1,000 admissions and rose to 6.2 infections per 1,000 admissions during the following 4-month period (P < .05). Patients who developed disease before and after decreased restriction of antibiotics were similar in terms of the mean number of antimicrobial agents administered and mean duration of therapy. The most commonly administered agents whose use preceded the onset of disease were cefazolin, trimethoprim-sulfamethoxazole, ampicillin, ticarcillin/clavulanate, and gentamicin (the latter drug was always used in combination with other agents). Immunoblot typing indicated that there was no association between C. difficile strains and administration of specific agents. There was no coincidental epidemic to account for the increased incidence of infection. The increased incidence of C. difficile disease is a potential problem that may occur following removal of extended-spectrum antimicrobial agents from formulary restriction status.

Low-cost, low-maintenance rearing of maggots in hospitals, clinics, and schools.

With the recent resurgence in the interest and use of maggot therapy, blow fly rearing can be expected to increase. The rearing of these necrophagous flies is technically simple, but can be expensive, malodorous, and wasteful of space. Although there are numerous references to maggot rearing in the older literature, they do not adequately address these specific problems. Therefore, we describe several of the simple, low-cost, and unobtrusive strategies for rearing blow flies that have proven useful in our hospital-based insectary.

Effects of seven antibiotics on the growth and development of Phaenicia sericata (Diptera: Calliphoridae) larvae.

Maggot therapy is used for patients with severe tissue destruction, who often receive antibiotics concurrently. Therefore, we studied the effects on maggots of ampicillin, cefazolin, ceftizoxime, clindamycin, gentamicin, mezlocillin, and vancomycin in concentrations of 1, 10, 100, and 1,000 times the average minimum bactericidal or bacteriostatic concentration against highly susceptible organisms. There was a reduction in larval survival in media with gentamicin at concentrations of 1,000 times the average pharmacologic level, or 4,000 micrograms/ml (2.7% survival), versus lower concentrations (80-88% survival). Maturation of the surviving pupae to adults also was decreased at this concentration. Media with cefazolin concentrations of 100 times the average bacteristatic level, or 800 micrograms/ml, also led to a significant decrease in larval survival (70% versus 80-88%). There were no differences in larval survival, rate of maturation, or pupal weights for organisms reared on media containing ampicillin, ceftizoxime, clindamycin, mezlocillin, or vancomycin. P. sericata matured normally--and thus can be used therapeutically--when exposed to standard pharmacologic levels of the 7 antibiotics tested. Furthermore, the methods employed herein may be helpful to forensic entomologists attempting to develop models for drug ingestion by maggots.

Helicobacter pylori: current perspectives.

Within the past decade, there has been an explosion of investigative activity and publications about Helicobacter pylori (H. pylori). Its role in gastroduodenal disease is becoming greater with definite etiologic association in chronic type B gastritis and a probable role in duodenal ulcer, a probable role in gastric ulcer, and possibly a factor in the development of interstitial type gastric carcinoma. Epidemiologic studies have shown H. pylori to be worldwide in distribution with higher prevalence rates and earlier initial infection rates in developing countries compared with industrialized nations. Person-to-person transmission appears to occur via the fecal-oral route. Pathogenesis of H. pylori-associated diseases remains unclear. We are, however, gaining insights into the role that H. pylori extracellular products play in damage to mucin and gastric epithelial cells as well as other gastroduodenal physiologic processes. Antimicrobial treatment, when effective, produces remission of mucosal pathologic changes and reduces duodenal recurrence rates. A major as yet unsolved problem is that there is no completely effective treatment regimen that consistently eradicates H. pylori in infected patients. Concepts of pathogenesis and future directions of research are discussed.

Campylobacter pylori interactions with gastric cell tissue culture.

Many investigators have reported that gastric mucosal biopsies of patients with chronic gastritis and peptic ulcer disease show the presence of Campylobacter pylori in a large majority of cases. Histologic examinations of such tissues indicate a close approximation of C. pylori with gastric surface epithelial cells. A recent report has described both adherence and cell invasion of gastric cells by C. pylori. Using a transmission electron microscope, we have examined the interaction between C. pylori, C. jejuni, and E. coli in vitro with a gastric cancer cell line, Kato III. Our results indicate marked toxicity of E. coli and moderate toxicity of C. jejuni for Kato III cells. C. pylori had only a minor effect on tissue culture viability. C. pylori was found to have a strong association with the Kato III cell membranes and evidence of occasional cell invasion. Both C. jejuni and E. coli showed no attachment or association with the Kato III cells. We interpret these findings as indicating that C. pylori may have a specific adhesion for gastric cells.

Evidence for gastric mucosal cell invasion by C. pylori: an ultrastructural study.

