Inflammatory response induced by Helicobacter pylori infection in lung.

Helicobacter pylori is a microorganism that in the last years has been associated with extragastric disorders such as respiratory diseases, however, its impact on lung is partially understood. The aim of this work was to study infection impact of H. pylori on the inflammatory markers expression at the pulmonary level using an animal model. Infection was performed by BALB/c wild type (WT) mice orotracheal instillation with 20 µl of 1 × 108H. pylori reference strain suspension once per day throughout 3 days. Inflammatory response was evaluated at 3, 7, 14, 21 and 30 days post infection. Lung was aseptically removed and pulmonary edema index values showed a significant change at 30 days of infection. Hematoxylin-Eosin (H-E) stain allowed to visualizing H. pylori presence in lung samples at 3 days of infection near the phagocytic cells or in the alveoli lumen. Bronchoalveolar lavage (BAL) was used for inflammatory response evaluation. Lactate dehydrogenase values showed a gradual increase in infected animals along infection time. Protein concentrations in mg/ml from BAL increased significantly at 7 days in infected animals. Macrophages viability obtained from BAL, decreased at the first moment of infection, maintaining constant values along contamination time. Results obtained demonstrate an inflammatory response in lung after orotracheal H. pylori infection and suggest that the pathogenic mechanism is strongly evidenced by tissue damage, endothelial dysfunction inflammatory mediators and markers expression at the pulmonary level.

Study of Helicobacter pylori infection on lung using an animal model.

Helicobacter pylori infection has been reported to be associated with extra-digestive disorders such as respiratory diseases; however, the impact of H. pylori on lung is incompletely understood. Inflammatory response is mediated by the release of cytokines, interferon, and enzymes such as metalloproteinases (MMPs). This may contribute to collagen accumulation during the early phase of infection. MMP expression is an important factor for the proliferation and infiltration of lung cells in the process of fibrosis formation. The aim of this work was to study the impact of the infection with H. pylori on lung using a mouse model. We looked for histological lesions of lung infected with the microorganism as well as the expression of inflammatory and of endothelial dysfunction markers. C57BL/6 wild type (WT) mice were infected by orotracheal instillation with 20 µl of 1 × 108H. pylori reference strain suspension once per day for 3 days. Animals infected and controls were sacrificed at 3, 7, 14, 21 and 30 days. The lung from mice were stained with Hematoxylin-Eosin (H-E), Masson's Trichromic and Periodic Acid Schifft (PAS) for histological study. Also, lipid hydroperoxides and enzime catalase (CAT) activity were determined. Expression level of multiple markers implicated in inflammation (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-4, IL-6, IL-8, IL-10; metalloproteinase MMP-9) and markers of endothelial dysfunction (I-CAM and V-CAM) was determined from lung tissues mRNA using RT-PCR. Results showed that H. pylori induced morphological changes in the lung tissue with recruitment of inflammatory cells and lung parenchymal cell degradation. The mRNA of IL-1ß and TNF-α; MMP-9, I-CAM and V-CAM increased at 3-7 days infections. Also, iNOS, IL-8 and Phosphocholine cytidyltransferase (CCT) increased with lung injury. Anti-inflammatory interleukin as: IL-4, and IL-10 increased at 7 and 14 days post infection respectively. The results obtained suggest that the pathogenic mechanism of H. pylori on lung could be strongly associated with lung injury as indicate the expression increased of inflammatory mediators and markers of endothelial dysfunction.

Evaluation of a serum-free transport medium supplemented with cyanobacterial extract, for the optimal survival of Helicobacter pylori from biopsy samples and strains.

