Tolerability and efficacy of hydrocolloid dressings in the treatment of venous leg ulcers under tropical conditions: an open prospective study.

In the Western world, hydrocolloid dressings are widely used in wound treatment. However, little is known about their tolerability and efficacy under tropical conditions. The purpose of this study was to assess the tolerability and efficacy of a hydrocolloid dressing in combination with short stretch compressive bandages under tropical conditions. Seventeen patients with venous leg ulcers attending an outpatient clinic in Surinam were enrolled in the study for a period of 6 weeks. Swabs for bacterial cultures were taken at the beginning and end of the study. All ulcers showed a good healing tendency. Percentage of granulation tissue in the ulcers improved from mean 27% at start to 92% at the end. Mean circumference at start was 9.9 cm, at the end 4.9 cm. Exudation diminished from moderate in six and severe in eight ulcers, to moderate in 10 and almost none in two ulcers. In general, the dressing was very well accepted, pain was never reported. Leakage was noticed 39 times in the 164 dressing changes. This study revealed no differences in the rate of bacterial infections or colonization of wounds compared with studies performed in temperate regions. Our data indicate that hydrocolloid dressings can be used under tropical conditions.

Accumulation of barley stripe mosaic virus is significantly reduced in transgenic wheat plants expressing a bacterial ribonuclease.

An rnc70 gene encoding a mutant bacterial ribonuclease III (RNase III) was introduced into wheat (Triticum aestivum cv. Bobwhite) by microprojectile bombardment. T1, T2, and T3 plants regenerated from three transgenic callus lines were challenged with barley stripe mosaic virus. Plants expressing RNase III exhibited a high level of resistance to the virus infection. This resistance was evidenced by the absence of virus symptoms and reduced accumulation of virions in these plants. The result demonstrates that this pathogen-targeted resistance strategy can be effectively employed in conferring resistance to viral diseases of cereal crops.

An efficient wheat transformation procedure: transformed calli with long-term morphogenic potential for plant regeneration.

A method for producing large numbers of transgenic wheat plants has been developed. With this approach, an average of 9.7% of immature embryo explants were transformed and generated multiple self-fertile, independently transformed plants. No untransformed plants, or escapes, were regenerated. This transformation procedure uses morphogenic calli derived from scutellum tissue of immature embryos of Triticum aestivum cv. Bobwhite co-bombarded with separate plasmids carrying a selectable marker gene (bar) and a gene of interest, respectively. Transformed wheat calli with a vigorous growth phenotype were obtained by extended culture on media containing 5.0 mg/l bialaphos. These calli retained morphogenic potential and were competent for plant regeneration for as long as 11 months. The bar gene and the gene of interest were co-expressed in T0 progeny plants. This wheat transformation protocol may facilitate quantitative production of multiple transgenic plants and significantly reduce the cost and labor otherwise required for screening out untransformed escapes.

Immunocytology shows the presence of tobacco etch virus P3 protein in nuclear inclusions.

Intracellular localization studies of various potyvirus proteins have been made in hope of finding clues to their function(s). Immunocytological studies localized many of the tobacco etch virus (TEV)-encoded proteins in infected cells. We used antiserum against the nonstructural P3 protein of TEV to determine the subcellular location of the P3 protein in ultrathin sections of virus-infected cells. Immunogold labeling with the antiserum showed labels associated with nucleoli, nuclei, or NIs, Absorption of antiserum with purified NIs or P3 protein resulted in no labeling. TEV NIs are known to contain a bifunctional genome-linked protein-viral proteinase (NIa-VPg) and RNA-dependent RNA polymerase (NIb). It appeared that the TEV P3 protein was a third nonstructural viral protein of NIs of TEV if the NIa-VPg is considered one protein. The presence of P3 in NIs was also supported by Western blot assays. P3 protein in the nucleolus and nucleus could indicate that it, too, is involved in early stages of viral replication.

A mammalian 2-5A system functions as an antiviral pathway in transgenic plants.

