Inhibition of viral replication in vitro by antiviral-treated amniotic membrane. Possible use of amniotic membrane as drug-delivering tool.

PURPOSE: To investigate if amniotic membrane (AM) incubated with antivirals can inhibit viral growth in vitro. METHODS: AM samples were incubated with a solution of acyclovir or trifluridine. The treated AM was placed onto monolayers of Vero cells, a continuous cell line from monkey kidney, infected with herpes simplex virus. Viral growth was assessed in comparison to control infected cells by direct examination with an inverted microscope at low magnification for the presence and extension of the typical cytopathic effect, or by estimation of viral genomes. RESULTS: AM soaked in acyclovir or trifluridine inhibited significantly the development of herpes simplex virus in cell cultures, based on the viral growth compared with controls. Non-treated AM did not significantly affect viral replication. CONCLUSIONS: Our preliminary in vitro data show that antiviral-treated amniotic membrane can inhibit viral replication. Therefore, the possibility to combine the previously published anti-inflammatory properties of AM with the capability to absorb antivirals and sustain drug release could be taken into consideration.

[Evaluation of the Yersinia enterocolitica pathogenicity through some phenotypic and genotypic characters: CRMOX agar positivity and presence of the ail gene]. / Valutazione della patogenicità di Yersinia enterocolitica attraverso alcuni caratteri fenotipici e genotipici: positività su CRMOX agar e presenza del gene ail.

Sixty-nine strains of Y. enterocolitica isolated from environmental and human matrices (waste water, food and faeces) were studied in order to evidence the presence of ail gene, calcium-dependency and Congo Red absorption for pathogenic strains identification. Out of 24 clinical strains, the ail gene was present in 21 (87%), among which 79% were CRMOX-positive as well. On the contrary, none of the 45 environmental strains showed the ail gene although only one (isolated from cooked vegetables) was CRMOX agar positive. Our results confirmed the importance of molecular methods to evidence the Y. enterocolitica pathogenic strains. However, our study pointed also the utility to consider the approach of classic bacteriology, like the subcoltivation on CRMOX agar to show calcium-dependency and Congo Red absorption. In particular, when dealing with environmental isolates, that medium will be useful as a preliminary screening to identify those isolates which need further research to indicate their pathogenic potential by the use of more complex but also more expensive molecular methods.

In-vitro activity of nisin against clinical isolates of Clostridium difficile.

Nisin is a cationic peptide produced by Lactococcus lactis. Its activity against clinical isolates of Clostridium difficile was compared to that of vancomycin and metronidazole by minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time-kill studies. Nisin was more active than the other agents, with a MIC90 of 0.256 mg/L and strong bactericidal activity. Nisin may be a promising agent for the management of C. difficile associated diarrhea.

Nontuberculous mycobacteria in hospital water systems: application of HPLC for identification of environmental mycobacteria.

Nontuberculous mycobacteria (NTM), ubiquitous in water environments, are increasingly recognized as nosocomial pathogens. Our study reports a one-year survey of the water system of two hospitals, A and B, in a small town near Florence, Italy. NTM were found throughout the study period in both settings, but B showed a significantly higher mycobacterial load. Mycobacterium gordonae and Mycobacterium fortuitum were the most frequent species isolated. Identification was carried out by conventional techniques and by high performance liquid chromatography (HPLC) analysis of cell wall mycolic acids. HPLC profiling could be used as a first-choice method for identification of environmental mycobacteria.

Impairment of gastric secretion modulation in duodenal ulcer and in long-term PPI treatment: quantitative morphologic findings and pathophysiologic implications.

Helicobacter pylori affects gastric secretion. This functional effect might have a morphometric counterpart. Therefore, the gastric cell secretory compartment was morphometrically assessed in different pathophysiologic conditions related to Helicobacter pylori infection. Nineteen Helicobacter pylori-positive nonduodenal ulcer subjects, 15 omeprazole chronically treated subjects, and 19 duodenal ulcer patients were studied against 19 controls. Somatostatin, gastrin, enterochromaffin-like, and parietal cell density was assessed in gastric biopsies. No differences in any cell type density were found between Helicobacter pylori-positive nonduodenal ulcer subjects and controls. On the contrary, differences were significant when comparing omeprazole and duodenal ulcer patients to controls (higher density of gastrin, enterochromaffin-like, and parietal cells, lower density of somatostatin cells). In duodenal ulcer a reversion to control values followed Helicobacter pylori eradication and ulcer healing. A direct linear correlation between enterochromaffin-like, gastrin, and parietal cell density was demonstrated. An almost complete map of mucosal cells involved in gastric secretion is provided by this study. The cell density pattern, identical to the omeprazole group, points to an impaired feedback control of secretion in duodenal ulcer. The reversion to control values after Helicobacter pylori eradication and ulcer healing demonstrates the pathogenetic role of Helicobacter pylori-host interaction in these changes.

