PROFESSOR JEAN DAUSSET\'S EDITORIAL IN ENGLISH
==============================================

Why a new medical journal? Why especially this new journal? What are its ambitions? What audience can it reach? Specialists, generalists or both simultaneously? It is intended to be a link between the international scientific community, in particular within the fields of life science and its technological application.

There is undoubtedly a niche to be occupied, an important role to be played to better link this community. I am delighted by the initiative taken by the promoters of this journal, who had the courage to enter the competitive field of scientific journals and to launch an exciting intellectual adventure.

For my part, I would like to briefly broach some medical questions that are dear to me, and that are sufficiently general to be the subject of an editorial.

Where indeed is medical practice going and what does the medicine of tomorrow hold for us? We have watched, amazed, the fantastic advances that are progressively entering current medical practice. The revolution we have witnessed has names, in particular: vaccination, developed by Pasteur, which still has new fields to conquer (until now there is no vaccine against malaria or against AIDS!). There is also antibiotic therapy, exploited to full advantage but beginning to run out of steam, showing its limitations because of the rapid rise of resistant microorganisms. It is only a few decades since developments in immunology have allowed the transplantation of organs or bone marrow. Transplantation---an old dream of humanity finally realized---whose successes themselves have led to many disappointments among the very many patients on the waiting lists, because of the shortage of the organs to be transplanted. It is nevertheless admirable that, in the whole world, 6 million individuals are ready to give their marrow to save patients, in particular those with leukemia. To this list of successes we should add the many victories in oncology due to improved monitoring of tumorigenic agents? And finally, we must not forget the methods of biological investigation and medical imaging, which have seen great improvements recently.

This panorama would be quite incomplete if I did not add the spectacular rise of medical genetics, which makes it possible to locate and to clone the genes responsible for many monogenic diseases and, in the near future, the genes that act in concert to produce the many polygenic diseases that afflict our industrialized societies.

Provided with this arsenal, the doctor, who is a specialist or general practitioner, can from now on offer to his patients increasingly effective therapies, often leading to the cure or amelioration of chronic diseases. Longevity continues to increase each year in the industrialized countries. It should be the same everywhere.

I initiated, many years ago, the concept of predictive medicine, based on frequent associations between some diseases and the alleles of genes of the HLA system. For example ankylosing spondylitis, associated with HLA-B27, or insulin dependent diabetes, associated with HLA-DR3/DR4. This concept has now been extended to the entire human genome and the number of genes of susceptibility to (or protection from) many diseases can only increase. Apart from the HLA system, we know of susceptibility to familial breast cancer, given by genes such as BRCA1 and BRCA2. This idea can only grow in importance, but there is a danger. There can be no question of detection of susceptibility genes without simultaneous development of a preventive or therapeutic counterpart. We should not, under any circumstances, warn a healthy person of an uncertain future, because in this case---and I emphasize this point---we are dealing with a risk of uncertain proportions, without a corresponding method of prevention or cure. It would be unforgivable to cause useless anxiety.

Whatever reservations we might have, the future direction is now clear. There are more and more specialties where tests of increased susceptibility to disease exist. These must be used for the benefit of the patient and not to his detriment.

The physician of the 21st century, if he or she applies these new concepts with prudence, could become the adviser of healthy patients, who are no longer ill. Each person will be able to manage his health capital, as he manages his banking capital; with full knowledge, guided by his doctor, of the developments of modern genetics.

Is it not interesting to note that medicine was, in its history, first of all curative, then preventive and finally predictive, whereas today the order is reversed: initially predictive, then preventive and finally, only in desperation, curative?

Professor Jean Dausset

Nobel Laureate


Background
==========

Reused and poorly sterilised equipment causes infection in developing countries. For example, unsterile injections result in 8 to 16 million new infections of Hepatitis B every year, according to one recent estimate \[[@B1]\]. Preventing disease transmitted by medical equipment in developing countries is a priority for health care providers, and global policy agencies such as the World Health Organization (WHO) and the United Nations Children\'s Fund (UNICEF) seek the most appropriate strategies to achieve sterile injections and surgical procedures.

For vaccinations, the WHO\'s and other agencies current strategy is to recommend auto-disable syringes for immunisation, and they aim for universal implementation of this by 2003 \[[@B2]\]. However, this strategy is not yet proven, the cost is high, delivery is not assured, and how they should be disposed of is not established. Some believe providers will continue to re-use injection equipment, and this is certainly likely where provision of auto-disable syringes are not assured. In addition, auto-disable technologies do not address the need for effective decontamination of equipment used in other procedures, such as general surgery, abortion and endoscopy.

Therefore, policies for decontamination of medical equipment are still required and it is important that these are grounded in good evidence that they are effective. As a rule, manufacturer\'s instructions for decontamination of equipment should always be followed but there may be many cases where this information is not available. The World Health Organisation reviewed their current decontamination guidelines and reported their findings in a document, which has not yet been published (N Sang,. O Kasiolo: Presentation of inconsistencies found in WHO documents. *World Health Organisation (unpublished)* 1997). From eight sets of different recommendations, they identified inconsistencies. For example, guidelines gave a range of different temperatures and times for steam and dry heat sterilisation; and \'bleach\' was recommended in some guidelines, and not in others (N Sang,. O Kasiolo: Presentation of inconsistencies found in WHO documents. *World Health Organisation (unpublished)* 1997). In addition, some WHO publications recommended alcohol and povidone iodine for equipment \[[@B3]\] but these agents are not included in other published hospital guidelines \[[@B4]\]; and boiling (generally considered not to kill bacterial spores) is recommended for sterilisation of equipment \[[@B3],[@B5]\].

The evidence base for the WHO guidelines is not explicit, but this is not unusual in this field. There are many guidelines and recommendations for effective decontamination practice including reports \[[@B6]\], government documents \[[@B7]\] and field manuals \[[@B5]\], with no reference to the data on which the recommendations are based.

We sought to synthesise current available evidence for the effectiveness of various procedures using explicit, pre-specified methods to help inform policies relevant to primary care settings in developing countries. As no explicit methods are recognised for this, we first developed the methods for synthesising this type of data. This paper describes the methods we used and illustrates how the synthesis works in practice, using the example of alcohol in relation to lipid viruses. We also summarise the findings from the analysis of bleach (sodium hypochlorite and similar chemicals), alcohol and povidone iodine. The full review of decontamination procedures can be downloaded from the Liverpool School of Tropical Medicine web site (Systematic Review of Medical Equipment Decontamination Practices -- <>).

Methods
=======

The Methods section is divided into three distinct sections: Inclusion Criteria, Search Strategy and Analysis. Studies were selected for inclusion in the review according to several explicit and pre-defined criteria. These criteria are described in detail here under headings of outcomes measured, types of study and the decontamination procedures under review. Following this, we describe the search strategy developed to identify all published studies that may meet these criteria. Finally, identified studies were assessed in detail to ascertain whether or not they actually met the inclusion criteria and the findings of each one summarised, compared and presented. We describe here the methods of analysis we used for this process and the criteria we used to compare studies.

1. Inclusion criteria
---------------------

### Outcomes

The outcomes sought were those relating to equipment decontamination. This is not easy to quantify. From the UK Department of Health guidelines, decontamination is defined as:

\'A process which removes or destroys contamination (defined as soiling or pollution with harmful, potentially infectious or other unwanted matter) and thereby prevents microorganisms or other contaminants reaching a susceptible site in sufficient quantities to initiate infection or any other harmful response. Three processes of decontamination are commonly used: sterilisation, disinfection and cleaning\' \[[@B7]\].

Decontamination procedures are defined by the types of microorganisms they kill or remove. Thus, a sterilising agent destroys all microbial life and a high-level disinfecting agent kills all microorganisms except large numbers of bacterial spores\[[@B4]\]. Microorganisms differ in their resistance to decontamination and we used indicator microorganisms from the groups shown (Table [1](#T1){ref-type="table"}) to represent the huge variety of potential contaminants. These indicators were chosen according to their particular resistance to decontamination (for example, picorna viruses) but some were also included on account of their particular medical significance in the area of equipment contamination (for example, HIV). The only decontamination outcome sought was the destruction of indicator microorganisms, demonstrated by a) negative microorganism culture *in vitro*, b) failure to demonstrate their viability in animal models or c) a negative result using sensitive molecular methods.

::: {#T1 .table-wrap}
::: {.caption}
######

Indicator microorganisms included as representative markers of the organism groups shown
:::

  **GROUP**       **INDICATORS**
  --------------- ------------------------------------------------------------------------------------------------------------------------
  **Bacterial**   **Spores**: *Bacillus sp., Clostridium*
                  **Tuberculous Mycobacteria**: (*M. bovis* &*M. tuberculosis*) **Non-Tuberculous Mycobacteria**: (other *Mycobacteria*)
                  **Vegetative**: *Pseudomonas, Enterobacteria, Staphylococcus*

  **Viral**       **Nonlipid (small)**: picorna viruses (e.g. Polio)
                  **Lipid (medium)**: HIV, Hepatitis B, herpes viruses

  **Fungal**      (e.g. *Trichophyton, Candida, Aspergillus*)

  **Other**       CJD
:::

It is clear that the ability to decontaminate equipment is dependant upon the initial level of contamination and we therefore needed a quantifiable benchmark against which to compare studies. The British standard protocol for testing disinfectants is the ability to show a 5log~10~ reduction in viable microorganisms (BS EN 1040: 1997 Chemical disinfectants and antiseptics. Basic bactericidal activity). We took this standard as a practical minimum requirement for studies to be included, considering it a reasonable level of kill while excluding as few relevant studies as possible.

### Types of study

Observational studies with microbiological outcomes in the laboratory and field. Studies of in-use procedures, procedures experimentally applied to indicator microorganisms and applied to artificially contaminated equipment were included. Studies written in any language other than English and studies prior to 1976 (the date from which electronic abstracts are included in Medline) were excluded. Given the volume of studies retrieved from the database, we considered it impractical to include data from earlier studies and studies that would need detailed translation.