It is now generally accepted that Campylobacter pylori is closely associated with peptic ulcer disease and chronic type B gastritis. Whether C. pylori is the direct etiologic cause of either or both of these illnesses remains unclear. Possible pathophysiologic effects of C. pylori are still a matter of debate and conjecture. Utilizing a small group of patients with gastric ulcers and chronic gastritis, we examined the ultrastructural relationship between C. pylori and gastric cells. Forty-eight percent of our gastric ulcer patients and 57% of our chronic gastritis patients had C. pylori in their lower corpus mucosa. Examination with the transmission electron microscope indicated a very close proximation by C. pylori to the surface epithelial cells strongly suggesting adherence. We also describe for the first time the invasion of gastric cells by C. pylori. Although an uncommon occurrence, we had repeated observations of C. pylori invading surface epithelial cells, parietal cells, and chief cells. Most of the intracellular C. pylori were intact but other forms appearing to be degenerating organisms were also seen. We suggest that cell invasion may be one mechanism by which C. pylori causes pathologic changes in the gastric mucosa. These observations may also explain why C. pylori chronically infects gastric cells and frequently recurs after treatment.

Stress during internship: a prospective study of mood states.

Mood changes of interns during the internship year were studied using the Profile of Mood States (POMS), a standardized adjective checklist. All 35 interns in the University of California, Irvine-Long Beach Medical Program completed the POMS at internship orientation and at five other times during the year. Of the six mood factors measured by the POMS, four changed significantly during the testing period. Anger-hostility scores were higher (p less than 0.01) in December than at orientation and remained so throughout the year. Tension-anxiety scores were higher (p less than 0.01) and fatigue-inertia scores were lower (p less than 0.01) at orientation than at any other time during the year. Vigor-activity scores were higher (p less than 0.01) at orientation than at the end of the year. Depression-dejection and confusion-bewilderment scores did not change significantly during the study period. Recognition of these mood changes is helpful for drawing the attention of house staff and faculty members to emotional stresses of training, and for identifying issues for discussion in intern support groups.

Opsonic activity of human ascitic fluid: a potentially important protective mechanism against spontaneous bacterial peritonitis.

The opsonic activity of 60 ascitic fluids from 47 patients was measured using a standard opsonophagocytic assay. Curve analysis of the opsonic activity compared to the ascitic fluid concentration of total protein, total hemolytic complement, C3 and C4 yielded correlation coefficients of 0.84 (p less than 0.001), 0.84 (p less than 0.001), 0.94 (p less than 0.001) and 0.92 (p less than 0.001), respectively. There appeared to be a threshold of concentration for each protein below which there was no killing of bacteria. Cirrhotic ascites had significantly (all p less than 0.001) lower concentrations of total protein and complement and less opsonic activity than noncirrhotic ascites (including malignant, cardiac and miscellaneous types). Perhaps it is the dilution of crucial antimicrobial proteins below a threshold which predisposes to spontaneous bacterial peritonitis.

The impact of routine admission chest x-ray films on patient care.

We evaluated the impact of routine chest x-ray films, obtained on admission, on the treatment of patients on internal medicine wards of the Veterans Administration Medical Center, Long Beach, California--a population known to have a high prevalence of cardiopulmonary disease. The reasons for ordering chest films were determined prospectively, and three Department of Medicine faculty members reviewed the charts of admitted patients to determine the impact of chest-film results on patient care. Routine chest x-ray films were ordered for 294 (60 per cent) of the 491 patients studied. Abnormalities were noted in 106 (36 per cent) of these 294 patients. The findings were previously known, chronic, and stable in 86 patients; they were new in only 20. Treatment was changed because of chest-film results in only 12 (4 per cent) of the patients. In only one of these patients would appropriate treatment probably have been omitted if a chest film had not been obtained, and the patient's outcome was not improved by the treatment instituted. We conclude that the impact of routine admission chest x-ray films on patient care is very small, even in a population with a high prevalence of cardiopulmonary disease. We recommend that such films not be ordered solely because of admission.

Bacterial meningitis in the elderly.

To assess the implications of meningitis in a more mature population, we reviewed the records of patients with meningitis: 71 aged 50 years and older and 138 patients aged 15 to 49 years. Among the older population, 54 (76%) had bacterial, nine (13%) had granulomatous, and eight (11%) had aseptic meningitis. Among the cases of bacterial meningitis in the older age group, Streptococcus pneumoniae accounted for 24% (13/54) and enteric bacilli accounted for 17% (9/54). Serious complications occurred in 38 elderly patients (70%) with bacterial meningitis, and mortality occurred in 24 (44%). In the younger age group with bacterial meningitis, the complication rate and mortality were 41% (13/32) and 13% (4/32), respectively. Meningitis in the elderly is likely to be bacterial and to cause greater morbidity and mortality.

The circadian blood pressure pattern in ambulatory normal subjects.

Ambulatory blood pressure (BP) monitoring using a portable noninvasive device capable of automatically measuring and recording BP every 7.5 minutes during a 24-hour study period was performed in 34 normal volunteers on 2 separate occasions, 2 to 8 weeks apart, to test the consistency of the whole-day blood pressure pattern. The average of all systolic BPs measured during the second study day was within 10 mm Hg of that measured during the first study day in 79% of the subjects, and the respective diastolic BP averages were within 5 mm Hg of each other in 65% of the subjects; 53% satisfied both of these criteria. The reproducibility of the circadian pattern of the BP was tested by dividing the 24-hour day into 12 consecutive 2-hour BP averages. When the corresponding 2-hour periods on the 2 study days were matched, there were strong correlations (r greater than 0.70) within most subjects for both the systolic and diastolic BP averages of the 2-hour periods (76% and 68% of subjects) and for the relative rank values of the periods (62% and 56%). Moreover, there were no significant differences between the averages (for all subjects together) on the 2 study days of the highest and lowest systolic and diastolic 2-hour BP values; similarly, the times at which these extreme values occurred on the 2 study days corresponded closely. Thus, in normal subjects there is a strong tendency for the circadian pattern and the actual levels of BP to be consistent between 24-hour study periods.