Metal uptake in a coastal diatom influenced by major nutrients (N, P, and Si).

The influence of major nutrient additions on trace metal uptake (Cd, Se, and Zn) in a coastal diatom Thalassiosira pseudonana was investigated. A short-term exposure (5 h) was employed to measure the concentration factor (ratio of metal concentration in the cells to metal concentration in the medium) which was used as a kinetic parameter to quantify the relative rate of metal uptake in the cells. Following an initial rapid surface sorption, a linear pattern of uptake over time was typical for Cd, Se and Zn, indicating that these metals may have been transported intracellularly during the short-term exposure period. N addition significantly increased the rates of Cd uptake in the cells. Although the concentration factor of Zn increased with N addition, statistical analysis indicated that N addition did not significantly affect the rate of Zn uptake in the cells. Se uptake in the cells was independent of N additions, but was lowered with increasing Si concentration. Si addition did not significantly affect Cd and Zn uptake. Similarly, P addition did not influence the rates of metal uptake in the cells. There was a significant correlation between the rate of uptake of Cd and Zn and the cell growth rate. This study demonstrated that water quality brought about by a change in nutrient condition could considerably influence the uptake of metals by marine phytoplankton.

Effects of major nutrient additions on metal uptake in phytoplankton.

We examined the influences of major nutrients (N, P, Si) on the accumulation of three trace metals [Cd, Se(IV), and Zn] in four species of marine phytoplankton (diatom, green alga, dinoflagellate, prasinophyte). Relative metal uptake was quantified by the kinetic measurements of metal concentration factor over a short exposure period. Our study demonstrated that nutrient addition significantly influenced the metal uptake rate and the cell growth rate in all four phytoplankton species. An increase in ambient N concentration considerably enhanced metal uptake by the cells. The dry weight concentration factor increased by 2.4-14.9 times for Cd, 1.1-4.0 times for Se, and 1.1-5.4 times for Zn in all four phytoplankton species with an addition of 176.4 microM N. The effects of P or Si addition on metal uptake and cell growth were less pronounced than the effects of N addition. Under most circumstances the rate of metal uptake increased exponentially with increasing cell growth rate constant. Only Se(IV) uptake in the diatom Phaeodactylum tricornutum was not correlated with cell growth rate. Se(IV) was not accumulated by the green algae Chlorella autotrophica at a high P concentration (7.2 microM), but appreciable accumulation was documented in cells inoculated without P addition. Our study therefore demonstrated that nutrient enrichments in many coastal waters can considerably affect trace metal uptake in phytoplankton and presumably metal trophic transfer in marine food chains.

Neuronal and glial advanced glycation end product [Nepsilon-(carboxymethyl)lysine]] in Alzheimer's disease brains.

The cellular distribution of an advanced glycation end product [Nepsilon-(carboxymethyl)lysine (CML)] in aged and Alzheimer's disease (AD) brains was assessed immunohistochemically. CML was localized in the cytoplasm of neurons, astrocytes, and microglia in both aged and AD brains. Glial deposition was far more marked in AD brains than in aged brains, and neuronal deposition was also increased. On electron microscopic immunohistochemistry, neuronal CML formed granular or linear deposits associated with lipofuscin, and glial deposits formed lines around the vacuoles. Neuronal and glial deposits were prominent throughout the cerebral cortex and hippocampus, but were sparse in the putamen, globus pallidus, substantia nigra, and cerebellum, with glial deposits being far more prominent in AD brains. The distribution of neuronal and glial deposits did not correspond with the distribution of AD pathology. The extent of CML deposits was inversely correlated with neurofibrillary tangle formation, particularly in the hippocampus. Most hippocampal pyramidal neurons with neurofibrillary tangles did not have CML, and most of the neurons with heavy CML deposits did not have neurofibrillary tangles. In the hippocampus, neuronal CML was prominent in the region where neuronal loss was mild. These observations suggest that CML deposition does not directly cause neurofibrillary tangle formation or neuronal loss in AD.

Influences of phosphate and silicate on Cr(VI) and Se(IV) accumulation in marine phytoplankton.