### Procedures included

We included decontamination procedures likely to be available in resource-poor settings and effective for the decontamination of \'critical\' and \'semicritical\' equipment (Table [2](#T2){ref-type="table"}). Critical and semicritical are terms used to describe equipment\'s need for decontamination and are linked to the subsequent use of the equipment. Thus, equipment that is used in normally sterile areas of the body (e.g. needles that penetrate the skin) has a \'critical requirement\' for decontamination and is described as critical equipment. Semicritical equipment does not penetrate skin or other tissue and the requirement for decontamination is classed as less (e.g. specula). To be effective for critical equipment, a procedure must destroy all microorganisms, but for semicritical equipment, all microorganisms except large numbers of bacterial spores should be destroyed \[[@B8]\]. Procedures requiring specialised apparatus or conditions (such as irradiation, ethylene oxide, gaseous formaldehyde and peracetic acid washer/disinfectors) were not looked at. Low-level disinfectants and antiseptics (such as quaternary ammonium compounds and phenolics) were not included, as these are not effective for critical and semicritical equipment.

::: {#T2 .table-wrap}
::: {.caption}
######

Procedures used for the decontamination of medical equipment and included in the review
:::

  ------------------------------------------------------
  **Heat-based**          **Chemical**
  ----------------------- ------------------------------
  Dry heat                Bleach (sodium hypochlorite)

  Pressurised steam       Formaldehyde

  Boiling and hot water   2% Glutaraldehyde\
                          hydrogen peroxide\
                          alcohol\
                          povidone iodine
  ------------------------------------------------------
:::

2. Search strategy
------------------

A search strategy for Medline was developed with advice from colleagues in the Cochrane Infectious Diseases Group in Liverpool. The full search strategy is listed in Table [3](#T3){ref-type="table"}. We used database-defined subject headings for decontamination procedures and indicator microorganisms. The terms used for procedures were \'*sterilization\'* and \'*disinfectants\'*. We combined the exploded versions of these terms (incorporating all terms listed as sub-headings) with specific procedure sub-headings such as *\'alcohols\'*. Some areas of disinfectant use were not related to medical equipment and these were excluded from the search using the exploded headings \'*histocytological preparation techniques\'*, \'*handwashing\'* and \'*sanitary engineering\'*.

::: {#T3 .table-wrap}
::: {.caption}
######

Sequence of search expressions used in Medline (1976-April 2000) as the search strategy
:::

  **No.**   **Search Expression**
  --------- ------------------------------------------------------------------
  1         exp disinfectants/
  2         exp hypochlorite/ or bleach.tw
  3         exp formaldehyde/
  4         exp glutaraldehyde/
  5         exp hydrogen peroxide/
  6         exp alcohols/
  7         exp povidone iodine/
  8         or/2--7
  9         exp sterilization/
  10        9 and (steam or heat or boil\$).tw
  11        (1 and 8) or 9 or 10
  12        exp histocytological preparation techniques/
  13        exp handwashing/
  14        exp sanitary engineering/
  15        11 not (12 or 13 or 14)
  16        exp bacillaceae/
  17        exp mycobacterium/ or mycobacterium.tw
  18        exp pseudomonas/ or pseudomonas.tw
  19        exp staphylococcus/ or staphylococcus.tw
  20        exp enterobacteriaceae/
  21        exp fungi/
  22        exp hiv/ or hiv.tw
  23        exp hepatitis viruses/ or hepatitis.tw
  24        exp picornaviridae/
  25        exp herpesviridae/ or herpes.tw
  26        exp creutzfeldt-jakob syndrome/ or creutzfeldt-jakob syndrome.tw
  27        exp cryptosporidium/ or cryptosporidium.tw
  28        or/16--27
  29        15 and 28
:::

The terms used for indicator microorganisms were those listed for each microorganism group. These were also exploded to include not only the relevant microorganisms but also any surrogates that may have been used experimentally. Text words for particular organism genera or diseases (e.g. *Herpes*) were also included in the search where relevant.

3. Analysis
-----------

### Assessment of studies for inclusion and quality

We obtained and assessed the full text of all relevant papers from the search. A table of excluded studies and the reasons for exclusions are in the full report. The most common reason for exclusion was that the number of microorganisms exposed to decontamination was not measured, meaning no comparative analysis was possible. However, where the outcomes of these studies provide useful information about the procedures, we refer to them.

The quality of included studies was assessed using criteria of a) quality of the procedure description, b) quality of the outcome description, c) quality of the study design and d) the number of observations for each recorded result. The form used is shown in Figure [1](#F1){ref-type="fig"}. Scores (out of a possible three) were given for each of these criteria and the total used to assign comparative reliability grades for each study. These grades were not definitive but allowed the comparison of studies within this review.

![Form used to assess the relative quality of studies identified by the systematic review search strategy. For each section (Arabic numerals), three options are given((i)-(iii)). The one that best describes the study being analysed is chosen and allocated as a score. At the bottom of the form, these scores are added (maximum possible score = 12) and the relative reliability of the study scored accordingly as high, medium or low. The form also allowed the noting of any other relevant information about the study described, such as the source of the micro-organism looked at.](1471-2334-2-4-1){#F1}

### Data extraction

We extracted the specific details of each procedure (such as time of exposure, concentration of chemical agent) and the level of kill it achieved for each indicator microorganism. In addition, we sought critical points of procedures, defined as procedural details that might explain variations in effectiveness of the same procedure when used in different studies. These we extracted systematically, and we particularly sought for details about:

a\) The form of the microorganisms used in the study (for example, were they in solution or dried to equipment?)

b\) the method of outcome assessment (for example, was it measured by culture or molecular means?).

c\) the study design (for example, did the study include controls?).

d\) Analysis of organic load (for example, was organic load present or its effect tested?).

### Data analysis

An example of the analysis of extracted data is shown in Figure [2](#F2){ref-type="fig"}. The results were grouped within procedures by the organism tested for, separating procedures between critical and semi critical equipment. Thus, studies using bacterial spores or CJD as indicator microorganisms were grouped as evidence for critical equipment. Studies using any other microorganisms were grouped as evidence for semicritical equipment. Each procedure was treated separately and so where one publication tested a range of procedures in the same experiment, each test was counted as a separate study.

![An example of the method by which results were presented in the review, showing the effectiveness of alcohol to kill lipid viruses. An overview of the relevant studies included in the review is given and a standard operating procedure iterated from these studies is described. The iterative process is then illustrated by classifying the studies as containing evidence for or against the standard procedure (Table A). Included in this assessment are possible reasons for disagreement between the studies shown (\'Possible sources of heterogeneity\'). One study in this group did not contain sufficient detail to classify it in these terms and the outcome is listed separately (Table B). The summary table of the evidence includes the standard procedure and comments about the strength of the evidence as well as factors that are identified in the studies as being essential for the desired procedure outcome (critical points).](1471-2334-2-4-2){#F2}

The studies identified used a wide range of different procedural specifications such as time of exposure to agent. To synthesise this information in a sensible way, we built up a representative set of specifications that were described as effective to destroy the particular indicator microorganisms. The specifications required to kill the most resistant microorganism were not always representative of the majority of studies. Therefore, the specifications effective for the majority of studies were chosen and each study classed as evidence for or against this standard. This process allowed flexibility to iteratively develop the standard specifications according to the findings of subsequent studies.

Once we had classed each study, we summarised in an adjacent column the possible reasons for disagreement between studies showing a procedure was effective, and those showing it was not effective (\"possible causes of heterogeneity\" in Table A, shown in Figure [2](#F2){ref-type="fig"}). In this way, major factors influencing procedure efficacy became clearer and these often coincided with the critical points described above.

Some included studies showed results that could not be described in these terms of \'evidence for\' or \'evidence against\' (for instance, during a time course of exposure to disinfectant, the period of measurement may stop short of the standard time specification iterated). These studies nevertheless contained valuable evidence or results that could be reasonably extrapolated and they are described and referred to when the effectiveness of a procedure is not clear from other studies.

### Presentation of results

For each procedure and level of decontamination, minimum conditions for effective decontamination were presented. These conditions are a product of the iterated standard specifications and any secondary outcomes that were shown to have a critical impact on the effectiveness of the procedure (critical points). An assessment of the evidence and the reviewer\'s conclusions were also presented.

Results
=======

Out of 3396 records that were retrieved using the search protocol in the Medline database (from 1976-April 2000), 88 published papers met the inclusion criteria for the review. Within these papers there were 135 studies of the effectiveness of the chosen procedures against the indicator microorganisms (Table [4](#T4){ref-type="table"}). The full results can be downloaded from the Liverpool School of Tropical Medicine website <>. We present here summaries of the results for alcohol, bleach, and povidone iodine.

::: {#T4 .table-wrap}
::: {.caption}
######

Description of included studies, showing numbers describing the different decontamination procedures included in the review
:::

  **Procedure**           **Total no. of studies**   **Equipment level**
  ----------------------- -------------------------- --------------------- ----
  **Dry heat**            2                          2                     0
  **Pressurised steam**   8                          6                     2
  **Boiling**             0                          0                     0
  **Bleach**              33                         10                    23
  **Formaldehyde**        8                          3                     5
  **Glutaraldehyde**      39                         12                    27
  **Hydrogen peroxide**   12                         3                     9
  **70% alcohol**         20                         0                     20
  **Povidone iodine**     13                         0                     13
:::

Alcohols
--------

The effectiveness of alcohols was assessed in 23 of the included studies. None of these studies assessed alcohol\'s effectiveness for critical equipment. For semicritical equipment, the standard procedure derived from the included studies was exposure to 70--80% ethanol or isopropanol for 5 minutes. Alcohols were particularly effective for lipid viruses (such as HIV) \[[@B9]-[@B11]\] and bacteria \[[@B12]-[@B14]\] but non-lipid viruses (such as hepatitis A) and mycobacteria may be more resistant, especially when dried to equipment\[[@B15],[@B16]\]. There was not enough evidence to give a more definite assessment. The presence of organic load did not impair effective decontamination with alcohol except in cases where contaminating material (such as dried serum) limited its access to microorganisms\[[@B10]\]. Results for alcohols applied to lipid viruses are shown in Figure [2](#F2){ref-type="fig"}, to illustrate the application of the review methods described.

Bleach
------

The effectiveness of bleach was assessed in 37 of the included studies; 10 effective for critical equipment and 27 for semicritical equipment. Comparison of these studies was made difficult by the different nomenclature used for \'bleach\'. As described, our definition of bleach includes sodium hypochlorite and similar chemicals. Bleach activity is regularly described in terms of concentration of available chlorine (% or mg per litre), parts per million (ppm) and ppm available chlorine. The most consistent terminology in the included studies was ppm available chlorine (used in 57% of papers identified) and the results are presented in these terms (for ppm, read ppm available chlorine). Where concentrations of available chlorine are quoted as percentages or mg per litre, these were converted to ppm for consistency. A formulation of 5,000 ppm available chlorine is equivalent to a 0.5% solution (of available chlorine).

