Publications

By using a high-throughput screening method, a mutant of a uropathogenic Escherichia coli strain affected in the rapA gene was isolated. The mutant formed normal-architecture biofilms but showed decreased penicillin G resistance, although the mutation did not affect planktonic cell resistance. Transcriptome analysis showed that 22 genes were down-regulated in the mutant biofilm. One of these genes was yhcQ, which encodes a putative multidrug resistance pump. Mutants with mutations in this gene also formed biofilms with decreased resistance, although the effect was less pronounced than that of the rapA mutation. Thus, an additional mechanism(s) controlled by a rapA-regulated gene(s) was involved in wild-type biofilm resistance. The search for this mechanism was guided by the fact that another down-regulated gene in rapA biofilms, yeeZ, is suspected to be involved in extra cell wall-related functions. A comparison of the biofilm matrix of the wild-type and rapA strains revealed decreased polysaccharide quantities and coverage in the mutant biofilms. Furthermore, the (fluorescent) functional penicillin G homologue Bocillin FL penetrated the mutant biofilms more readily. The results strongly suggest a dual mechanism for the wild-type biofilm penicillin G resistance, retarded penetration, and effective efflux. The results of studies with an E. coli K-12 strain pointed to the same conclusion. Since efflux and penetration can be general resistance mechanisms, tests were conducted with other antibiotics. The rapA biofilm was also more sensitive to norfloxacin, chloramphenicol, and gentamicin.

BACKGROUND

The role of exposure to substances in the workplace in new-onset asthma is not well characterised in population-based studies. We therefore aimed to estimate the relative and attributable risks of new-onset asthma in relation to occupations, work-related exposures, and inhalation accidents.

METHODS

We studied prospectively 6837 participants from 13 countries who previously took part in the European Community Respiratory Health Survey (1990-95) and did not report respiratory symptoms or a history of asthma at the time of the first study. Asthma was assessed by methacholine challenge test and by questionnaire data on asthma symptoms. Exposures were defined by high-risk occupations, an asthma-specific job exposure matrix with additional expert judgment, and through self-report of acute inhalation events. Relative risks for new onset asthma were calculated with log-binomial models adjusted for age, sex, smoking, and study centre.

FINDINGS

A significant excess asthma risk was seen after exposure to substances known to cause occupational asthma (Relative risk=1.6, 95% CI 1.1-2.3, p=0.017). Risks were highest for asthma defined by bronchial hyper-reactivity in addition to symptoms (2.4, 1.3-4.6, p=0.008). Of common occupations, a significant excess risk of asthma was seen for nursing (2.2, 1.3-4.0, p=0.007). Asthma risk was also increased in participants who reported an acute symptomatic inhalation event such as fire, mixing cleaning products, or chemical spills (RR=3.3, 95% CI 1.0-11.1, p=0.051). The population-attributable risk for adult asthma due to occupational exposures ranged from 10% to 25%, equivalent to an incidence of new-onset occupational asthma of 250-300 cases per million people per year.

INTERPRETATION

Occupational exposures account for a substantial proportion of adult asthma incidence. The increased risk of asthma after inhalation accidents suggests that workers who have such accidents should be monitored closely.

BACKGROUND

In vivo studies on HIV-1 pathogenesis and testing of antiviral strategies have been hampered by the lack of an immunocompetent small animal model that is highly susceptible to HIV-1 infection. Since native rodents are non-permissive, we developed transgenic rats that selectively express the HIV-1 receptor complex, hCD4 and hCCR5, on relevant target cells. These animals display a transient low-level plasma viremia after HIV-1YU-2 infection, demonstrating HIV-1 susceptibility in vivo. However, unlike macrophages, primary CD4 T-cells from double-transgenic animals fail to support viral spread ex vivo. To identify quantitative limitations or absolute blocks in this rodent species, we quantitatively assessed the efficiency of key steps in the early phase of the viral replication cycle in a side-by-side comparison in infected cell lines and primary T-cells from hCD4/hCCR5-transgenic rats and human donors.

RESULTS

Levels of virus entry, HIV-1 cDNA synthesis, nuclear import, and integration into the host genome were shown to be remarkably similar in cell lines and, where technically accessible, in primary T-cells from both species. In contrast, a profound impairment at the level of early HIV gene expression was disclosed at the single-cell level in primary rat T-cells and most other rat-derived cells. Macrophages were a notable exception, possibly reflecting the unique transcriptional milieu in this evolutionarily conserved target cell of all lentiviruses. Importantly, transient trans-complementation by ex vivo nucleofection with the Tat-interacting protein Cyclin T1 of human origin markedly elevated HIV gene expression in primary rat T-cells.

CONCLUSION

This is the first study that has quantitatively determined the efficiency of consecutive steps in the HIV-1 replication cycle in infected primary HIV target cells from a candidate transgenic small animal and compared it to human cells. Unlike cells derived from mice or rabbits, rat cells complete all of the early steps in the HIV-1 replication cycle, including provirus integration in vivo, with high efficiency. A deficiency in gene expression was disclosed at the single cell level and could be counteracted by the human pTEFb transcription complex factor Cyclin T1. Collectively, these results provide the basis for the advancement of this transgenic rat model through strategies aimed at boosting HIV-1 gene expression in primary rat CD4 T-cells, including human Cyclin T1 transgenesis.