Evaluation of two string tests for obtaining gastric juice for culture, nested-PCR detection, and combined single- and double-stranded conformational polymorphism discrimination of Helicobacter pylori.

We have compared two gastric string tests for obtaining gastric juice for culture of Helicobacter pylori and for nested-PCR detection and PCR-based combined single- and double-stranded conformational polymorphism (SDSCP) discrimination of infecting strains. String test specimens were obtained from one seropositive volunteer for 13 consecutive weeks. The distal 10 cm of each string was suspended in 1 ml saline and quantitatively cultured. An additional nine volunteers with histories of upper-gastrointestinal complaints were given a string test for culture and nested-PCR assay. H. pylori isolates and/or gastric juice from each volunteer were extracted for DNA and analyzed by PCR-based SDSCP. Quantitative culture showed that the Entero-test was four times as sensitive as the Gastro-test but was more prone to contamination by oral flora. However, the two string tests are equally sensitive by PCR assays. Thus, the Gastro-test is more suitable for culture detection of H. pylori, since it is less prone to oral contamination and its shorter length is better tolerated. SDSCP analysis of H. pylori DNA from four PCR-positive volunteers without requiring culture showed four distinct profiles, indicating different infecting strains. SDSCP analysis of strains isolated before and after treatment of one volunteer had the same SDSCP profile, suggesting endogenous reinfection by the same strain.

Inhibiting early activation of tissue nuclear factor-kappa B and nuclear factor interleukin 6 with (1-->3)-beta-D-glucan increases long-term survival in polymicrobial sepsis.

BACKGROUND: Recent data implicate the activation of nuclear factor-kappa B (NF-kappa B) and nuclear factor interleukin 6 (NF-IL6) as important steps in the pathophysiologic mechanisms of adult respiratory distress syndrome and systemic inflammatory response syndrome. METHODS: This study evaluated the effect of immunomodulating polysaccharides on transcription factor activation, cytokine expression, and mortality in a murine cecal ligation and puncture (CLP) model. ICR/HSD mice were treated with glucan (50 mg/kg) 1 hour before or 15 minutes after CLP. Liver and lung tissue were harvested at 3 hours and mortality trends were observed for 20 days. RESULTS: CLP increased liver and lung NF-kappa B and NF-IL6 nuclear binding activity as well as tumor necrosis factor-alpha and interleukin 6 messenger RNA levels at 3 hours. Pretreatment or posttreatment with glucans inhibited tissue NF-kappa B and NF-IL6 nuclear binding activity and tissue cytokine messenger RNA levels. Prophylaxis with glucan phosphate or scleroglucan increased (P < .001) long-term survival (20% CLP vs 65% glucan phosphate, 75% scleroglucan). Posttreatment with glucan phosphate also increased (P < .05) long-term survival (20% vs 65%). CONCLUSIONS: Pretreatment or posttreatment with biologic response modifiers decreased tissue transcription factor nuclear binding activity and cytokine message in liver and lung of septic mice. Inhibiting early transcription factor activation and cytokine message expression correlates with improved outcome in polymicrobial sepsis as denoted by increased long-term survival.

Early activation of hepatic NFkappaB and NF-IL6 in polymicrobial sepsis correlates with bacteremia, cytokine expression, and mortality.

BACKGROUND: The role of transcription factor activation in the pathophysiology of sepsis syndrome has not been established. This study investigated the relation between tissue nuclear factor kappaB (NFkappaB) and nuclear factor interleukin 6 (NF-IL6 or C/EBP) activation and bacteremia, inflammatory cytokine expression, and mortality in a murine model of cecal ligation and puncture (CLP). METHODS: Transcription factor activation was assessed by the electrophoretic mobility shift assay. Cytokine mRNA levels were established by reverse transcription-polymerase chain reaction and quantified by scanning densitometry. Bacteremia was evaluated by standard aerobic and anaerobic microbiologic methods. RESULTS: CLP stimulated hepatic NFkappaB activation at 2, 3, 4, 5, 6, and 8 hours compared with control and sham-operated mice. Hepatic NFkappaB activation during CLP peaked at 4 hours (1114% vs. no surgery, 609% vs. sham). Hepatic NF-IL6 activation was observed at 3, 4, and 6 hours after CLP. Hepatic and splenic levels of tumor necrosis factor-alpha and IL-6 mRNA were also elevated after CLP. Bacteremia in CLP mice consisted of Bacteroides species and to a lesser extent facultative gram-negative bacilli and group D Enterococcus. CONCLUSIONS: Early activation of hepatic and splenic NFkappaB and NF-IL6 positively correlates with tissue cytokine mRNA expression and mortality in a surgical model of polymicrobial sepsis. The data suggest that transcription factor activation is an early event in the pathophysiology of sepsis.