For critical equipment, a standard procedure of 5000 ppm sodium hypochlorite bleach for 5 minutes (pH = 7--8) was sufficient to kill bacterial spores. For semicritical equipment, the standard procedure of 1000 ppm for 10 minutes was found to be sufficient. From the critical points described, effective decontamination is compromised by the presence of various organic matter \[[@B17]-[@B19]\], a very large number of microorganisms \[[@B20]\] and situations where microorganisms are dried to equipment \[[@B15]\]. Mycobacteria and viruses are particularly resistant to the standard procedure for semicritical equipment when they are dried to a surface. These factors highlight the necessity of cleaning equipment before using hypochlorite and this is also a critical point that is described \[[@B21]\]. In addition, it should be remembered that bleach can be corrosive to metal equipment and this may be an important consideration for its use.

Povidone iodine
---------------

The effectiveness of povidone iodine was assessed in 15 of the included studies. As with alcohol, none of these studies assessed its effectiveness for critical equipment. For semicritical equipment, the standard procedure of a concentration giving 1% available iodine for 15 minutes was found to be sufficient. Bacteria and mycobacteria were particularly susceptible to this procedure but viruses may be more resistant \[[@B22],[@B23]\]. There were not enough studies using viruses to determine a definite effectiveness or non-effectiveness for povidone iodine.

Discussion
==========

The method
----------

Decontamination guidelines often give no reference to the source of their recommendations \[[@B7]\] and very rarely justify or qualify any evidence that is cited \[[@B4]\]. The evidence is difficult to assess, therefore, where inconsistencies occur between guidelines (N Sang,. O Kasiolo: Presentation of inconsistencies found in WHO documents. *World Health Organisation (unpublished)* 1997). The review described is an attempt to systematically gather the relevant evidence for effective decontamination and reproducibly assess it so that decisions may be made as to the most relevant procedure to use.

We have developed methods to assess and analyse observational data where there is little standardisation in outcome measurement between studies. The novel means by which we have analysed the quality of this data has allowed the comparison of studies that lack the consistency and robustness of randomised controlled trials. The protocol we have used is also flexible enough to allow the review to be updated as more studies become available, much like a Cochrane Review. This approach may have applications in other areas of guideline appraisal and could be developed to provide a solid evidence base for other practices that may have been adopted.

This report summarises evidence systematically gathered for bleach, alcohol and povidone iodine. There is good evidence for the effectiveness of bleach as a decontaminating agent for both critical and semicritical equipment and the standard procedural specifications derived using our methods support WHO guidelines. Similarly, the findings for alcohol also agree with WHO publications, though it is not recommended to the same extent as bleach. The exclusion of povidone iodine from the majority of decontamination guidelines (N Sang,. O Kasiolo: Presentation of inconsistencies found in WHO documents. *World Health Organisation (unpublished)* 1997) is supported by the evidence here, since the iodophor has uncertain activity against viruses.

In order to manage the data and provide a valid analysis, certain stated bias was included. The first bias was using a cut-off time for inclusion of studies. As already described, 1966 to the present day is the limit of electronic databases and we limited this further to only include studies post-1976. This was mainly for practical reasons, to keep the volume of studies manageable and also to limit chasing irrelevant studies whose titles sound full of promise but for which no abstract is provided (abstracts are not included for pre-1976 records in Medline). The introduction of language bias was also for practical reasons. Many studies in languages other than English were potentially eligible for inclusion but they would have required very detailed translation in order to assess the precise de contamination methods used. Despite these limits, we consider that the number of studies retrieved were sufficient to develop representative specifications.

One of the main areas for exclusion of relevant studies was their quantification or description of indicator microorganism destruction. In order to compare the potency of decontamination procedures between studies, we set the ability to show at least a 5log reduction in microorganisms as an inclusion limit. In reality, effective decontamination is achieved when there is no infection passed on and this will be influenced by the infective dose of the contaminating microorganism and the susceptibility of the host, as well as the initial level of contamination. These factors will be different for every situation and so decontamination, as such, cannot be quantified in a laboratory. Setting a minimum level of demonstrable destruction allows the comparison of data from diverse studies and is simply a tool to aid the analysis of data in this review.

Setting such a limit obviously excluded studies that were otherwise relevant and, it could be argued, introduced misleading bias. Where there was heterogeneity in the evidence for a particular microorganism/procedure combination, however, these excluded studies were checked for data that may influence the outcome. In every case, such data did not challenge the standard specifications iterated from the included studies, suggesting this was a valid and useful cut-off point to incorporate.

The explicit methods used to search for studies and assess them for inclusion enable this review to be updated. The methods we have used to analyse the procedural specification data also allow updating of the results by further iteration. By grouping evidence as for and against the standard set, reasons for disagreement between new studies and the standards developed in this review should be immediately apparent.

Though the levels of evidence for each procedure were mixed, the results of this review agree with current decontamination guidelines, often reached by consensus and not systematically. This approach, therefore, provides a means by which observational and non-standardised data can be systematically summarised to give formal evidence for guidelines. The implications of the results for the WHO are to standardise decontamination guidelines across the various arms of the organisation. These methods of systematic review and analysis may enable evidence-based guidelines to be made in similar areas where data cannot be pooled or assessed using standard methods.

Conclusions
===========

Comparing the evidence with guidelines
--------------------------------------

The standard procedures iterated for bleach differed from the WHO guidelines that included this agent (N Sang,. O Kasiolo: Presentation of inconsistencies found in WHO documents. *World Health Organisation (unpublished)* 1997) but did not contradict them. Guidelines only gave a recommendation for critical equipment and this was neat bleach (generally 52,000 ppm) for 30 seconds or 5000 ppm bleach for 10 minutes. Procedures for alcohol and povidone iodine are not described in the guidelines reviewed (N Sang,. O Kasiolo: Presentation of inconsistencies found in WHO documents. *World Health Organisation (unpublished)* 1997) but are mentioned elsewhere in WHO publications. The procedures derived here show some scope for their use as disinfectants but there is not enough available evidence to demonstrate their effectiveness against all the indicator microorganisms. In recommending alcohol, one WHO publication describes it as not effective against non-lipid viruses and this is supported by the evidence here\[[@B3]\]. The same publication recommends iodophors as effective for both non-lipid viruses and bacterial spores but the evidence for povidone iodine here disputes this.

There are several considerations to be made when choosing a particular means of decontamination and this review focuses only on the microbiological effectiveness of agents. Other factors include cost and the compatability of decontamination agents with particular pieces of equipment. Manufacturer\'s guidelines for decontamination should always be followed where possible as these will take into account risks of equipment corrosion through inappropriate decontamination.

Critical points of the procedures
---------------------------------

The factors that impacted the effectiveness of procedures most frequently in the review were the presence of organic material and the drying of microorganisms to a surface before decontamination. These factors may either neutralise the potency of the decontamination agent being used or protect the target microorganisms from exposure to the agent. Thus, the critical point common to the majority of the procedures was the need to clean equipment before decontamination, and this is specifically mentioned or quantified in many of the studies. This demonstrates that, for the procedures described here, the application of a decontamination procedure is as important as the choice of procedure.

Competing interests
===================

None declared

Authors\' contributions
=======================

WS wrote the protocols, identified the studies, conducted the data extraction, and synthesised the data. PG identified the topic and advised on methods development, data extraction, interpretation and writing of the paper. TH helped identify the relevant questions, outcomes and indicators and advised on the literature search. All authors contributed to revisions and finalising of the paper and the report.

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

<>

Acknowledgements
================

A panel of specialists helped at the protocol and review stage by providing important technical advice and suggestions. We are very grateful for their input. They were: Barbara Stilwell (WHO), John Lloyd (PATH), Mary Catlin (PATH), Anthony Battersby (FBA systems), Craig Mackintosh (Clatterbridge Hospital). The panel are not responsible for the content, and the data presented and the views expressed are entirely those of the authors.

This research was supported by the Department for International Development (UK) through their support to the Effective Health Care Alliance Programme based at the Liverpool School of Tropical Medicine.


Background
==========

DNA of prokaryotic and eukaryotic cells and their viruses is often modified by methylation, carried out by S-adenosyl-L-methionine (AdoMet)-dependent DNA methyltransferases (MTases). Since a particular nucleotide sequence may exist in its methylated or unmethylated form, methylation can be regarded as an increase of the information content of DNA, which serves a wide variety of biological functions. In Eukaryota, DNA methylation plays a role in crucial regulatory processes, such as regulation of gene expression, embryonic development, genomic imprinting, and carcinogenesis (reviewed in ref. \[[@B1]\]). In Prokaryota, DNA methylation can be involved in DNA mismatch repair, regulation of gene expression, and control of timing of DNA replication (reviewed in ref. \[[@B2]\]). However, the majority of prokaryotic MTases are paired with a restriction endonuclease of cognate sequence specificity, together forming restriction-modification (RM) systems. RM systems are thought to serve as defense mechanisms that protect the cell against invasion of foreign genetic elements such as phages and plasmids \[[@B3]\]. It has been also suggested that RM systems are maintained in evolution because they participate in generating bacterial diversity by promoting homologous recombination \[[@B4]\] or because they act as as \"selfish\" genetic elements that undergo extensive horizontal transfer \[[@B5]\]. These three hypotheses are contrasting, but not mutually exclusive.

MTases can be divided into three different groups on the basis of the chemical reactions they catalyze: generating N6-methyladenine (m^6^A), N4-methylcytosine (m^4^C), and C5-methylcytosine (m^5^C). It has been suggested that m^4^C and m^6^A MTases (collectively termed \"N-MTases\") may be more closely related to each other than to m^5^C MTases \[[@B6]\]. Nevertheless, subsequent analyses showed that the relationships between these groups of proteins are quite complicated and their evolution may have involved several independent conversions of the reaction specificity \[[@B7]-[@B9]\]. Amino acid sequence alignments of DNA MTases revealed several conserved motifs, of which I-VIII and X are common to most subfamilies, and a region of essentially higher variability \[[@B6],[@B10]\]. Based on the results of X-ray crystallography of members of all three groups and structure-based multiple sequence alignment, motifs IV-VIII were assigned to the active-site subdomain, motifs X and I-III to the AdoMet-binding subdomain, and the variable region was recognized as a separate domain, implicated in recognition of the target sequence (and termed TRD for target-recognition domain) (reviewed in refs. \[[@B11],[@B12]\]). Structural studies on m^5^C, m^4^C, and m^6^A MTases demonstrated that the TRDs of these proteins are structurally dissimilar and most likely were acquired in independent gene fusion events \[[@B11]\].