The purpose of this work was to evaluate the use of an alternative transport medium supplemented with a cyanobacterial extract (CE), free of animal derivatives, to preserve the viability of Helicobacter pylori strains during long-term transportation and allow its recovery from biopsy samples. The transport media evaluated were Mueller-Hinton broth 0.3% agar (MH) and 0.3% of CE (MH-CE). MH broth 5% fetal calf serum (FCS) was used as the reference medium (MH-FCS). Biopsy samples from 134 patients, H. pylori NCTC 11638 and six clinical isolates were studied. A higher recovery (p ≤ 0.001) at 4°C was obtained in MH-CE than in MH-FCS after 96 h of storage. Only MH-CE allowed recovery after 120 h. The H. pylori recovery at room temperature after 96 h was higher (p ≤ 0.005) in MH-CE than in MH-FCS. Similar survival rates were observed in biopsy samples conserved in MH-CE and MH-FCS at 4°C. The recovery after 48 h at room temperature in MH-CE was higher (p ≤ 0.05) than MH-FCS and was the only medium allowing recovery after 72 h. The MH-CE medium is a simple, inexpensive and animal derivatives-free transport medium that can be used to preserve H. pylori viability and its recovery from biopsy samples.

Antimicrobial activity of Artemisia douglasiana and dehydroleucodine against Helicobacter pylori.

ETHNOPHARMACOLOGICAL RELEVANCE: The increasing resistance of Helicobacter pylori to antibiotics demands the search for novel compounds from plant based sources. Artemisia douglasiana Besser is widely used in Cuyo region (Argentina) as folk medicine for the treatment of gastric ailments. AIM OF STUDY: Based on our previous studies that Artemisia douglasiana exert cytoprotective actions against ethanol-induced gastric mucosal injury we assayed the anti-Helicobacter pylori effect of the Artemisia douglasiana extract and its active compound, dehydroleucodine. MATERIALS AND METHODS: The in vitro anti-bacterial activity of Artemisia douglasiana extract and its active compound, dehydroleucodine were determined against one standard strain and six clinical isolates of Helicobacter pylori by using the agar dilution methods. RESULTS: The results showed that both dehydroleucodine and Artemisia douglasiana extract had activity against the microorganism with MICs between 1-8 and 60-120 mg/L, respectively. CONCLUSIONS: Artemisia douglasiana may be a useful alternative treatment strategy principally in eradication of metronidazole and clarithromycin-resistant strain.

Genotypic, phenotypic, and clinical characteristics of isolates of Helicobacter pylori from San Luis, Argentina.

The vacA and cagA genotypes of Helicobacter pylori exhibited distinct geographic distribution and correlation with severity of disease. In the above genotypes (obtained from 150 H. pylori-positive patients--139 with gastritis, 10 with ulcer and 1 patient with gastric cancer) combinations vacA s1/m1 and s2/m2 were detected using PCR in 75 and 25% of isolates, respectively, in patients with chronic gastritis. The of s1/m1 and s2/m2 combinations were also detected from ulcers (60 and 40%, respectively). The cagA was detected in 30% of isolates. Concentrated culture supernatants of 7 (64%) out of 11 H. pylori strains induced vacuolization in Vero cells in titers ranging from 1:5 to 1:40. The vacA s1 genotype was significantly associated with, but not predictive of the presence of vacuolating cytotoxin activity and the cagA gene.

Antimicrobial activity of aqueous extracts of Larrea divaricata Cav (jarilla) against Helicobacter pylori.

We studied here the effect of aqueous extracts of Larrea divaricata Cav on the growth of Helicobacter pylori. Results show that cold extract, infusion, decoction and simulated digestion had inhibitory activity at 0.04-0.1 mg/l against clarithromycin and metronidazole susceptible and resistant H. pylori strains. These results support the popular use of L. divaricata Cav in gastric disturbances and prompt further research to characterize these compounds with a therapeutic potential against gastric ulcers and gastric cancer associated with H. pylori.

[Detection of resistance to clarithromycin in clinical isolates of Helicobacter pylori from children and adults]. / Detección de resistencia a claritromicina en aislamientos clínicos de Helicobacter pylori de niños y adultos.