Mood changes during the internship.

A prospective study using two standardized psychological tests, the Profile of Mood States (POMS) and the Self-Rating Depression Scale (SDS), was conducted in an effort to quantify the emotional changes experienced by internal medicine house staff members during the internship. In contrast to instruments used in previous investigations of this type, the POMS and the SDS are standardized tests with proven reliability and validity. The six mood factors measured, "tension-anxiety," "depression-dejection," "anger-hostility," "vigor-activity," "fatigue-inertia," and "confusion-bewilderment," are reported to be among those factors most often affected by the internship experience. Twenty-three interns completed both tests at four-month intervals during one academic year. One-way analysis of variance for repeated measures revealed that the level of only anger-hostility of the mood factors changed significantly during the year. The intensity of this factor increased between the first and third testing periods before dropping at the end of the year. In contrast to findings in previous studies, the depression and fatigue factors did not increase during the year. By characterizing interns' reactions to the stresses of postgraduate medical education, standardized psychological tests can contribute to improved understanding of these reactions and to more intelligent planning of support systems.

Circadian blood pressure patterns in ambulatory hypertensive patients: effects of age.

Circadian blood pressure monitoring was performed in 50 untreated ambulatory hypertensive patients to study the effects of age on the pattern and variability of blood pressure and heart rate. Casual blood pressure, measured in the morning, was greater than the average of the blood pressures measured at 7.5 minute intervals for 24 hours (148 +/- 2/95 +/- 2 and 137 +/- 2/88 +/- 2 mm Hg, p less than 0.001). The correlation between casual systolic pressure and the 24 hour average was stronger (p less than 0.05) in younger (less than 55 years of age) patients (r = 0.69, n = 24, p less than 0.001) than in older patients (r = 0.42, n = 26, p less than 0.1). Similarly, diastolic pressures correlated more strongly (p less than 0.05) in younger patients (r = 0.71, p less than 0.001) than in older patients (r = 0.43, p less than 0.05). Variability of systolic pressure, defined as the standard deviation of all readings obtained during 24 hours, was greater than that of diastolic pressure (16.7 and 13.1 mm Hg, respectively, p less than 0.001). Moreover, the variability of systolic pressure was greater in older than in younger patients (18.1 and 15.2 mm Hg, respectively, p less than 0.01). The variability of diastolic pressure was slightly but not significantly greater in older patients (13.7 and 12.5 mm Hg, not significant). The circadian pattern of blood pressure, expressed as averages of readings obtained during consecutive 2 hour intervals, was similar in the two age groups. However, the level of systolic pressure was consistently higher (p less than 0.01) and that of both diastolic pressure and heart rate consistently lower (p less than 0.01) in older patients. Thus, ambulatory circadian blood pressure monitoring reveals significant changes in blood pressure levels and its variability with age; the casual blood pressure does not accurately reflect these changes. Longer periods of blood pressure monitoring are required for accurate assessment of the characteristics of hypertension in the aged.

A representative value for whole-day BP monitoring.

Shorter-term alternatives to 24-hour monitoring of BP were analyzed in six hypertensive patients. Average BPs during the 24-hour period were lower than averages during a two-hour morning period or those based on either three readings or a single reading taken at 8 AM. The systolic and diastolic BP two-hour averages correlated more strongly with the 24-hour averages than did the three-reading or single-reading alternatives. Thus, the average of serial BP readings obtained during a two-hour morning period is a consistent predictor of the whole-day BP and represents it more closely than the conventionally used single reading.

Reproducibility of the whole-day blood pressure pattern in essential hypertension.

The reproducibility of the whole-day blood pressure, measured by an automated non-invasive device at 7.5 minute intervals, was examined in 6 hypertensive patients in a quiet in-hospital environment. During a repeat study performed two weeks after the baseline monitoring period, the average 14-hour systolic blood pressure had changed significantly in 3 patients and the diastolic blood pressure in 2. If the day was divided into 12 consecutive 2-hour periods, systolic blood pressure averages during corresponding periods of the two studies correlated significantly in 3 of the 6 patients, and diastolic blood pressure averages in 5. After a third 24-hour monitoring period, carried out in 4 of the 6 patients, it was found that the baseline study correlated more strongly with the second repeat study than with the first, and that the correlations between the two latter studies were the strongest of all. Thus, even during relatively non-stimulatory conditions the 24-hour blood pressure is not constant in all patients; there is a tendency, however, towards greater reproducibility of the whole-day blood pressure patterns as patients become more familiar with the monitoring procedure.