DNA MTases have been subdivided into 6 classes (α, β, γ, ζ, and the hypothetical δ and ε ; Figure [1a](#F1){ref-type="fig"}) according to the possible linear arrangements of three modules: the AdoMet-binding subdomain, the active site subdomain, and the variable TRD \[[@B6]\]. All of the enzymes preserve the same spatial arrangement of the motifs and α, δ, and ε form a circularly permuted set as does β, γ, and ζ (Figure [1b](#F1){ref-type="fig"}). The majority of DNA N-MTases fall into the α, β, and γ classes, with no *bona fide* γ-m^4^C MTases known. M.*Ngo*MXV \[[@B13]\] and its close homolog M.*Lmo*A118I \[[@B14]\] are the only experimentally characterized m^4^C MTases, whose architecture is very similar to γ-m^6^A MTases. Nevertheless, these remarkably small MTases (153 aa), as well as their uncharacterized homologs identified in sequence databases, lack both the classical TRD and the region corresponding to motif X and therefore can be regarded as \"minimal\" members of the family \[[@B13]\]. Another group of small (\~175 aa) MTases, which are similar to γ-m^6^A MTases but lack motif X and the TRD, are the atypical Dam (m^6^A) MTases encoded by several phages, including HP1, VT-2, and T1 \[[@B15]\].

![Conserved fold and variable topology of the MTase domain, **a)** The linear organization of six classes of amino-MTases (α-ζ) postulated by Malone et al. \[[@B6]\], m^5^C MTases (the prevailing archetypal topology labeled as m^5^C, and the two minor classes as ζ and DRM2), and the minimal MTases lacking the discrete TRD. The AdoMet-binding region is shown as a blue arrow, the catalytic region is shown as a red arrow, conserved motifs are labeled accordingly **b).** The topology diagram: triangles represent β-strands, circles represent a- and 3~10~-helices, connecting lines represent loops; the thick lines correspond to the loops on the catalytic face of the protein, which harbor residues involved in cofactor binding and catalysis and most likely in DNA-binding. Elements forming the AdoMet-binding pocket are colored blue; elements forming the target base-binding/catalytic pocket are colored red. Circled Roman numerals represent nine motifs, the key motifs I and IV are shown in bold and underlined. Orange arrows show the topological breakpoints (N/C for generation of N- and C-termini) and sites of TRD insertion characteristic for the individual classes of MTases. The elements characteristic for the β-class MTases are shown in magenta, the corresponding elements found in M.*Mwo*I (δ-class) and TVN1413 (ζ-class) are shown in green and cyan, respectively.](1471-2148-2-3-1){#F1}

Most m^5^C MTases resemble the N-MTases of the γ class, with the only difference being their motif X localized at the C-terminus instead of the N-terminus. However, a few m^5^C MTases have been described with unusual permutations (Figure [1](#F1){ref-type="fig"}). To my knowledge, M.*Bss*HII is the only DNA MTase, for which the ζ architecture has been convincingly confirmed by experiment \[[@B16]\] and homology modeling \[[@B17]\]. In addition, a novel class of permuted m^5^C MTases typified by DRM2 have been recently identified in plants; this prediction has also been supported by threading of the permuted sequence onto the common fold \[[@B18]\]. Some DNA MTases contain terminal extensions and various insertions \[[@B6]\], but it has never been demonstrated that they are related to known or predicted TRDs in other proteins.

DNA MTases are only one of the numerous families of remotely related enzymes that exhibit a common fold \[[@B11],[@B19]\]. Other members of the superfamily methylate a variety of chemically diverse molecules, including various RNAs, proteins, lipids, small molecules, etc. While all members of the superfamily share the same structural core, only DNA MTases vary in the order of conserved motifs. The rest show the order X, I, II, III, IV, V, VI, VII, VIII and only protein-arginine MTases lack the last three motifs \[[@B19]\]. The question thus arises, why do DNA MTases, but not MTases in general, exhibit sequence permutations within the same structural framework?

Two models have been proposed to explain sequence permutations arising during the evolution of N-MTases \[[@B9],[@B20]\]. Jeltsch argued that the process of domain permutation needs duplication and in-frame fusion of the MTase gene, producing one enzyme with two catalytic domains \[[@B20]\]. Subsequent introduction of a new start codon in the middle of the first gene copy and a stop codon at the equivalent position in the second gene copy would then result in a circularly permuted variant. For instance, the ζ- or β-like permutants could arise from a hypothetical tandem γγ-class MTase. This model corresponds to the widely accepted concept that a permuted protein may arise naturally from tandem repeats by extraction of the C-terminal portion of one repeat together with the N-terminal portion of the subsequent repeat, so long as the protein\'s N and C termini are in close spatial proximity (reviewed in ref. \[[@B21]\]). Although the idea itself offers a plausible explanation for the origin of permutants within many protein families, the only known duplicated m^6^A MTases are the type IIS enzymes of the αα-class, whose permutation would eventually produce enzymes of the δ or ε class that have not been identified to date. The TRDs of known MTases from β and γ classes are unrelated \[[@B22],[@B23]\], hence it is unlikely that simple conversions \"from γγ to β \" or \"from ββ to γ \" have occurred in nature. Furthermore, the N- and C-termini of M.*Taq*I, the only γ-m^6^A MTase whose 3D structure is known, are quite distant in space \[[@B22]\]. Still, this scenario may be valid for enzymes that have not been identified yet, or whose sequences have not been studied in enough detail.

It has been hypothesized (ref. \[[@B9]\]) that the permuted DNA MTase variants were generated by intra- or intergenic rearrangements of gene fragments (i.e. \"module shuffling\"; reviewed in refs. \[[@B24],[@B25]\]) that left no evidence of duplication intermediates. However, only in one case (M.*Bss*HII) has it been possible to reconstruct a possible evolutionary history of shuffled fragments \[[@B17]\]. Moreover, no examples of N-MTases in different classes are known, whose TRDs are markedly similar. Hence, no convincing examples of permutations of an entire N-MTase molecule have been identified to date. The reported permutations in N-MTases concern only the segments within the catalytic domain, while the unrelated TRDs were acquired or evolved independently in distinct classes \[[@B20]\]. Identification of closely related DNA MTases with homologous TRDs that nevertheless lie in different classes might help decide between the two given hypotheses and shed light on how the different classes of DNA MTases arose.

Results
=======

Candidates for N-MTases in the midst of the process of permutation were sought amongst all DNA MTases, whose sequences were available from REBASE \[[@B26]\]. Those sequences exhibiting deviations from the typical spacing between the conserved motifs or unusual extensions at the termini were chosen for detailed analysis. Within some extensions, several known or predicted DNA-binding domains were identified (to be published elsewhere), however only two pairs of MTase sequences were found that fit the criteria of similarity between the AdoMet-binding subdomain, the active site subdomain, and the TRD, without conservation of their co-linearity.

One MTase that emerged from the analysis was the M.*Mwo*I protein, which recognizes an interrupted palindrome GCNNNNNNNGC and methylates one of the cytosines in each strand, generating m^4^C \[[@B27]\]. M.*Mwo*I (GenBank record 2961238) was earlier classified as a member of the β-class, however it exhibits quite unusual length of 668 aa, which is approximately twice the length of a typical β-class MTase \[[@B6]\], and it lacks the variable insertion between motifs VIII and X, corresponding to the TRD in β-class MTases \[[@B9]\].

BLAST searches and threading analysis (see Methods) revealed that the N-terminal part of M.*Mwo*I (aa 1--270) aligns very well with the catalytic and the AdoMet-binding subdomains of β-class MTases. However, in β-class MTases these subdomains are separated by the TRD, which in M.*Mwo*I is replaced by only a few residues (Figures [2a](#F2){ref-type="fig"}, [3a](#F3){ref-type="fig"}). Comprehensive results of the threading analysis of the M.*Mwo*I sequence are available at the URL <>, the alignments of the N-terminal region are essentially identical to those reported previously \[[@B9]\]. Remarkably, The C-terminal region of M.*Mwo*I revealed no similarities to other sequences, with one prominent exception, namely the predicted TRD of the m^4^C MTase M.*Sfi*I (GenBank entry 2761010) with the BLAST expectation (e) value of 10^-3^. Further database searches using the sequence of M.*Sfi*I and the isolated fragments of putative TRDs confirmed that the three major subdomains of M.*Mwo*I and M.*Sfi*I exhibit significant sequence similarity, but the linear order of these elements differs between them. If this prediction is correct, M.*Mwo*I should be classified as the first member of the δ-class, rather than the β-class (Figures [1](#F1){ref-type="fig"}, [2a](#F2){ref-type="fig"}). It is noteworthy that using either PSI-BLAST or threading, no significant sequence or structural similarities of the TRD of M.*Mwo*I and M.*Sfi*I could be detected to TRDs of other MTases and generally to sequences of other proteins. The additional sequence region of M.*Mwo*I (aa 434--497), which may be regarded as a linker between the N-terminal catalytic domain and the newly identified C-terminal TRD, also showed no matches to any sequences in the database. Hence, the determination of which arrangement of subdomains, that of M.*Mwo*I or M.*Sfi*I, corresponds to the ancestral state must await discovery of their homologs.