Several methods have been used to detect clarithromycin resistance. Agar dilution is now recommended by the NCCLS (susceptible to clarithromycin: MIC<=0.25 mg/l; intermediate resistance: MIC=0.5 mg/l; resistant: MIC>=1 mg/l), and the detection of mutations involved in resistance is used in many laboratories. We analyzed 36 clarithromycin-resistant strains isolated from children and 30 from adults. In vitro susceptibility to clarithromycin was determined by an agar dilution method. DNA from the isolates was extracted using the method published by Ge and Taylor. A2142G and A2143G mutations were identified by PCR-RFLP. A 1.4 Kpb of the 23S rRNA gene was amplified and digested using MboII or BsaI restriction enzymes to detect mutations. The prevalence of the A-G transition mutation at position 2143 was higher in the children (80.55%) than in the adult patients (46.66%) (p<0.05); however, the prevalence of the mutation at position 2142 was higher in adults than in children (36.66% vs. 5.55%; p<0.05). In children, a higher MIC (2-64 mg/l) was observed when the A-G mutation was detected at position 2143. However, in adult patients higher MICs were observed when the A-G mutation was detected at position 2142.

[Helicobacter pylori infection diagnosis in dyspeptic patients from the City of San Luis, Argentina, using invasive methods]. / Diagnóstico de infección por Helicobacter pylori en pacientes dispépticos de la Ciudad de San Luis, Argentina, utilizando métodos invasivos.

In this study we present a relationship between different gastroduodenal pathologies and Helicobacter pylori infection. We used four diagnosis invasive methods for H. pylori infection: urease test (UT), histopathology (H), Gram stain (G) and culture (C). The upper gastrointestinal endoscopy of 300 dyspeptic patients showed that 71.6% had erosive congestive gastropathies, 13.6% had duodenopathies, 5.6% had gastric ulcer, 6.3% had duodenal ulcer and 2.6% had probable gastric neoplasia. We also correlated the data of water intake source with the pathologies. The percentage of infected patients with H. pylori was determined using: a) two simultaneous reference tests (UT and H), 54.3%, b) each test UT = 55.0%, H = 59.0%, G = 51.3%, and C = 43.0%. Sex, age and the source of water ingested did not show statistically significant differences.

Diagnóstico de infección por Helicobacter pylori en pacientes dispépticos de la Ciudad de San Luis, Argentina, utilizando métodos invasivos / Helicobacter pylori infection diagnosis in dyspeptic patients from the City of San Luis, Argentina, using invasive methods

In this study we present a relationship between different gastroduodenal pathologies and Helicobacter pylori infection. We used four diagnosis invasive methods for H. pylori infection: urease test (UT), histopathology (H), Gram stain (G) and culture (C). The upper gastrointestinal endoscopy of 300 dyspeptic patients showed that 71.6 had erosive congestive gastropathies, 13.6 had duodenopathies, 5.6 had gastric ulcer, 6.3 had duodenal ulcer and 2.6 had probable gastric neoplasia. We also correlated the data of water intake source with the pathologies. The percentage of infected patients with H. pylori was determined using: a) two simultaneous reference tests (UT and H), 54.3, b) each test UT = 55.0, H = 59.0, G = 51.3, and C = 43.0. Sex, age and the source of water ingested did not show statistically significant differences.(AU)

Diagnóstico de infección por Helicobacter pylori en pacientes dispépticos de la Ciudad de San Luis, Argentina, utilizando métodos invasivos / Helicobacter pylori infection diagnosis in dyspeptic patients from the City of San Luis, Argentina, using invasive methods

In this study we present a relationship between different gastroduodenal pathologies and Helicobacter pylori infection. We used four diagnosis invasive methods for H. pylori infection: urease test (UT), histopathology (H), Gram stain (G) and culture (C). The upper gastrointestinal endoscopy of 300 dyspeptic patients showed that 71.6 had erosive congestive gastropathies, 13.6 had duodenopathies, 5.6 had gastric ulcer, 6.3 had duodenal ulcer and 2.6 had probable gastric neoplasia. We also correlated the data of water intake source with the pathologies. The percentage of infected patients with H. pylori was determined using: a) two simultaneous reference tests (UT and H), 54.3, b) each test UT = 55.0, H = 59.0, G = 51.3, and C = 43.0. Sex, age and the source of water ingested did not show statistically significant differences.