The influences of major nutrient additions (P, Si, or N) on the accumulation of chromate [Cr(VI)] and selenite [Se(IV)] in three species of marine phytoplankton, including two diatoms (Thalassiosira pseudonana, and Skeletonema costatum), and one green alga (Chlorella autotrophica) were examined. Accumulation rate was quantified by measurements of metal concentration factor in algal cells over a short 5-h exposure period. Our results suggested that Se(IV) was little bioavailable to C. autotrophica and S. costatum, but was highly bioavailable to diatom T. pseudonana. Se(IV) accumulation in both diatoms and green alga was inversely dependent on the ambient P and Si concentration. Concentration factor decreased by 2.4-8.1 and 1.5-4.6 times with the additions of 7.2 microM P or 105.6 microM Si, respectively, after 5 h of exposure. Bioconcentration factors of Se(IV) in these species determined under laboratory conditions, which generally employ high nutrient concentrations, may not be applicable to those prevailing in the field. In contrast to Se(IV), Cr(VI) accumulation in the green alga and two diatoms was not related to the ambient P concentrations, but was inversely dependent on the ambient Si concentrations. These data demonstrated that the influences of major nutrients on anionic metal accumulation were highly species-specific, both for metals and phytoplankton. Nutrient enrichments in many water bodies may potentially affect the accumulation of anionic metals, and thus their biological transport and fate in these environments.

HPLC does not differentiate Mycobacterium paratuberculosis from Mycobacterium avium.

HPLC, which is gaining its place as identification tool in mycobacteriology laboratories, has been proposed to distinguish Mycobacterium paratuberculosis from Mycobacterium avium. We had reported no significant difference between M. avium and M. paratuberculosis reference strain ATCC 19698. Because of the advantages offered by such a method, we enlarged our observations to include more isolates of M. paratuberculosis. Within the double cluster of peaks obtained by both M. avium and M. paratuberculosis, we could not find a consistent difference typical of M. paratuberculosis. Therefore, the present study confirmed that M. avium and M. paratuberculosis could not be distinguished by HPLC, raising doubts of a straightforward use of HPLC to identify M. paratuberculosis.

[Ipsilateral central-type facial palsy and contralateral hemiparesis associated with unilateral medial medullary infarction: a case report].

Clinical pictures of medial medullary syndrome are variable, depending upon the extent of the lesion. Facial palsy has rarely been observed even in medullary infarction. However, central-type facial palsy is usually found contralaterally to the infarct area at the level of the rostral medulla. In the present report, we discuss the pathogenesis of the neurological manifestations in a 57-year-old man with hypertension. The patient presented with mild left facial palsy of central type, right hemiparesis, paresthesia, with deep sensory disturbance of the right extremities. An MRI of the brain showed an infarction localized in the medial region of the left upper medulla. Although the exact course of the supranuclear facial pathways remains controversial, the ipsilateral central facial palsy in this patient is considered to have two possible causes: the interruption of aberrant fibers of the corticobulbar tract, which branch off and swing back at the level of the upper-middle medulla, or the disruption of recurrent ascending fibers from the contralateral pyramidal tract, through decussation.

Helicobacter pylori and duodenal ulcer: evidence for a histamine pathways-involving link.

OBJECTIVES: A "gastrin link" has been suggested to explain the statistically relevant association between Helicobacter pylori and duodenal ulcer. Given the well known, although not entirely clarified, relationships between gastrin and histamine, the purpose of this study was to assess whether gastric mucosal histamine pathways and, more specifically, histamine-storing cells are involved in the Helicobacter pylori-duodenal ulcer route. METHODS: Fasting serum gastrin, gastric mucosal histamine content, and mucosal density of both enterochromaffin-like cells and mast cells were compared in 11 H. pylori-positive, non-duodenal ulcer subjects, in 16 duodenal ulcer patients (all H. pylori positive), and in 11 H. pylori-negative control subjects. RESULTS: Fasting serum gastrin concentration and mucosal histamine content were significantly higher in the duodenal ulcer group than in controls, whereas H. pylori-positive, non-ulcer subjects had values that were intermediate between those of the other two groups. Enterochromaffin-like cell density was significantly greater in duodenal ulcer patients than in the other groups. CONCLUSIONS: These results demonstrate the involvement of histamine pathways in H. pylori infection and duodenal ulcer. The most original finding in this study was that enterochromaffin-like cell density is three times greater in duodenal ulcer patients than in H. pylori-positive, non-ulcer subjects. This could explain the previous report of an exaggerated acid response to gastrin in duodenal ulcer patients when compared with H. pylori-positive, non-ulcer subjects and thus provide further insight into the pathogenesis of ulcers.

Helicobacter pylori and cell proliferation of the gastric mucosa: possible implications for gastric carcinogenesis.