![Rearrangements in the primary sequence of the two pairs of permuted MTases **a)** Relationships between segments of M.*Sfi*I and M.*Mwo*I **b)** Relationships between segments of M.*Tha*I and M.*Tvo*ORF1413P.](1471-2148-2-3-2){#F2}

![Sequence alignment of β-MTases with the permuted MTases: **a)** M.*Mwo*I (δ-class) **b)** M.*Tvo*ORF1413P (ζ-class). Blue numbers indicate the sequence coordinates (residue numbers). Red numbers in parentheses indicate the size of terminal extensions or loops, which were omitted for clarity. Identical residues are highlighted in black, conservative substitutions are highlighted in gray. Conserved motifs are labeled according to the nomenclature described by Malone et al \[[@B6]\]. Secondary structure elements shown below the alignment were deduced from the M.*Pvu*II coordinates or predicted for the TRD of M.*Sfi*I/M.*Mwo*I.](1471-2148-2-3-3){#F3}

M.*Sfi*I recognizes the sequence GGCCNNNNNGGCC, which belongs to a broader set of sequences recognized by the GCNNNNNNNGC-specific M.*Mwo*I. It is not known, how these enzymes recognize such a lengthy sequence with a non-specific spacer. Nonetheless, it can be imagined that the TRD of M.*Sfi*I evolved from the TRD of M.*Mwo*I by acquisition of new contacts to bases outside and inside the GC pair (N-\>G)GC(N-\>C)NNNNN(N-\>G)GC(N-\>C) or conversely, the stringent DNA-recognition specificity of the M.*Sfi*I-like TRD was relaxed to give rise to the less specific M.*Mwo*I. In the absence of protein-DNA co-crystal structures for the β-class of MTases and lack of suitable structural templates for modeling the TRD structure in M.*Sfi*I and M.*Mwo*I, prediction of the detailed protein-DNA contacts is unfeasible. However, I hope that the finding reported herein will prompt mutagenesis experiments -- it is tempting to speculate that swapping the predicted TRDs between M.*Mwo*I and M.*Sfi*I will result in an exchange of specificities.

A second MTase that came from the initial screen was M.*Tvo*ORF1413P, interpreted as a member of the γ-class in ReBase \[[@B26]\]<>, but exhibiting an extension of over 150 aa located N-terminally to motif X instead of the C-terminal extension after motif VIII required by the structure of the γ-class. In a BLAST search initiated with the M.*Tvo*ORF1413P sequence, M.*Tha*I was reported as the best hit, with a highly scored alignment of the AdoMet-binding region (e-value 5\*10^-14^) and a quite poorly scored (0.049) alignment of the catalytic region. However, M.*Tha*I is a member of the β-class and these two regions of similarity are swapped in the primary sequences of the two MTases (Figures [2b](#F2){ref-type="fig"}, [3b](#F3){ref-type="fig"}). BLAST searches initiated with the N- and C-terminal parts of M.*Tvo*ORF1413P showed that its C-terminal region scores better (e-value 6\*10^-4^) when aligned with the catalytic domain of another CGCG-specific β-m^4^C MTase, M.*Tma*I (GenBank record 4980829), a close relative of M.*Tha*I (data not shown).

Threading analysis of the M.*Tha*I sequence revealed its perfect compatibility with the M.*Rsr*I and M.*Pvu*II structures (results are available at <>, allowing homology modeling of the M.*Tha*I structure (Figures [3b](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"}). These results reveal that the TRD of M.*Tha*I is shorter than TRDs of M.*Rsr*I and M.*Pvu*II, which may be an indication that in this enzyme some DNA-binding residues migrated to other loops \[[@B9]\]. On the other hand, threading of M.*Tvo*ORF1413P revealed that its C-terminus corresponds to the last β-strand of the common MTase core (motif VIII; Figure [1](#F1){ref-type="fig"}), strongly arguing against its assignment to the γ-class, which requires the presence of the TRD C-terminal to this element (threading results are available at <>, <> and <> with the three entries corresponding to the full length sequence, the N-terminal part and the C-terminal parts, respectively). Instead, all threading algorithms reported that the N-terminus of M.*Tvo*ORF1413P matches perfectly the additional β-strand (motif IX-N) in M.*Rsr*I and M.*Pvu*II and this region, along with the predicted TRD, aligns quite well with motif IX-N and the TRD of M.*Tha*I (Figure [3b](#F3){ref-type="fig"}). It is noteworthy, that for the core regions, the predicted secondary structure agreed very well both between M.*Tha*I and M.*Tvo*ORF1413P, and between these MTases and the experimentally determined structures of M.*Rsr*I and M.*Pvu*II (for details see the above mentioned links to MetaServer results). For the 42 N-terminal residues of M.*Tvo*ORF1413P no similarity to known sequences or structures could be demonstrated, and modeling based on N-terminally extended threading alignments resulted in misfolded structures; it is therefore possible that this region forms an elaboration of the common fold, which is unique to M.TvoORF1413P.

![Cartoon diagrams depicting the structure of homology models of **a)** M.*Tha*I **b)** M.*Tvo*ORF1413P. Secondary structure elements are colored according to the linear order -- from blue (N-terminus) to red (C-terminus). The termini of the models are labeled; the N-terminal 51 aa of M.*Tha*I and 38 aa of M.*Tvo*ORF1413P, as well as an insertion comprising aa 195--220 of M.*Tvo*ORF1413P were not modeled.](1471-2148-2-3-4){#F4}

The MTase activity of M.*Tvo*ORF1413P remains to be demonstrated. However, its close homolog has been recently identified, which exhibits a genuine  MTase activity (Drs. M.A. Abdurashitov and S.K. Degtyarev, personal communication). M.*Bst*F5I-4, whose sequence remains unpublished, is evidently homologous to M.*Tvo*ORF1413P over the region including the predicted N-terminal TRD, as well as motifs IX-N, X, and I-VIII (BLAST e-value 3\*10^-20^, 26% identical plus 23% conservatively substituted residues; with 60% identical residues in the predicted TRD, i.e. the 10 aa loop preceding motif IX-N; data not shown). It cannot be ruled out that the small TRD of M.*Tha*I, M.*Bst*F5I-4, and M.*Tvo*ORF1413P harbors only a fraction of specificity determinants and that other loops on the catalytic face of the protein contribute to specific DNA recognition. Nevertheless, according to the classical definition of the TRD (the variable region between motifs VIII and X \[[@B6],[@B28]\]), the presented results of sequence analysis and structure prediction suggest that the common ancestor of ζ-class MTases M.*Bst*F5I-4 and M.*Tvo*ORF1413P evolved from M.*Tha*I (β-class member) by sequence permutation.

Discussion
==========

Sequence analysis resulted in identification of two novel cases of sequence permutation in DNA MTases, and demonstration for the very first time, that  MTases of different classes may exhibit significant sequence similarity not only in the catalytic domain, but also in the TRD. This finding suggests that the analyzed gene pairs diverged relatively recently, permitting a test of the hypothesis that the observed rearrangements occurred according to the \"permutation-by-duplication\" model \[[@B20]\] or to the alternative model, involving intragenic relocation of gene segments. If sequences resembling fragments of one of the DNA MTase analyzed herein were identified in its own neighborhood, this would provide strong evidence that gene duplication occurred. It would also suggest that this particular MTase is a permuted version of its homolog, whose neighborhood is free from duplicated fragments, rather than the opposite.

Regrettably, the neighborhood of the *Sfi*I and *Mwo*I RM systems is unknown, however the context of *Tha*I RM system and M.*Tvo*ORF1413P can be analyzed using the complete genome sequences of *Thermoplasma acidophilum*\[[@B29]\] and *T. volcanium*\[[@B30]\], respectively. The genome sequences of both *Thermoplasma* species flanking M.*Tha*I and M.*Tvo*ORF1413P (10 000 base pairs in each direction) were compared using the BLAST-family programs at the level of DNA and putative translations in all open reading frames (with stop codons translated as missing characters, e.g. \"X\"). No evidence of sequences similar to the genes encoding these two MTases were found, except for another putative  MTase M.*Tvo*ORF1416P located 2640 bp 5\' to M.*Tvo*ORF1413P. M.*Tvo*ORF1416P is a typical α-class member and exhibits significantly higher similarity to α-MTases such as M.*Psp*GI (BLAST e-value 2\*10^-73^) or M.*Mva*I (e-value 3\*10^-67^) than to M.*Tvo*ORF1413P (insignificant e-value 5.3). Therefore, the two MTases should be regarded as remote homologs of each other and as members of different phylogenetic lineages \[[@B9]\] that met rather accidentally in the *T. volcanium* chromosome rather than as products of recent duplication of one gene.

The lack of evidence supporting the gene duplication mechanism in the case of MTases from *Thermoplasma* is not entirely convincing, especially since no direct evidence supporting the alternative \"cut-and-paste\" mechanism can be provided by sequence analysis. Hence, the events leading to M.*Sfi*I(β)-M.*Mwo*I(δ) and M.*Tha*I- (β) M.*Tvo*ORF1413P (ζ) permutations were reconstructed based on both mechanisms (Figures [5](#F5){ref-type="fig"}, [6](#F6){ref-type="fig"}, [7](#F7){ref-type="fig"}, and [8](#F8){ref-type="fig"}). For the sake of simplicity, it was assumed that the unique δ and ζ-class members were in these cases generated by permutation of common β-class members, however an analogous reconstruction could be carried out assuming the opposite directionality of rearrangements, leading to similar conclusions.

![Diagram illustrating a putative evolutionary history of the M.*Sfi*I-M.*Mwo*I gene pair according to the \"permutation-by-duplication\" mechanism. Conserved motifs and the TRD are labeled. Protein sequence regions corresponding to the AdoMet-binding subdomain, the catalytic subdomain, the TRD, and non-conserved segments of unknown function are depicted in green, blue, magenta and grey, respectively. Grey labels indicate non-functional regions that are presumably able to fold in solution and do not interfere with the folding of the functional unit. Red vertical bars denote ends generated by new start or stop codons or the boundaries of intragenic deletions. Deleted segments are marked with \"Δ\". Putative promoters are indicated as pink arrows.](1471-2148-2-3-5){#F5}

![Diagram illustrating a putative evolutionary history of the M.*Tha*I-M.*Tvo*ORF1413P gene pair according to the \"permutation-by-duplication\" mechanism. Labels follow the legend to Figure [5](#F5){ref-type="fig"}.](1471-2148-2-3-6){#F6}

![Diagram illustrating a putative evolutionary history of the M.*Sfi*I-M.*Mwo*I gene pairs according to the \"cut-and-paste\" mechanism. Red vertical bars denote ends generated by endo- or exo-nucleolytic cleavage (red \"PacMan\" faces). Red arrows delineate points of insertion of gene fragments. Other labels follow the legend to Figure [5](#F5){ref-type="fig"}.](1471-2148-2-3-7){#F7}

![Diagram illustrating a putative evolutionary history of the M.*Tha*I-M.*Tvo*ORF1413P gene pair according to the \"cut-and-paste\" mechanism. All labels follow the legends to Figures [5](#F5){ref-type="fig"} and [7](#F7){ref-type="fig"}.](1471-2148-2-3-8){#F8}

Figures [5](#F5){ref-type="fig"} and [6](#F6){ref-type="fig"} shows possible histories leading to both permutations according to the gene duplication mechanism. The order of the sequence motifs remains conserved between M.*Sfi*I and M.*Mwo*I, but these two MTases differ in that a large segment bearing the TRD appears in the middle of the former but in the C-terminus of the latter, hence permutation of these two proteins is not circular (Figure [5](#F5){ref-type="fig"}). To produce M.*Mwo*I, duplication of M.*Sfi*I would have to be followed by both creation of a novel stop codon to eliminate the region encoding the \"new\" catalytic subdomain of the C-terminal repeat and deletion of the regions corresponding to the \"old\" TRD and the \"new\" AdoMet-binding subdomain. It is quite unlikely that all these changes occurred in a single event, and their occurrence in a series of steps would seem inevitably to produce a nonfunctional intermediate, which would require two steps to regain activity. If these changes did occur gradually, the product of gene duplication, in which one repeat retained the AdoMet-binding subdomain but lost the catalytic subdomain (or conversely), would expose the hydrophobic core of the remaining nonfunctional subdomain to the solvent. Folding and enzymatic function of such \"1 & ½\" mutant would be probably heavily compromised. The function of M.*Sfi*I and M.*Mwo*I is to protect the chromosome from being cleaved by the cognate ENase. Hence, it seems rather unlikely that the host cell would survive such a series of unlikely events passing through functionally compromised intermediates.