Diagnóstico de infección por Helicobacter pylori en pacientes dispépticos de la Ciudad de San Luis, Argentina, utilizando métodos invasivos. / [Helicobacter pylori infection diagnosis in dyspeptic patients from the City of San Luis, Argentina, using invasive methods]

In this study we present a relationship between different gastroduodenal pathologies and Helicobacter pylori infection. We used four diagnosis invasive methods for H. pylori infection: urease test (UT), histopathology (H), Gram stain (G) and culture (C). The upper gastrointestinal endoscopy of 300 dyspeptic patients showed that 71.6

had erosive congestive gastropathies, 13.6

had duodenopathies, 5.6

had gastric ulcer, 6.3

had duodenal ulcer and 2.6

had probable gastric neoplasia. We also correlated the data of water intake source with the pathologies. The percentage of infected patients with H. pylori was determined using: a) two simultaneous reference tests (UT and H), 54.3

, b) each test UT = 55.0

, H = 59.0

, G = 51.3

, and C = 43.0

. Sex, age and the source of water ingested did not show statistically significant differences.

Sensitive methods for determination of free digitoxin concentration using digitoxin immunoassays: demonstration of elevated free digitoxin concentration caused by digitoxin-phenytoin interaction by applying these new techniques.

Digitoxin is very strongly bound to serum albumin. Although free digitoxin is pharmacologically active, it is not monitored because of the lack of a sufficiently sensitive technique. The concentration of free digitoxin in the protein-free ultrafiltrate is usually below the detection limit of digitoxin immunoassays. A modified technique is described by which free digitoxin can be routinely monitored using commercially available immunoassays. The fluorescence polarization immunoassay for determining total digitoxin concentration requires that 100 microL of serum be treated with 300 microL of methanol to precipitate proteins. It is demonstrated that free digitoxin can easily be measured by adding 100 microL of methanol to 300 microL of ultrafiltrate, thus improving the sensitivity of the assay three-fold. The free digitoxin concentration can easily be calculated by dividing the observed value by 3. An attempt to use only ultrafiltrate (no methanol added) caused significant bias in the result, probably as a result of a matrix problem. The chemiluminescent assay for digitoxin does not require any specimen pretreatment and requires only 10 microL of serum. The program was modified and used 50 microL of ultrafiltrate to improve the sensitivity of the free digitoxin assay. If the chemiluminescent assay is used to measure free digitoxin, the true free digitoxin concentration can be calculated by dividing the observed value by 4.3. The free digitoxin concentrations were comparable in eight patients receiving digitoxin as measured by both methods. To show an application of this technique, two serum pools were prepared from patients receiving digitoxin and supplemented with various concentrations of phenytoin. A significant increase in free digitoxin concentration was observed because of the displacement of digitoxin from protein binding sites by phenytoin.

[Isolation of Helicobacter pylori from dental plaque]. / Aislamiento de Helicobacter pylori en placa dental.

It has been suggested that oral dissemination might be the major transmission vehicle for Helicobacter pylori, and that dental plaque might act as its reservoir. The presence of H. pylori was investigated in 62 odontological male and female patients (average age: 35 years old). Samples were taken from supragingival plaque, placed in 0.3 ml of thioglycolate broth, cultured within 12 h in Mueller-Hinton agar with the addition of 5-7% of sheep blood and antibiotic supplement, and incubated at 37 degrees C in microaerophilia for 5-7 days. Typical colonies were identified by gram, urease, oxidase and catalase. H. pylori was detected in a 15 year-old patient suffering from gastric acidity (1.61% positivity index). The medium used facilitated recovery of the agent from a sample abundant in germs. H. pylori was not recovered from the same patient 12 months later, suggesting that there might have been a transitory passage by gastric reflux or that the bacterium was acquired from an exogenous source.