Early activation of pulmonary nuclear factor kappaB and nuclear factor interleukin-6 in polymicrobial sepsis.

BACKGROUND: Transcription factor activation may be a pivotal step in the pathophysiology of sepsis syndrome and adult respiratory distress syndrome. This study investigated the activation of lung nuclear factor kappaB (NFkappaB) and nuclear factor interleukin-6 (NF-IL6) and how they correlate to proinflammatory cytokine expression and mortality in a murine model of cecal ligation and puncture (CLP). METHODS: Polymicrobial sepsis was induced by CLP. Transcription factor activation was assessed at 0, 1, 2, 3, 4, 5, 6, 8, and 24 hours after CLP by the electrophoretic mobility-shift assay. Lung cytokine mRNA levels were established by reverse transcriptase-polymerase chain reaction. RESULTS: CLP induced pulmonary NFkappaB activation at 3, 4, and 8 hours (p < 0.05). Lung NFkappaB activation peaked at 3 hours (533% vs. no surgery, 2,900% vs. sham treatment) after CLP. Supershift analysis revealed a predominance of p50 subunits in the lung nuclear extracts of septic mice 3 hours after CLP, indicating the presence of p50 homodimer. In contrast, liver nuclear extracts from septic mice indicated the presence of both p65 and p50 subunits at 3 hours. Lung NF-IL6 activation (p < 0.05) was observed at 4 hours (649% vs. no surgery, 296% vs. sham treatment) and 6 hours after CLP. Lung tumor necrosis factor-alpha mRNA levels were increased (p < 0.05) at all time intervals after CLP. Lung IL-6 mRNA levels were increased at 3, 6, and 8 hours after CLP. CONCLUSION: Early activation of lung NFkappaB and NF-IL6 and lung cytokine mRNA expression correlated with mortality in polymicrobial sepsis. Although IL-6 mRNA levels correlated with NFkappaB and NF-IL6 activation, tumor necrosis factor-alpha mRNA levels did not, in that they preceded transcription factor activation. These data suggest a potential role for NFkappaB and NF-IL6 activation in the initiation and propagation of acute lung injury.

Identification of H. pylori in saliva by a nested PCR assay derived from a newly cloned DNA probe.

A novel probe was developed from genomic DNA of Helicobacter pylori ATCC type strain 43629. It hybridized with all 73 H. pylori clinical isolates tested but not with any of 183 non-H. pylori DNAs in dot blot hybridization. Typing tests revealed 41 different HaeIII-digestion patterns from 57 H. pylori strains tested. Based on the sequence of the probe, a nested PCR was developed that detected as little as 2 fg of H. pylori DNA or approximately equivalent to one cell. No PCR products were amplified from any of 21 non-H. pylori strains tested. Using this nested PCR, H. pylori DNA was detected in 33 of 45 (73%) saliva samples collected from patients with gastric H. pylori infection. These data suggest that the probe is useful for typing H. pylori and that the nested PCR is a valuable tool for detecting H. pylori DNA in saliva.

Systemic hemodynamic, neurohormonal, and renal effects of a steady-state infusion of human brain natriuretic peptide in patients with hemodynamically decompensated heart failure.

BACKGROUND: Human brain natriuretic peptide (hBNP) is a promising agent for the treatment of decompensated cardiac failure. However, the systemic hemodynamic, neurohormonal, and renal effects of hBNP have been incompletely studied in human heart failure. METHODS AND RESULTS: The effects of a continuous 4-hour infusion of hBNP were determined in 16 decompensated heart failure patients in an invasive, randomized, double-blind, placebo-controlled study. Patients were evaluated during three 4-hour study periods: baseline, treatment (placebo [n = 4] versus hBNP 0.025 or 0.05 microgram/kg/min [n = 12]), and post-treatment. Urinary volume losses were replaced hourly to separate the vasodilatory and diuretic effects of hBNP. Two patients in the hBNP group were excluded from the analysis because of adverse events. hBNP significantly (P < .001) reduced right atrial pressure and pulmonary capillary wedge pressure by approximately 30% and 40%, respectively. hBNP also significantly lowered systemic vascular resistance from 1722 +/- 139 to 1101 +/- 83 dynes.s.cm-5 (P < .05). These unloading effects of hBNP produced a 28% increase in cardiac index (P < .05) with no change in heart rate. Compared to placebo, hBNP decreased plasma norepinephrine and aldosterone. Renal hemodynamics were unaffected by hBNP; however, most patients were resistant to its natriuretic effect. CONCLUSIONS: 1) The predominant hemodynamic effects of hBNP were a decrease in cardiac preload and systemic vascular resistance. 2) hBNP also improved cardiac output without increasing heart rate. 3) Plasma norepinephrine and aldosterone levels decreased during hBNP infusion. 4) hBNP is pharmacologically active and has potential in the therapy for decompensated heart failure.