Resistance to virus infections in higher vertebrates is mediated in part through catalysis of RNA decay by the, interferon-regulated 2-5A system. A functional 2-5A system requires two enzymes, a 2-5A synthetase that produces 5'-phosphorylated, 2',5'-linked oligoadenylates (2-5A) in response to double-stranded RNA, and the 2-5A-dependent RNase L. We have coexpressed these human enzymes in transgenic tobacco plants by using a single plasmid containing the cDNAs for both human RNase L and a low molecular weight form of human 2-5A synthetase under control of different, constitutive promoters. Expression of the human cDNAs in the transgenic plants was demonstrated from Northern blots, by specific enzyme assays, and by immunodetection (for RNase L). Infection of leaves, detached or in planta, of the coexpressing transgenic plants by tobacco mosaic virus, alfalfa [correction of alfafa] mosaic virus, or tobacco etch virus resulted in necrotic lesions. In contrast, leaves expressing 2-5A synthetase or RNase L alone and leaves containing the plasmid vector alone produced typical systemic infections. While alfalfa mosaic virus produced lesions only in the inoculated leaves regardless of the concentration of virus in the inoculum, high, but not low, levels of tobacco etch virus inoculum resulted in escape of virus to uninoculated leaves. Nevertheless, there was a substantial reduction of tobacco etch virus yield as measured by ELISA assay in the coexpressing transgenic plants. These results indicate that expression of a mammalian 2-5A system in plants provides resistance to virus infections.

Impact of metronidazole resistance on the eradication of Helicobacter pylori.

We investigated how often Helicobacter pylori was resistant to metronidazole before treatment in 283 H. pylori positive patients and whether the in vitro susceptibility to metronidazole could predict the clinical outcome of several drug regimens containing metronidazole. Metronidazole susceptibility was tested using either disc diffusion or plates containing metronidazole. Metronidazole-resistant strains were found in 41% of patients before their first anti-H. pylori treatment course. In patients with metronidazole-susceptible isolates, eradication was achieved in 74% after colloidal bismuth subcitrate (CBS) with metronidazole and in 91% after triple therapies consisting of CBS, metronidazole and amoxicillin or tetracycline. Dual therapies of 1 or 4 weeks' duration and triple therapies lasting 1 week were ineffective in patients with metronidazole-resistant strains. A 4-week course of triple therapy could still eradicate H. pylori in 68% of patients with metronidazole-resistant strains, but at the cost of significant side-effects. It is concluded that metronidazole resistance is common and is generally associated with a poor outcome of anti-H. pylori therapies containing metronidazole. Alternative drug schedules are urgently needed for patients with metronidazole-resistant H. pylori.

Structural proteins of three viruses in the Potyviridae adhere only to their homologous cylindrical inclusions in mixed infections.

Immunoelectron microscopy of serial sections of leaf cells of wheat doubly infected by wheat streak mosaic virus and Agropyron mosaic virus or wheat spindle streak mosaic virus showed that structural proteins or virions associated only with homologous cylindrical inclusions (CI) or with self in aggregates. CI protein of WSMV was seen firmly attached to, and extended through, a plasmodesma. Based on CI protein similarities with transport protein families, membrane-associated secretion proteins, other transport proteins in the GTPase superfamily, and the here reported findings, it is suggested that CI proteins could serve as cell-to-cell movement proteins in the Potyviridae.

Molecular cloning and sequencing of the coat protein gene of a Nebraskan isolate of tobacco necrosis virus: the deduced coat protein sequence has only moderate homology with those of strain A and strain D.

A partial nucleotide sequence spanning the coat protein (CP) gene of a Nebraskan isolate of tobacco necrosis virus (TNV-NE) has been determined. The sequence contains at least four open reading frames (ORFs). The 5'-terminal ORF encodes a protein that has 86% and 38% homology with the polymerases of strains A (TNV-A) and D (TNV-D), respectively. The second and third ORFs probably encode 10.7 kDa and 6.2 kDa proteins (p 10.7 and p 6.2). These are respectively 90% and 96% amino acid homologous encoded by similar ORFs in TNV-A but only 26% and 20% homologous with those in TNV-D. The fourth 3'-proximal ORF encodes the 30.3 kDa CP. The amino acid sequence of TNV-NE CP is only 51% and 44% homologous to those of TNV-A and TNV-D, respectively. Thus, the CP genes of TNV-NE, TNV-A, and TNV-D are quite different. Like the sequences to the 5' side of the CP gene, that of TNV-NE is more closely related to TNV-A than to TNV-D.

Catalase negative mutants of Helicobacter pylori.

Nine strains of Helicobacter pylori have been isolated exhibiting spontaneous mutations with a loss of catalase activity. Growth characteristics in vitro were unaffected by the mutation showing that catalase is not essential for growth of Helicobacter pylori. Parent strains and mutants could not be distinguished morphologically from each other when compared by electron microscopy. Restriction endonuclease digestion with HindIII, separated in an 0.7% agarose gel in TBE buffer, showed each pair to be highly related to each other. SDS-PAGE separation of proteins from four mutants and parent strains showed that all mutants lacked a 57 kDa protein. The partial N-terminal sequence of this protein shows homology with maize catalase and may be the subunit of the Helicobacter pylori catalase tetramer. It is concluded that catalase negative mutants of Helicobacter pylori occur spontaneously in vitro, but have not yet been observed in vivo. The paucity of such catalase negative strains in clinical specimens could mean that catalase is a virulence factor in vivo that puts mutants at a selective disadvantage.