Compared to M.*Sfi*I and M.*Mwo*I, evolution of M.*Tvo*ORF1413P from M.*Tha*I seems more likely (Figure [6](#F6){ref-type="fig"}), since in this \"classical\" case of circular permutation requires only removal of the terminal regions by formation of new start and stop codons. However, in this case deletion of terminal subdomains, or their large parts, would also have to be concurrent, otherwise nonfunctional intermediates could arise, leading to cell death due to insufficient protection against the cognate ENase. If only one of the repeats in the original tandem ββ fusion protein is damaged, deletion of the remaining nonfunctional part would most likely restore the highly active, single copy version of the parent MTase. It is noteworthy that the tandem duplication mechanism offers no stage, at which evolutionary pressure would result in optimization of a poorly active intermediate specifically towards the permuted version.

Another problem with the scenario involving tandem ββ MTase fusion is that such fusions have never been reported to occur. In the X-ray structure of the β-class member M.*Rsr*I, the two identical subunits make quite extensive contacts (a loss of 1799.3 Ang\*\*2 of solvent accessible surface area per chain upon complex formation; see URL: <>, suggesting that the dimeric structure of this MTase is biologically relevant \[[@B23]\]. The TRD and the active site reside on opposite sides of the M.*Rsr*I monomer, but the unique dimeric configuration brings the TRD of one subunit near the active site of the other, indicating that dimerization may be required for recognition and methylation to occur. The N- and C-termini of M.*Rsr*I are located close to each other in the monomer (8.8 A), but the C-terminus of one monomer is located on the opposite side of the dimer in respect to the N-terminus of the other monomer, separated by a distance of 74 A in a straight line. If the configuration observed in the crystal structure of M.*Rsr*I is representative for other members of the β-class that use two cooperating MTase domains, covalent joining of the termini of these domains would require a very long linker peptide, looping around the dimer. Hence, tandem fusion seems disrupting for cooperation of two β-class MTases within the dimer.

An alternative \"cut-and-paste\" mechanism (Figures [7](#F7){ref-type="fig"}, [8](#F8){ref-type="fig"}), inspired by the observed genomic rearrangements associated with the presence of restriction endonucleases \[[@B5],[@B31]\], involves generation of a functional gene from fragments. In one scenario (Figures [7](#F7){ref-type="fig"} and [8](#F8){ref-type="fig"}, left panels), the fragments may be generated due to combined action of various endo- and exonucleases that partially degrade the DNA fragment encoding the MTase gene, thereby producing recombingenic ends. If degradation occurs, at least two copies of the MTase gene must be present in the cell in order to reconstruct all important regions. Another scenario (Figures [7](#F7){ref-type="fig"} and [8](#F8){ref-type="fig"}, right panels) involves precise action of a sequence-specific endonuclease, which fortuitously cleaves the MTase gene in the regions corresponding to linkers between the TRD and the two subdomains of the catalytic domain. In this case, one or more copies of the MTase gene may be present in the cell. These two scenarios are not mutually exclusive, provided that the fragments resulting from any type of cleavage or degradation span all regions necessary for the MTase activity, and are able to recombine with each other or use \"sticky ends\" to ensure ligation.

The \"cut-and-paste\" mechanism (Figures [7](#F7){ref-type="fig"}, [8](#F8){ref-type="fig"}) differs from the \"permutation-by-duplication\" mechanism (Figures [5](#F5){ref-type="fig"}, [6](#F6){ref-type="fig"}) in that it involves a momentary stage at which there are no intact, active MTase gene copies in the cell. The accompanying ENase gene might be fragmented as well, leading to elimination of the RM system from the cell. However, if the ENase remains active for a certain period of time, only those cells survive in which the MTase gene is restored from fragments. Such MTase may exhibit various deletions, duplications of certain regions and rearrangements, as long as these modifications allow the protein to provide protection against the ENase. The selection pressure will result in rapid optimization of the MTase function, most likely to the nearest maximum in the fitness landscape. With a certain probability, the permuted gene copy will arise, and under such \"all or nothing\" conditions, its sequence will be optimized towards the modification activity sufficient to protect the host\'s chromosome. It is worth mentioning that in the short term the ENase may remain active and provide selective pressure for restoring expression of the MTase even if its own gene has been destroyed.

A hybrid mechanism can be envisaged in which the complete, fully functional MTase gene undergoes recombination with a fragment of a MTase gene (not shown). This mechanism has limitations similar to that of the \"permutation-by-duplication\" mechanism in that the newly fused fragment must not compromise the function of the original protein. However, there is no specific reason why a part of the original domain should be deleted with a higher frequency than the new fragment and why the latter scenario should be selected for, unless the alternative fragments are not identical and the new fragment encodes a function, which may increase the fitness of the protein.

The scenarios shown in Fig. [5a](#F5){ref-type="fig"} require that the TRD can function autonomously, outside of its original structural environment. For some TRDs, at least, this is the case (reviews: \[[@B2],[@B12]\]). Not only is the movement of TRDs within a MTase plausible, but the exchange of TRDs between MTases can provide different specificities and thereby functional advantage \[[@B32]\]. Indeed,  MTases that methylate more than one specific DNA target owing to the presence of several TRDs at various locations of the enzyme have been identified \[[@B33],[@B34]\], and shuffling of TRDs have been suggested to occur among both mono- and multispecific  MTases \[[@B35]\]. However, while the unrelated TRDs of many structurally characterized MTases form structurally autonomous domains, the TRDs of two members of the β-class, M.*Rsr*I \[[@B23]\] and M.*Pvu*II \[[@B36]\], form only an amendment of the common fold, which is quite unlikely to behave as an independently folded and functionally autonomous unit (review: \[[@B12]\]).

The putative TRD of M.*Tvo*ORF1413P and M.*Tha*I is most likely too small and too poorly structured to be regarded as an independent domain (Figures [3b](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"}). However, the putative TRD of M.*Sfi*I and M.*Mwo*I is much longer and comprises at least four predicted helices (Figure [3a](#F3){ref-type="fig"}), therefore it cannot be excluded that it may form an independently folded, functional unit. It has been demonstrated that  MTase M.*Aqu*I comprises two independent polypeptides corresponding to the catalytic domain and the TRD, which associate in solution to form a functional enzyme \[[@B37]\]. Correspondingly, one of the scenarios of evolution of the M.*Mwo*I enzyme involves temporary separation of the gene fragments encoding the catalytic domain and the TRD (Figure [7](#F7){ref-type="fig"}). According to this scenario, the linker region between the catalytic domain and the TRD originated from the initially non-coding sequence that initially separated the two functional units. It seems likely that covalent linkage of the two domains by the newly established linker increased the fitness of the rearranged MTase.

The \"cut-and-paste\" scenario offers an explanation for the sequence permutations being observed in DNA MTases and not among ENases. This results from the asymmetry of selection for restoring methylation function and restriction function after the corresponding genes are fragmented. In other words, the newly permuted proteins are probably poor enzymes, but the ENase provides strong selective pressure for optimization of the MTase function, while in the second case selective pressure is relatively weak (functional ENases probably provide only a minor selective advantage and they are not required for protection of the host \"against\" the MTase). However, it cannot be excluded that ENases are simply not amenable to any sequence permutation for structural reasons, since these proteins exhibit different fold than MTases.

Interestingly, sequence permutations have been observed only amongst DNA MTases \[[@B1]\], but not in other MTase families (enzymes acting on RNA, proteins, small molecules, etc.), despite their common structure. To date, no explanation has been offered for this peculiarity, even though it raised considerable interest in the field \[[@B11]\]. It is tempting to speculate that the rearrangements observed amongst DNA MTases but not other MTases are induced \[[@B5]\] by the increased exposure of their genes to the repertoire of various nucleases encoded by different hosts during horizontal transfer events common among the RM systems (J. Elhai, personal communication). High frequency of such events was inferred from sequence analyses \[[@B5],[@B38]\]. If this hypothesis is correct, interaction with the mechanisms of defense against alien genetic elements encoded by various Prokaryotes may be also responsible for permutations of entire domains within type I and type III RM systems and a plethora of combinations of various domains in many \"non-classical\" RM system subtypes (review: \[[@B12]\]).

Summary
=======

Analyses of the evolutionary scenarios presented herein favor the \"cut-and-paste\" mechanism or the hybrid mechanism (fusion of an intact MTase with the TRD) for the M.*Sfi*I(β)-M.*Mwo*I(δ) rearrangement and the original \"permutation-by-duplication\" mechanism or the \"cut-and-paste\" mechanism (rather than their hybrid) for the M.*Tha*I-(β) M.*Tvo*ORF1413P(ζ) rearrangement. Even though in these and probably other cases, certain scenarios may seem more likely than the other, none of them can be ruled out completely. The presented mechanisms are not mutually exclusive, and all have probably played significant roles in the generation of permuted MTases.