Combined oral positive inotropic and beta-blocker therapy for treatment of refractory class IV heart failure.

OBJECTIVES: We sought to assess the effects of combined oral positive inotropic and beta-blocker therapy in patients with severe heart failure. BACKGROUND: Patients with severe, class IV heart failure who receive standard medical therapy exhibit a 1-year mortality rate >50%. Moreover, such patients generally do not tolerate beta-blockade, a promising new therapy for chronic heart failure. Positive inotropes, including phosphodiesterase inhibitors, are associated with increased mortality when administered over the long term in these patients. The addition of a beta-blocker to positive inotropic therapy might attenuate this adverse effect, although long-term oral inotropic therapy might serve as a bridge to beta-blockade. METHODS: Thirty patients with severe heart failure (left ventricular ejection fraction [LVEF] 17.2+/-1.2%, cardiac index 1.6+/-0.1 liter/min per m2) were treated with the combination of oral enoximone (a phosphodiesterase inhibitor) and oral metoprolol at two institutions. Enoximone was given at a dose of < or = 1 mg/kg body weight three times a day. After clinical stabilization, metoprolol was initiated at 6.25 mg twice a day and slowly titrated up to a target dose of 100 to 200 mg/day. RESULTS: Ninety-six percent of the patients tolerated enoximone, whereas 80% tolerated the addition of metoprolol. The mean duration of combination therapy was 9.4+/-1.8 months. The mean length of follow-up was 20.9+/-3.9 months. Of the 23 patients receiving the combination therapy, 48% were weaned off enoximone over the long term. The LVEF increased significantly, from 17.7+/-1.6% to 27.6+/-3.4% (p=0.01), whereas the New York Heart Association functional class improved from 4+/-0 to 2.8+/-0.1 (p=0.0001). The number of hospital admissions tended to decrease during therapy (p=0.06). The estimated probability of survival at 1 year was 81+/-9%. Heart transplantation was performed successfully in nine patients (30%). CONCLUSIONS: Combination therapy with a positive inotrope and a beta-blocker appears to be useful in the treatment of severe, class IV heart failure. It may be used as a palliative measure when transplantation is not an option or as a bridge to heart transplantation. Further study of this form of combined therapy is warranted.

Differentiation of Helicobacter pylori strains directly from gastric biopsy specimens by PCR-based restriction fragment length polymorphism analysis without culture.

Recent studies have shown the usefulness of PCR-based restriction fragment length polymorphism (RFLP) analysis for differentiating Helicobacter pylori strains isolated by culture. For this study, a PCR-based RFLP assay was developed for directly typing H. pylori strains from gastric biopsy specimens. Nineteen gastric biopsy specimens obtained from patients undergoing endoscopy for gastrointestinal complaints were cultured for isolation of H. pylori. Genomic DNA preparations from these gastric biopsy specimens and the corresponding H. pylori isolates were tested by our PCR-based RFLP assay. The 1,179-bp H. pylori DNA fragments amplified by the PCR assay were digested with the restriction enzymes HhaI, MboI, and AluI and analyzed by agarose gel electrophoresis. HhaI, MboI, and AluI digestion produced 11, 10, and 6 distinguishable digestion patterns, respectively, from the 19 H. pylori isolates tested and generated 13, 11, and 6 different patterns, respectively, from the 19 gastric biopsy specimens. The patterns from 13 of the 19 gastric biopsy specimens matched those of the H. pylori isolates from the corresponding patients. The patterns from the remaining six biopsy specimens appeared to represent infection by two strains of H. pylori; the pattern of one strain was identical to that of the isolate from the corresponding patient. By combining all the restriction enzyme digestion patterns obtained by using HhaI, MboI, and AluI, we observed 19 distinct RFLP patterns from the 19 specimens. The results suggest that the PCR-based RFLP analysis method may be useful as a primary technique to identify and distinguish H. pylori strains directly from gastric biopsy specimens without culture of the organisms.

A newly developed PCR assay of H. pylori in gastric biopsy, saliva, and feces. Evidence of high prevalence of H. pylori in saliva supports oral transmission.