Cylindrical inclusion bodies of wheat streak mosaic virus and three other potyviruses only self-assemble in mixed infections.

Potyviruses produce cylindrical inclusions (CIs) in the cytoplasm of infected cells. Immunogold labelling and electron microscopy of embedded and sectioned wheat and maize cells doubly infected by different potyviruses revealed no mixing of inclusion proteins in CIs. The viruses were wheat streak mosaic virus (WSMV), agropyron mosaic virus and hordeum mosaic virus, in wheat, and WSMV and maize dwarf mosaic virus in maize. The three viruses in wheat were indistinguishable morphologically and in ultrastructural features but can be separated by serology and host range. The absence of phenotypic mixing in CIs showed that in the presence of CI proteins of other potyviruses, assembly was either highly virus-specific, or that no opportunity existed for CI proteins to assemble into hybrid CIs in mixed infections.

Genotypical variation of Campylobacter pylori from gastric mucosa.

In a previous study, the recurrence of the Campylobacter pylori infection after apparently successful antibacterial therapy was determined to be due to recrudescence rather than reinfection. Although the DNA patterns of pre- and posttreatment isolates were very similar, we detected minor differences between the two patterns in about one third of the patients. These differences were not artifacts, but originated in the coexistence in the stomach of (sub)populations of bacteria with slightly different chromosomal DNAs, plasmids, or both. The presence of such (sub)populations was probably caused by mutation in vivo, as mutation in vitro was demonstrated in one patient after the original isolate was subcultured 10 times. Minor differences were not correlated with a difference in susceptibility to the antibiotic(s) that was used. An additional conclusion of this investigation was that the results of plasmid analysis should be interpreted very carefully when this method is used as an epidemiologic marker in the investigation of C. pylori infections.

Patient-to-patient transmission of Campylobacter pylori infection by fiberoptic gastroduodenoscopy and biopsy.

Three instances of subclinical reinfection with Campylobacter pylori were observed in two successfully treated patients during follow-up of C. pylori gastritis. The reinfections occurred 1 month and 21 months (patient 1) and 32 months (patient 2) after the completion of antibacterial treatment. Sequential measurement by ELISA of serum IgG antibody levels to the microorganism showed a significant increase in two of the three instances of reinfection. Patient-to-patient transmission was proved by restriction enzyme analysis of bacterial DNA. Between patients the endoscope had been mechanically cleaned using a detergent and treated with 70% ethanol. The risk of gastroscopic cross-infection with C. pylori was estimated by retrospective analysis of the data of 281 negative examinations (107 in 47 initially negative patients and 174 in 37 cured patients). The frequency in uninfected patients of documented endoscopic transmission of C. pylori infection was 1.1% in this study, corresponding with three iatrogenic acquisitions of manifest infection for every 1000 gastroduodenoscopies in our clinic.

Detection of Campylobacter pylori in stomach tissue by DNA in situ hybridisation.

A non-radioactive DNA in situ hybridisation (DISH) protocol was developed. It requires the use of biotinylated Campylobacter pylori DNA as the probe to detect C pylori DNA in routinely embedded stomach biopsy specimens. In sequential tissue samples from a 58 year old woman with recurrent chronic active gastritis the C pylori probe hybridised with bacteria whenever they were histologically visible. When no bacteria were visible histologically, hybridisation was negative with one exception. In a single biopsy specimen without visible C pylori, hybridisation occurred with the surface of the antrum epithelium, while control hybridisation with a heterologous probe remained negative. From a parallel biopsy specimen taken at the same time the C pylori culture was positive. It is concluded that DISH, although more laborious than routine staining techniques, may be more sensitive in that it detects bacteria very easily, even when sections are not counterstained or when they are present in low numbers, and that bacteria which do hybridise are unequivocally identified as C pylori and not Campylobacter-like organisms.

14C-urea breath test in C pylori gastritis.