OBJECTIVES: Helicobacter pylori infection is recognized as a risk factor for gastric adenocarcinoma. "Mitogenesis increases mutagenesis," so the effects of H. pylori infection on the gastric mucosal proliferative compartment have been investigated. METHODS: In 25 H. pylori-positive and 19 H. pylori-negative subjects, epithelial cell proliferative activity and the pattern of the proliferative compartment were separately evaluated in relation to both the different type of mucosa (antrum and corpus) and the H. pylori positivity/negativity after 3H-thymidine labeling. RESULTS: Both mucosal cell kinetics and the pattern of the proliferative compartment in the antrum appeared different from those of the corpus. Comparing H. pylori-positive and H. pylori-negative subjects, differences were detected only in the total number of cells in the antrum, whereas all of the cell kinetics parameters, except the labeling index, were greater in the corpus of the former group. A superficialization of the proliferative compartment was shown in H. pylori-positive subjects. Changes were more evident in subjects with more severe gastritis but were also present in H. pylori-positive subjects without corpus gastritis. CONCLUSIONS: These results show that H. pylori infection is associated with modifications in the proliferative compartment of the gastric mucosa. Both infection per se and chronic gastritis seem to be relevant for such changes.

Gastric mucosal histamine storing cells. Evidence for different roles of mast cells and enterochromaffin-like cells in humans.

Gastric mucosal histamine content, enterochromaffin-like cell density, and mast cell density were studied in 13 subjects under omeprazole therapy, 13 partially gastrectomized subjects with a Billroth II reconstruction, 10 partially gastrectomized subjects with a Roux-en-Y reconstruction, and 9 control subjects. Histamine content was significantly greater both in the subjects with higher gastrinemic levels (omeprazole-treated subjects) and those with more abundant enterogastric reflux (Billroth II subjects) than in controls. Enterochromaffin-like cell density was significantly greater in the omeprazole subjects than in each of the other groups. Mast cell density was significantly greater in Billroth II subjects than in controls. Serum gastrin levels, mucosal histamine content, and enterochromaffin-like cell density were positively correlated. Gastrin was not correlated to mast cell density. These results support the existence of different control pathways for enterochromaffin-like and mast cells. Moreover, they suggest that enterochromaffin-like cells and mast cells are involved in the regulation of gastric secretion and in gastric mucosal injury-repair mechanisms, respectively, due to histamine release.

Betalactam therapy and intestinal flora.

Betalactams, mainly when orally administered, may lead to intestinal flora modifications related to their spectrum of activity, rate of absorption and degradation. therefore it is important to investigate the possible influence of recently developed oral cephem derivatives on normal human microflora. We have investigated the impact on normal human intestinal flora in a 10-day course with cefetamet-pivoxil (CET, 500 mg BID) in comparison to cefixime (CFX, 400 mg qD) or cefuroxime axetil (CA, 250 mg BID) in 24 patients suffering from acute exacerbation of chronic bronchitis. Stool specimens were taken before (day 0), at the end (day 10) and 14 days after treatment (day 24) and quali-quantitative microflora composition was determined with a detection limit of 10 CFU/g dry weight. Treatment with CET caused slight and non-significant modifications of normal intestinal flora. On the contrary CFX and CA significantly affect Enterobacteriaceae and clostridia with a concomitant increase in enterococci for CFX. With both CFX and CA there was a new appearance of Salmonella spp. as well as Clostridium difficile in 4 and 2 cases, respectively. Therefore CET seems to affect normal bowel flora minimally in comparison to other oral cephalosporins. This aspect might contribute to the low incidence of GI related side effects in patients treated with CEt for longer than 1 week.

[Pseudomonas aeruginosa antibodies in patients with cystic fibrosis: clinical implications]. / Anticorpi anti-Pseudomonas aeruginosa in pazienti con fibrosi cistica: implicazioni cliniche.

Patients chronically colonised by P. aeruginosa develop a pronounced antibody response against P. aeruginosa that can be used to discriminate between superficial colonization and chronic infection. Anti-P. aeruginosa antibodies fail to afford protection against this pathogen; moreover high levels of antibodies are correlated with a poor prognosis. We investigated the significance of anti-P. aeruginosa antibodies (precipitins) by crossed immunoelectrophoresis (CIE) in 94 patients attending the Cystic Fibrosis Center of Florence. The highest numbers of precipitins were found in serum from patients chronically colonized in comparison to those patients who were transiently or not colonized. A negative correlation was found between the number of precipitin peaks and clinical conditions, evaluated with Schwachman score, and the number of precipitins and pulmonary functions. In summary, anti-P. aeruginosa antibodies fail to protect against P. aeruginosa bronchopulmonary infections, and are correlated to a more severe disease. Based on our experience, P. aeruginosa antibodies can be considered a reliable index of lung damage.