We have recently developed a new PCR assay for the detection of H. pylori. In this study, the polymerase chain reaction (PCR) assay was used to detect H. pylori in 88 gastric biopsy, 85 saliva, and 71 fecal specimens from 88 patients. H. pylori infection was confirmed in 71 of 88 patients by culture and/or histological stain of gastric biopsies. Serum IgG antibody to H. pylori was also measured and resulted in 97% sensitivity and 94% specificity. H. pylori DNA was detected by the PCR assay in gastric biopsy specimens from all 71 patients (100% sensitivity) with proven gastric H. pylori infection but not from 17 noninfected patients (100% specificity). In saliva specimens, H. pylori DNA was identified in 57 of the 68 patients (84%) with proven gastric H. pylori infection and in three of the 17 patients without gastric H. pylori infection. However, the PCR assay was only able to detect H. pylori DNA in the feces from 15 of 61 patients (25%) with proven gastric H. pylori infection and one of the 10 patients without gastric H. pylori infection. The results show that the PCR assay is reliable for detecting the presence of H. pylori in gastric biopsy and saliva specimens. The data indicate that H. pylori exists in a higher prevalence in saliva than feces and that the fecal-oral route may be an important means of transmission of this infection in developing countries but not as significant as previously suspected in the developed countries. It is likely that the oral-oral route is more prominent.

High prevalence of Helicobacter pylori in saliva demonstrated by a novel PCR assay.

AIMS: To investigate the prevalence of Helicobacter pylori in the saliva of patients infected with this bacterium. METHODS: A novel polymerase chain reaction (PCR) assay was developed to detect H pylori in saliva and gastric biopsy specimens from patients undergoing endoscopy. RESULTS: Our PCR assay amplified a 417 base pair fragment of DNA from all 21 DNAs derived from H pylori clinical isolates but did not amplify DNA from 23 non-H pylori strains. Sixty three frozen gastric biopsy and 56 saliva specimens were tested. H pylori specific DNA was detected by PCR in all 39 culture positive biopsy specimens and was also identified from another seven biopsy specimens which were negative by culture but positive by histology. H pylori specific DNA was identified by PCR in saliva specimens from 30 (75%) of 40 patients with H pylori infection demonstrated by culture or histological examination, or both, and in three patients without H pylori infection in the stomach. CONCLUSION: The results indicate that the oral cavity harbours H pylori and may be the source of infection and transmission.

Comparative hemodynamic effects of OPC-18790 and dobutamine in patients with advanced heart failure.

OPC-18790 (Otsuka America Pharmaceutical, Rockville, MD), a novel positive inotropic agent, produces titratable hemodynamic benefits in patients with advanced heart failure. In such patients, OPC-18790 has been shown to acutely increase the cardiac index, while reducing systemic vascular resistance and left ventricular filling pressure, without an associated increase in heart rate. This study was performed to compare the acute hemodynamic effects of OPC-18790 and the beta-adrenergic receptor agonist, dobutamine, in patients with advanced heart failure. OPC-18790 and dobutamine were compared on successive days in 13 patients with worsening New York Heart Association class III or IV heart failure. The mean (+/- SEM) left ventricular ejection fraction was 15 +/- 2% (range, 6-29%). Pretreatment hemodynamics were: heart rate, 96 +/- 2 beats/min; mean arterial pressure, 77 +/- 3 mmHg; cardiac index, 1.80 +/- 0.10 L/min/m2; pulmonary capillary wedge pressure, 27 +/- 1 mmHg; mean pulmonary arterial pressure, 41 +/- 2 mmHg; and systemic vascular resistance, 1,732 +/- 152 dynes.s/cm5. At infusion rates yielding comparable increases in the cardiac index (5 micrograms/kg/min for 2 hours for each drug), OPC-18790 produced significantly more favorable effects on heart rate (-2 +/- 3% vs 11 +/- 4%; P = .01), pulmonary capillary wedge pressure (-32 +/- 4% vs -17 +/- 8%; P = .04), mean pulmonary arterial pressure (-14 +/- 3% vs 6 +/- 11%; P = .06), stroke volume index (48 +/- 8% vs 29 +/- 7%; P = .02), stroke work index (70 +/- 11 vs 42% +/- 12%; P = .03), and rate pressure product (2 +/- 4% vs 14 +/- 4%; P = .05). The hemodynamic profile for OPC-18790 differs from dobutamine, with OPC-18790 exhibiting no increase in heart rate, greater preload reduction, and an increase in cardiac performance at a lower estimated metabolic cost.

Eighty years of occupational medicine in Australia.

Australia has been backward in providing safe conditions for workers. Occupational physicians have had to battle indifference, if not active opposition. Political expedience, not science, has tended to determine outcomes.