14C-urea breath test was used to detect Campylobacter pylori colonisation in 129 consecutive non-ulcer dyspepsia patients. Fasting patients were given 3 microCi (110 kBq) of 14C-labelled urea after a test meal. Breath samples were collected at 10 minute intervals for 90 minutes and the C-14 activity was counted on a liquid scintillation analyser. Urea derived 14CO2 appears in the exhaled breath of Campylobacter pylori culture positive individuals within 20-30 minutes. Likelihood analysis revealed a most favourable cut off level of [0.07% dose 14C-urea/mmol CO2] multiplied by body weight at t = 40 minutes, to separate culture positive from culture negative subjects. Using this upper limit of normal, a positive likelihood ratio of 50 and a negative likelihood ratio of 0.05 was calculated. Sensitivity of the test was 95% and specificity 98%. The 14C-urea breath test is a simple, sensitive and non-invasive test, that detects viable C pylori microorganism and semiquantitatively assesses the bacterial load of C pylori colonisation. Administration of a single dose of colloidal bismuth subcitrate resulted in a rapid decrease in 14CO2 excretion, so this test can be used to confirm eradication of the bacterium in therapeutic trials without endoscopy, or need for culture.

Campylobacter pyloridis-associated chronic active antral gastritis. A prospective study of its prevalence and the effects of antibacterial and antiulcer treatment.

To determine the clinical importance of Campylobacter pyloridis infection, its association with gastric inflammation, and the response to drug therapy, patients with a duodenal or gastric ulcer (n = 63), patients with nonulcer dyspepsia (n = 240), and asymptomatic volunteers (n = 34) were studied. In a prospective longitudinal study, the type, intensity, and distribution of inflammation in antral biopsy specimens were correlated with the presence of C. pyloridis. Campylobacter pyloridis was cultured from antral biopsy specimens in 98% of the ulcer patients, 70% of the nonulcer dyspepsia patients, and 20% of the asymptomatic volunteers. The dependency of chronic active gastritis on the presence of C. pyloridis was shown by an association of gastritis with positive culture and healing of gastritis with negative culture after various therapeutic regimens. Spontaneous disappearance of C. pyloridis never occurred. Colloidal bismuth subcitrate, amoxicillin, and the combination of colloidal bismuth subcitrate and amoxicillin were effective therapies in eradicating C. pyloridis. Recolonization with the same bacterial subtype and recurrence of gastritis frequently occurred within 1 mo after initial eradication. In this study we demonstrate ultimate normalization of gastric mucosa after successful eradication of C. pyloridis. Especially complete normalization of gastric mucosa after amoxicillin monotherapy provides additional strong evidence for a true cause-effect relationship between C. pyloridis colonization and gastritis.

Identification of Campylobacter pyloridis isolates by restriction endonuclease DNA analysis.

Campylobacter pyloridis isolates recovered from gastric biopsy specimens of 16 patients were examined by restriction endonuclease DNA analysis with HindIII. For 8 of these 16 patients two different isolates were compared to study the persistence of the colonizing strains and the stability of their DNA digest patterns during a period of 2 years (two patients), the identity or nonidentity of different colony types within one culture (two patients), and the nature of the relapses after apparently successful antibacterial therapy (four patients). The isolates from the 16 patients all produced different DNA digest patterns. Comparison of the two different isolates recovered from the same patients showed that these isolates were identical in all eight cases. Laboratory subculturing of a C. pyloridis strain (10 times) did not change its DNA digest pattern. These results indicate the stability of the DNA digest patterns and a marked variability of these patterns among isolates from different patients. Using restriction endonuclease DNA analysis, we found the persistence in the stomach of the same C. pyloridis strain during a period of 2 years and the identity of different colony types within one culture. The relapses after apparently successful antibacterial treatment could be attributed to recrudescence rather than reinfection. Restriction endonuclease DNA analysis is a sensitive and useful method for identifying C. pyloridis isolates.

The role of colloidal bismuth subcitrate in gastric ulcer and gastritis.

The pathophysiology of gastric ulcer and gastritis has been related to an increase in damaging factors such as duodeno-gastric reflux and anti-inflammatory drugs. A decreased capacity for mucosal prostaglandin generation is highly likely. Acid secretion is usually normal or even reduced. Colloidal bismuth subcitrate (CBS) has been shown to increase gastric mucosal defence. In controlled trials a clear superiority over placebo has been demonstrated. Amalgamation of the data shows an average healing rate of about 70% in gastric ulcer, which is statistically significantly higher than that for cimetidine in comparative trials. The therapeutic gain (the difference between the healing rate of drug and placebo in a direct comparison) of CBS is high compared with other anti-ulcer agents. In campylobacter gastritis we have shown that the microorganism is very sensitive to CBS, and its eradication results in histological improvement.