Methods
=======

The PSI-BLAST algorithm \[[@B39]\] was used to search the non-redundant version of current sequence databases (nr) and the publicly available complete and incomplete genome sequences at the NCBI website <>. All genuine and putative N-MTase sequences available from REBASE \[[@B26]\] were submitted as queries with default parameters. Protein structure prediction was carried out using the MetaServer available at <>, which combines several secondary structure prediction and threading methods (ref. \[[@B40]\] and references therein). These threading methods compare the query sequence (the target) with a library of structures (templates) and return 10 alignments that scored best according to the implemented criterion of compatibility. The results are evaluated by the Pcons server \[[@B41]\], which compares the models and the associated scores and produces a ranking of potentially best predictions (target-template alignments). Based on the results produced by the MetaServer, homology modeling was carried using the SWISS-MODEL/PROMOD II server \[[@B42]\]. Model evaluation was carried out using the PROSA II \[[@B43]\] program integrated with PROMOD II, suggesting that the stereochemistry and energetic parameters of the models were acceptable.

Acknowledgements
================

I would like to thank Jeff Elhai, who as a non-anonymous reviewer provided extensive comments, and raised constructive criticism of the \"cut-and-paste\" mechanism, which lead to considerable improvement of the manuscript, Dr. Ichizo Kobayashi for critical reading and comments on the initial version of the manuscript and Drs. Murat Abdurashitov and Sergey Degtyarev for supplying unpublished data. This work was supported by KBN (grant 6P04 B00519).


Background
==========

Respiratory syncytial virus (RSV) belongs to the Pneumovirinae subfamily of the Paramyxovirodae family of enveloped single-stranded negative sense RNA viruses. RSV infection of the lower respiratory tract cells results in cell death and sloughing into the lumen of the respiratory tree. Worldwide, RSV is the leading cause of infant mortality from respiratory infections and is so highly contagious that by age two nearly all children have been infected. RSV infection in infancy cause severe bronchiolitis and pneumonia and may predispose children to the subsequent development of asthma, the most common chronic illness of childhood \[[@B1]\]. Many studies have indicated that chemokines can play an important role in the onset and severity of asthma and it has been shown that RSV infection of lung epithelial cells increases chemokine production, although the mechanisms involved are largely unknown \[[@B2]-[@B5]\].

The chemotactic cytokines, or chemokines, compose a large superfamily of small structurally related polypeptides that play important roles in host defense by recruiting specific subsets of leukocytes to sites of inflammation and injury \[[@B6]\]. Chemokines have been associated with a number of inflammatory diseases and conditions, including asthma, sepsis, inflammatory bowel disease, and adult respiratory distress syndrome \[[@B7]-[@B9]\]. The chemokine superfamily can be divided into two major groups based on the position of the first two of four-conserved cysteine residues at the amino terminus, which are either adjacent (CC subfamily) or separated by one amino acid (CXC subfamily). The CXC chemokines such as IL-8 were originally identified as potent activators and chemoattractants for neutrophils, whereas the CC chemokines such as MCP-1 and RANTES mostly attract monocytes and eosinophils respectively \[[@B10]\]. Chemokines are secreted in a stimulus-and cell type-specific manner \[[@B11]-[@B17]\] and are regulated primarily at the level of gene transcription \[[@B18]-[@B24]\]. The transcriptional promoters of IL-8, RANTES and MCP-1 contain binding sites for the redox-responsive transcription factor NF-κB, which has been shown to be important for their regulation by viral infections and cytokines \[[@B18],[@B20],[@B23],[@B25]-[@B34]\].

We previously demonstrated that the chemokines IL-8, MCP-1 and RANTES are differentially regulated in A549 airway epithelial cells \[[@B35]-[@B38]\]. To further elucidate the mechanisms of chemokine expression in A549, we have compared the induction of IL-8, MCP-1 and RANTES by RSV infection with that of TNFα. Our findings suggest that RSV induction of chemokine gene expression involves a redox-sensitive NF-κB signaling mechanism that differs from that mediated by TNFα and involving predominantly the Rel A subunit of NF-κB.

Materials and methods
=====================

Materials
---------

Dulbecco\'s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), Dulbecco\'s phosphate buffered saline (DPBS), antibiotic/antimycotic, 1% trypsin/EDTA, Hanks Balanced Salt Solution (HBSS) and TRIZOL were purchased from Invitrogen Gibco Cell Culture (Carlsbad, CA). N-acetyl-L-cysteine, dexamethasone, glycerol and MTT tetrazolium salt were obtained from Sigma (St. Louis, MO). TNFα was obtained from R&D systems (Minneapolis, MN). ELISA kits were purchased from Pierce Endogen (Rockford, IL). Human CK5 RiboQuant ribonuclease protection assay kit was purchased from BD Pharmingen (San Diego, CA). \[α-^32^P\]UTP (250 μCi) was obtained from Perkin Elmer Life Sciences (Boston, MA). Gel shift assay system was purchased from Promega (Madison, WI). \[γ-^32^P\]ATP (500 μCi) was obtained from ICN (Costa Mesa, CA). Antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The A549 cell line and RSV Long strain were obtained from the American Type Culture Collection (Rockville, MD).

Virus stock growth and maintenance
----------------------------------

RSV, Long strain, was grown on HEp-2, a human tracheal epithelial cell line. Cells were grown to 50% confluence in DMEM containing 7% FBS and 1% antibiotic/antimycotic. After two washes with 1X DPBS, a minimal volume of RSV, at a multiplicity of infection (MOI) of 1 or greater, containing less than 1% FBS was added. The virus and cells were incubated for 2 hours, after which DMEM was added to bring culture to normal growth volume and 7% FBS. Cultures were then incubated for 48 hours. All plates were scraped and contents were transferred to 50 ml conical tubes. The virus/cell cocktail was vortexed briefly and large debris was removed in a tabletop centrifuge. RSV was purified by ultracentrifugation through 30% glycerol in HBSS. RSV stocks were resuspended in HBSS containing 0.5% bovine serum albumin and 100 mM magnesium sulfate and frozen at -70° C.

Cell culture, infection and treatments
--------------------------------------

The A549 human type II lung carcinoma cell line was grown and maintained in DMEM containing 7% FBS and 1% antibiotic/antimycotic. For treatment, cells were grown to approximately 75% confluence, washed once with 1X DPBS and incubated overnight with DMEM containing 1% FBS. Cultures were then washed twice with 1X DPBS and pretreated with inhibitors DEX or NAC in serum-free DMEM to final concentrations as indicated in the figures. RSV and TNFα were added to the DMEM/inhibitor mix to final concentrations as indicated in the figures. One hour after agonist addition, FBS was added to 1% concentration for the duration of the experiment.

Protein secretion
-----------------

After addition of RSV and TNFα, cells were cultured for 24 h. Supernatants were collected and spun at high speed to remove cellular debris. The resulting suspension was frozen at -70°C until assayed following manufacturer\'s instructions by chemokine-specific ELISA (Endogen).

RNase protection
----------------

RNase protection assays (RPA) were performed using an RPA kit purchased from BD Pharmingen. In brief, total RNA was isolated from stimulated A549 cells with TRIZOL. The hCK-5 multiprobe template set was used to synthesize RNA probes for the chemokines lymphotactin, RANTES, IP-10, MIP-1α, MIP-1β, IL-8, MCP-1, and I-309 as well as the house-keeping genes L32 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) labeled with \[α-^32^P\]UTP using T7 RNA polymerase. 3 × 10^5^ cpm of labeled probe were hybridized with 10 μg of total RNA for 16 h at 56°C. MRNA-probe hybrids were treated with RNase cocktail and phenol-chloroform extracted. Protected hybrids were resolved on a 6% denaturing polyacrylamide sequencing gels and exposed to a Molecular Dynamics detection screen overnight. Laser densitometry was performed using a Molecular Dynamics Storm scanner system (Molecular Dynamics, Inc., Sunny vale, CA).

DNA binding studies
-------------------

Electrophoretic mobility shift assays (EMSA) were performed essentially as described previously \[[@B39]\]. Briefly, nuclear protein extracts (7 μg protein) prepared form A549 cells by the method of Osborn *et al*\[[@B40]\] were incubated with 5 × 10^5^ cpm (\~0.1 ng) of ^32^P end-labeled consensus NF-κB oligonucleotide (5\'-AGTTGAGGGGACTTTCCCAGGC-3\') probe contained in the Promega gel shift assay system for 20--30 min at room temperature in 10 μl reaction volume using the kit provided reaction buffer. To demonstrate binding specificity, 100-fold molar excess (10 ng) of a specific or non-specific oligonucleotide (5\'-ATTCGATCGGGGCGGGGCGAGC-3\') was included in the binding reaction. Protein-DNA and protein-DNA-antibody complexes were resolved in 5% polyacrylamide gels preelectrophoresed for 30 min at room temperature in 0.25X TBE buffer (22.5 mM Tris-borate and 0.5 mM EDTA, pH 8.3). Gels were dried and exposed to radiographic film with an intensifying screen at -70°C.

NF-kappaB subunit specific ELISA
--------------------------------

NF-κB subunits Rel A (p65) and NF-κB1 (p50) were quantified using Trans-AM™ transcription factor assay kit from Active Motif (Carlsbad, CA). The assay is essentially an ELISA in which the consensus NF-κB binding site (5\'-GGGACTTTCC-3\') is immobilized onto the 96 well plate. Nuclear cell extracts (3 μg) were added to the wells and assayed for either Rel A or p50 binding as per the manufacturer\'s instructions. Optical density was determined on a spectophotometer at 450 nm.

Results
=======

RSV induction of chemokine expression differs from that induced by TNFalpha
---------------------------------------------------------------------------

As shown in Fig. [1](#F1){ref-type="fig"}, RSV infection of A549 epithelial cells induces the production and secretion of the chemokines IL-8, MCP-1, and RANTES. In A549 epithelial cells substantial MCP-1 was constitutively expressed whereas little or no spontaneous IL-8 or RANTES was detected. However, upon RSV infection (MOI = 3) a significant increase in chemokine production and secretion was observed 24 hr post-infection for each of the chemokines.