Chronic gastritis associated with infection due to Helicobacter pylori in southern Appalachian veterans with dyspepsia.

A prospective study of infection due to Helicobacter pylori in 104 Southern Appalachian veterans with upper gastrointestinal symptoms revealed a prevalence of 67%. There was no age difference observed between those with and without H pylori. The organism was shown to be present in 12 of 13 patients with duodenal ulcer (92%), 6 of 10 with gastric ulcer (60%), and 52 of 81 with nonulcer dyspepsia (64%). Using culture as standard, the urease test showed a sensitivity of 76% and specificity of 100%, while the biopsy stain had a sensitivity of 97%. The presence of acute inflammation in the antrum and body of the stomach closely correlated with the presence of the organism. Somewhat at variance with previous studies, our study indicated that H pylori in the body mucosa was strongly associated with chronic superficial gastritis with and without acute inflammation. Such a finding may reflect the natural history of infection due to H pylori and the advanced age of our patients.

Isolation of Helicobacter pylori from saliva.

Helicobacter pylori was grown in low numbers from the saliva of one of nine patients who were positive for gastric H. pylori. The saliva-derived isolate from this patient was identical to the antral biopsy-derived isolate from the same patient and differed from isolates cultured from the antral biopsies of all other patients by soluble-protein electrophoresis, restriction endonuclease DNA analysis, and Southern blot hybridization. This is the first observation, to our knowledge, of the recovery of viable H. pylori from saliva.

A highly specific and sensitive DNA probe derived from chromosomal DNA of Helicobacter pylori is useful for typing H. pylori isolates.

HindIII-digested DNA fragments derived from an EcoRI-digested 6.5-kb fragment of chromosomal DNA prepared from Helicobacter pylori ATCC 43629 (type strain) were cloned into the pUC19 vector. A 0.86-kb insert was identified as a potential chromosomal DNA probe. The specificity of the probe was evaluated by testing 166 non-H. pylori bacterial strains representing 38 genera and 91 species which included aerobic, anaerobic, and microaerophilic flora of the upper and lower gastrointestinal tracts. None of the 166 non-H. pylori strains hybridized with this probe (100% specificity), and the sensitivity of this probe was also 100% when H. pylori isolates from 72 patients with gastritis and with the homologous ATCC type strain were tested by dot blot hybridization. The capability of this probe for differentiating between strains of H. pylori was evaluated by Southern blot hybridization of HaeIII-digested chromosomal DNA from 68 clinical isolates and the homologous ATCC type strain of H. pylori. Fifty-one unique hybridization patterns were seen among the 69 strains tested, demonstrating considerable genotypic variation among H. pylori clinical isolates. We propose that this probe would be of significant value for conducting epidemiologic studies.

Increased prevalence of Helicobacter pylori antibodies among nurses.

BACKGROUND: Numerous studies have suggested that Helicobacter pylori infection in asymptomatic subjects is transmitted from person to person. Its prevalence is higher in the institutionalized setting. If that is the case, persons involved in patient care should have a higher prevalence of the infection. METHODS: We estimated the prevalence of H pylori antibodies among groups of asymptomatic medical and nursing staff and compared them with volunteer blood donors of similar age and sex. RESULTS: One hundred fifty-eight nurses and aides, 59 residents, 46 senior medical students, and 22 senior nursing students were enrolled in this study. Serum samples were tested for IgG antibodies against H pylori by enzyme-linked immunosorbent assay. Sixty-two (39%) of 158 nurses were found to be positive for antibodies to H pylori compared with 114 (26%) of 441 specimens from the blood donor group. Within the youngest age group (20 to 34 years), 13 (25%) of 51 nurses were positive for H pylori antibodies compared with 19 (13%) of 143 age-matched serum samples from the blood donor group. Within the middle age group (35 to 49 years), 32 (39%) of 83 nurses were positive for H pylori antibodies vs 43 (26%) of 167 age-matched blood donors. In the oldest age group (> 50 years), 17 (71%) of 24 nurses were positive for H pylori antibodies compared with 52 (40%) of 131 age-matched blood donors. Twenty-three (27%) of 86 nurses with 1 to 15 years of occupational exposure were positive for H pylori antibodies compared with 40 (56%) of 72 nurses with more than 15 years of occupational exposure. CONCLUSIONS: Nurses have an increased prevalence of H pylori antibodies that is significantly higher than the comparable prevalence of volunteer blood donors and is evident in the youngest age group. In addition, the increased prevalence is related to a longer duration of patient exposure in the nursing group.