Helicobacter pylori potentiates histamine release from rat serosal mast cells induced by bile acids.

In the present study we have experimentally addressed the effects of Helicobacter pylori on the bile acid capability of histamine release. Bile acids alone were confirmed to be able to induce in vitro histamine release from rat serosal and mucosal mast cells. On the contrary, no significant histamine release was obtained when incubating any Helicobacter pylori preparations alone with mast cells. However, histamine release induced by bile acids was significantly enhanced, without any significant increase in lactate dehydrogenase activity, when whole washed or formalin-killed bacterial cells or crude cell walls were incubated with mast cells in the presence of cholic (0.3 mM), deoxycholic (0.3 mM), or lithocholic (0.3 mM) acids, chenodeoxycholylglycine (0.3 mM), and deoxycholyltaurine (3 mM). The electron microscopic features of mast cells incubated with Helicobacter pylori were consistent with an exocytotic secretion. The release of histamine induced by 0.3 mM deoxycholic acid in the presence of Helicobacter pylori was inhibited by the preincubation of the cells with dimaprit (an H2 agonist) and potentiated by the H2 antagonist, ranitidine. The current results suggest a link between human Helicobacter pylori infection and histamine release and a possible involvement of gastric mucosal mast cells in the pathogenesis of Helicobacter pylori-associated gastritis.

Helicobacter pylori potentiates histamine release from serosal rat mast cells in vitro.

Helicobacter pylori seems to be involved in the etiology of peptic ulcer and chronic gastritis. Histamine is fundamental in gastric secretion modulation, and some features of H. pylori-associated gastritis (edema, vasodilatation, inflammatory cell infiltration) are typical of the histamine-mediated response. This in vitro study has been undertaken as a preliminary step, in order to find a possible link between H. pylori and histamine release. H. pylori isolated from gastric biopsies has been tested as whole washed bacterium, whole formalin-killed bacterium, and crude cell wall preparation with serosal mast cells obtained by density gradient centrifugation or elutriation from peritoneal and pleural washings of male Wistar albino rats. Histamine release was assayed fluorimetrically. No significant histamine release was obtained by testing the various bacterial preparations alone with mast cells. On the contrary, whole washed cells, whole formalin-killed cells and crude cell walls from H. pylori have been found to potentiate compound 48/80 or calcium ionophore A23187-induced histamine release. Crude cell walls showed the highest activity, whereas filtered supernatants from broth cultures constantly appeared inactive. The enhancement in histamine release differed between the different strains. The present in vitro study, which shows the potentiating effect of H. pylori on histamine release, may prove interesting; however, at present, clinical implications cannot be inferred and further studies as well as in vivo demonstrations are needed.

Reflux-related gastric mucosal injury is associated with increased mucosal histamine content in humans.

BACKGROUND: Experimental studies in the dog and the rat have shown histamine involvement in reflux-related gastric mucosal injury. However, no definite demonstrations of a link between reflux-related gastric mucosal injury and mast cell mediators exist in humans. METHODS: The relationships between reflux, gastric mucosal histamine content, and gastric histology were assessed in partially gastrectomized subjects presumptively with high (11 Billroth II subjects) and low reflux levels (9 total biliary diversion subjects), respectively. Findings were compared with those in a control group consisting of 8 endoscopically and histologically proven normal subjects. RESULTS: Bile acid quantity and concentration in the gastric aspirates were significantly greater in Billroth II subjects than in total biliary diversion subjects. Significantly higher cumulative scores for foveolar hyperplasia, mucosal edema, capillary dilatation and congestion, and smooth muscle fibers in the lamina propria were found in Billroth II subjects than in total biliary diversion subjects. Mucosal histamine content as well as mast cell density and degranulation differed significantly between Billroth II and the other two groups. CONCLUSIONS: These results represent the first demonstration in humans of an association between mast cell mediators and chemical gastric mucosal injury.

Detection of Helicobacter pylori-like organisms in the stomach of some food-source animals using a monoclonal antibody.

To investigate a possible animal reservoir of Helicobacter pylori, 15 pigs, 15 rabbits and 5 cows slaughtered for consumption were studied. Raised serum IgG levels were found in 93% of the pigs and 87% of the rabbits, but levels were normal in the cattle. In the controlled testings three of 22 humans had elevated IgG to H pylori and in all three H pylori was detected by the use of monoclonal antibody. Helicobacter pylori were identified in gastric brushings by a monoclonal antibody in 8 out of 10 pigs and 7 out of 10 rabbits. This study suggests an animal reservoir of Helicobacter pylori which may be of importance in human infection.