Aislamiento de Helicobacter pylori en placa dental / Isolation of Helicobacter pylori in dental plaque

Se ha sugerido que la diseminación oral sería la principal vía de transmisión de Helicobacter pylori y la placa dental podría actuar como reservorio. Se investigó su presencia en 62 pacientes odontológicos de ambos sexos, edad promedio 35 años. Las muestras, tomadas de placas supragingival, colocadas en 0,3 ml de caldo tioglicolato, fueron cultivadas dentro de las 12 h en agar Mueller-Hinton con 5-7 por ciento de sangre de oveja y suplemento antibiótico e incubadas a 37ºC en microaerofilia 5-7 días. Las colonias típicas se identificaron por gram, ureasa, oxidasa y catalasa. H. pylori fue detectado en un paciente de 15 años que padecía acidez gástrica (índice de positividad 1,61 por ciento). El medio utilizado facilitó la

Aislamiento de Helicobacter pylori en placa dental / Isolation of Helicobacter pylori in dental plaque

Se ha sugerido que la diseminación oral sería la principal vía de transmisión de Helicobacter pylori y la placa dental podría actuar como reservorio. Se investigó su presencia en 62 pacientes odontológicos de ambos sexos, edad promedio 35 años. Las muestras, tomadas de placas supragingival, colocadas en 0,3 ml de caldo tioglicolato, fueron cultivadas dentro de las 12 h en agar Mueller-Hinton con 5-7 por ciento de sangre de oveja y suplemento antibiótico e incubadas a 37§C en microaerofilia 5-7 días. Las colonias típicas se identificaron por gram, ureasa, oxidasa y catalasa. H. pylori fue detectado en un paciente de 15 años que padecía acidez gástrica (índice de positividad 1,61 por ciento). El medio utilizado facilitó la recuperación del agente desde una muestra abundante en gérmenes. No se aisló H. pylori del mismo paciente 12 meses después, sugiriendo un pasaje transitorio por reflujo gástrico o adquisición de la bacteria de una fuente exógena

Aislamiento de Helicobacter pylori de pacientes con trastornos gastroduodenales en San Luis (Argentina) / Helicobacter pylori isolated from patients with gastrointestinal tract diseases in San Luis (Argentina)

Se investigó la presencia de Helicobacter pylori en 50 pacientes con trastornos gastroduodenales que concurrieron a dos centros de salud de la ciudad de San Luis. De cada paciente se tomaron cuatro muestras de biopsia de mucosa de antro gástrico, dos de ellas destinadas al estudio histológico y dos al análisis bacteriológico: observación al Gram, prueba de ureasa y cultivo. Helicobacter pylori se detectó en 38 (76 por ciento) de los pacientes mediante el estudio histológico y en 30 (60 por ciento) por la tinción de Gram. De estos últimos, 28 (93 por ciento) dieron positiva la prueba de ureasa coincidiendo con un número significativo de bacterias. El 80 por ciento (24/30) de las muestras positivas al Gram mostró un buen desarrollo microbiano en los medios de cultivo de Mueller-Hinton y de Skirrow indistintamente. Se recomienda la prueba de ureasa como una alternativa de diagnóstico: rápida, económica y efectiva cuando hay una cantidad suficiente de bacterias. (AU)

Aislamiento de Helicobacter pylori de pacientes con trastornos gastroduodenales en San Luis (Argentina) / Helicobacter pylori isolated from patients with gastrointestinal tract diseases in San Luis (Argentina)

Se investigó la presencia de Helicobacter pylori en 50 pacientes con trastornos gastroduodenales que concurrieron a dos centros de salud de la ciudad de San Luis. De cada paciente se tomaron cuatro muestras de biopsia de mucosa de antro gástrico, dos de ellas destinadas al estudio histológico y dos al análisis bacteriológico: observación al Gram, prueba de ureasa y cultivo. Helicobacter pylori se detectó en 38 (76 por ciento) de los pacientes mediante el estudio histológico y en 30 (60 por ciento) por la tinción de Gram. De estos últimos, 28 (93 por ciento) dieron positiva la prueba de ureasa coincidiendo con un número significativo de bacterias. El 80 por ciento (24/30) de las muestras positivas al Gram mostró un buen desarrollo microbiano en los medios de cultivo de Mueller-Hinton y de Skirrow indistintamente. Se recomienda la prueba de ureasa como una alternativa de diagnóstico: rápida, económica y efectiva cuando hay una cantidad suficiente de bacterias.