![**RSV induces chemokine protein secretion from A549 cells.** 2 × 10^5^ A549 cells were infected by RSV (MOI = 3) for 24 hr and supernatants assessed for IL-8, MCP-1 and RANTES by chemokine specific ELISA. The histogram shows the mean pg/ml of protein detected from three independent experiments. The error bars show the standard deviation from the mean.](1471-2334-2-5-1){#F1}

To determine whether the chemokine induction was mediated by increased chemokine gene expression we assessed the kinetics of mRNA synthesis by chemokine specific RNase protection. A549 cells were infected with RSV (MOI = 1) or stimulated with TNFα (100 ng/ml) and total RNA isolated at various times post-treatment over a 24 hr time course. As shown in Fig. [2](#F2){ref-type="fig"}, RSV rapidly induced IL-8 and MCP-1 with steady-state mRNA readily detected as early as 1 hr and continued to increase over the 24 hr time course. In contrast, RANTES mRNA was not detected until 4 hrs post infection after which its expression rapidly increased also reaching a maximum at 24 hrs. The RSV induction kinetics differed dramatically from the induction kinetics for TNFα in the same experiment. Whereas RSV induction of the chemokines did not reach a maximum until 24 hrs, TNFα induction was maximal between 4 and 8 hrs and was either unchanged or lower at 24 hrs post-infection.

![**Chemokine mRNA expression induced by RSV and TNFα.** 2 × 10^7^ A549 cells were either infected by RSV (MOI = 1) or stimulated with 100 ng/ml TNF-α. Total RNA was isolated at 1,2, 4, 8, and 24 h and assessed for chemokine expression by RNase protection. Chemokine expression was quantified by densitometry as described in the Methods. The graphs show the time course of chemokine mRNA after normalizing to GAPDH (lowest band in the gel). Shown is representative of 3 independent experiments.](1471-2334-2-5-2){#F2}

The magnitude of induction also differed between TNFα and RSV infection. In general TNFα was a stronger inducer of chemokine expression. This difference was also reflected in the amount of chemokine secreted from the cells (data not shown). Together, these data indicate that RSV infection of A549 epithelial cells induces chemokine expression with distinct kinetics suggesting the mechanism of RSV induction differs from that of proinflammatory cytokines.

NF-kappaB inhibitors differentially effect RSV and TNFalpha induction of chemokine expression
---------------------------------------------------------------------------------------------

IL-8, MCP-1 and RANTES gene expression are regulated by the redox responsive transcription factor NF-κB \[[@B18],[@B20],[@B23],[@B25]-[@B33],[@B41]-[@B44]\]. The NF-κB signaling pathways have been shown to be differentially affected by antioxidants and glucocorticoid steroids \[[@B45]\]. To determine whether RSV and TNFα induction of chemokine expression might be mediated by different NF-κB signaling pathways we pretreated A549 cells with the glutathione precursor N-acetyl-L-cysteine (NAC) or the synthetic glucocorticoid dexamethasone (DEX). Twenty-four hours post infection, total RNA was isolated and steady state mRNA expression analyzed by chemokine specific RNase protection. As shown in Fig. [3](#F3){ref-type="fig"}, the two inhibitors had very different effects on chemokine gene expression. Most strikingly, RSV induced chemokine expression was more sensitive to NAC, whereas TNFα induced chemokine expression was conversely more sensitive to DEX. Thus, NAC preferentially inhibited chemokine expression induced by RSV, while DEX predominantly inhibited chemokine expression induced by TNFα.

![**Chemokine mRNA inhibition in A549 cells.** 2 × 10^7^ A549 cells were pretreated with 5 mM N-acetyl-L-cysteine (NAC) or 500 nM dexamethasone (DEX). After 1 hr the pretreated cells were either infected with RSV (MOI = 1) or stimulated with TNFα (100 ng/ml) for 24 hr. The graphs show the percent inhibition for each chemokine mRNA after normalization to GAPDH. Shown is representative of two independent experiments.](1471-2334-2-5-3){#F3}

Differential inhibition of TNFalpha and RSV induced NF-kappaB
-------------------------------------------------------------

NF-κB binds to the chemokine promoter as either homo- or heterodimers composed of Rel A (p65) and NF-κB1 (p50). Initially we detected TNFα and RSV induction of NF-κB by EMSA (KA Roebuck and LR Carpenter unpublished data) but because multiple gel shift complexes were induced it was not possible to distinguish the individual contributions of Rel A and p50 to the formation of these complexes. Therefore to assess the induction of the Rel A and p50 subunits individually in A549 nuclear cell extracts, we used a subunit specific NF-κB binding ELISA system that utilizes anti-Rel A and anti-p50 antibodies. As shown in Fig. [4](#F4){ref-type="fig"}, Rel A and p50 subunits of NF-κB are differentially expressed and induced in A549 epithelial cells. In untreated cells, no Rel A (solid bars) was detectable whereas in resting cells constitutive levels of p50 were observed (open bars), suggesting that unstimulated A549 epithelial cells contain p50 homodimers, which with regard to transcriptional activity have been shown to be functionally inert or inhibitory. In contrast, cells stimulated by TNFα (Fig. [4A](#F4){ref-type="fig"}) or infected by RSV (Fig. [4B](#F4){ref-type="fig"}) markedly induced Rel A, the transcriptionally active subunit of NF-κB. In addition, TNFα was also able to induce p50 by about 5-fold whereas RSV increased the constitutive p50 level by only about 50%. The induction of Rel A and p50 were specific since competition with either wild type or mutant consensus NF-κB oligonucleotide respectively inhibited or not the RSV and TNFα induced binding activity. These data demonstrate that TNFα is a potent inducer of both Rel A and p50 whereas RSV primarily induces the Rel A subunit of NF-κB.

![**Induction of Rel A (p65) and NF-κB1 (p50) by RSV and TNFα is differentially inhibited by NAC and DEX.** 2 × 10^7^ A549 cells were pretreated with 5 mM N-acetyl-L-cysteine (NAC) or 500 nM dexamethasone (DEX). After 1 hr the pretreated cells were either stimulated with TNFα (100 ng/ml) or infected with RSV (MOI = 1) for 2 hr. Nuclear extracts (3 μg) were prepared and assessed for Rel A (p65, solid bars) or NF-κB1 (p50, open bars) binding activity using a Trans-AM™ transcription factor assay kit from Active Motif (Carlsbad, CA) as per the manufacturer\'s instructions. Graph shows the NF-κB activation results (OD~450nm~) from A549 cells stimulated with TNFα (A) or infected with RSV (B). An excess of either wildtype (wt) or mutant (mut) NF-κB oligonucleotide provided by the kit was included in the binding reactions to demonstrate NF-κB binding specificity. Note that NAC and DEX inhibits RSV induced p65 and p50 whereas the inhibitors had no effect on TNFα induced NF-κB binding activity.](1471-2334-2-5-4){#F4}

To determine whether RSV and TNFα activate NF-κB differently, we examined the effects of NAC and DEX on Rel A and p50 binding activity. As shown in Fig. [4](#F4){ref-type="fig"}, Rel A and p50 subunits are differentially inhibited by NAC and DEX which have been shown to inhibit NF-κB by distinct mechanisms. Both NAC and DEX were unable to inhibit TNFα induction of either Rel A or p50. In contrast, in RSV infected cells both DEX and NAC inhibited Rel A and p50 binding activity. However, the inhibitory effects were most striking for Rel A suggesting the inhibitors primarily target the RSV induction of Rel A.

The differential effects of NAC on TNFα and RSV induced NF-κB binding activity correlated with its effects on chemokine gene expression (Fig. [3](#F3){ref-type="fig"}) and protein secretion (data not shown) suggesting that RSV induces IL-8, MCP-1 and RANTES through redox-sensitive NF-κB binding complexes. DEX also showed differential effects on NF-κB binding activity with DEX preferentially inhibiting RSV induction of NF-κB. However, the effects of DEX on NF-κB binding induced by TNFα did not correlate with its effects on chemokine expression, suggesting in TNFα activation of chemokine gene expression DEX targets another mechanism of chemokine induction not involving NF-κB.

Discussion
==========

In this study, we examined the role of NF-κB in the activation of chemokine gene expression in response to TNFα and RSV infection. The inhibitor studies correlating chemokine gene expression and NF-κB binding activity indicate that RSV, in contrast to TNFα, induces IL-8, MCP-1 and RANTES expression in A549 epithelial cells through a redox-sensitive NF-κB signaling pathway that appears to involve predominately Rel A. IL-8, MCP-1 and RANTES are known redox regulated genes and our results are consistent with the findings that oxidant tone can regulate chemokine gene expression in RSV infected airway epithelial cells \[[@B46]-[@B48]\].

NF-κB activation is controlled by its inhibitory protein I-κBα which when phosphorylated on ser32 and ser36 marks it for degradation by the 26S proteosome complex. Thus I-κB is a potential regulatory target for the differential induction of NF-κB. Although we have not directly examined the role of I-κB, Fiedler *et al*\[[@B49],[@B50]\] have reported differential effects of RSV and TNFα on I-κB activity in A549 epithelial cells. In addition, Bitko and Barik \[[@B51]\] have demonstrated that another I-κB protein (I-κBβ) may also contribute via a redox-sensitive pathway to NF-κB activation by RSV.

In addition to NF-κB dependent regulation there appears to also be NF-κB independent activation of chemokine expression since DEX inhibited TNFα induced chemokine expression but not NF-κB binding activity. Along these lines, it has been shown that RSV activation of chemokine gene promoters involves multiple inducible transcription factors including AP-1 \[[@B52],[@B53]\], which is also a redox sensitive transcription factor important in chemokine gene expression \[[@B28],[@B36],[@B54]\]. AP-1 can also be inhibited by DEX and has been shown to cooperate with NF-κB to mediate RSV induction of IL-8 \[[@B46],[@B55]\].

The differential effects of NAC and DEX on chemokine gene expression and NF-κB activation suggest that the mechanisms of RSV and TNFα activation of NF-κB and chemokine expression differ possibly involving distinct NF-κB signaling mechanisms. TNFα rapidly and potently activates Rel A and p50 subunits of NF-κB through a kinase-mediated phosphorylation cascade involving a high molecular mass signaling complex called the IKK complex \[[@B56]\]. TNFα activation of the multiprotein IKK complex results in the serine phorsphorylation of I-κB and the subsequent activation of NF-κB \[[@B57]\].

Consistent with the idea of RSV and TNFα activating distinct NF-κB signaling pathways, we found that the NF-κB inhibitor NAC had differential effects on chemokine gene expression. In contrast to TNFα, RSV induced chemokine expression was sensitive to NAC particularly the Rel A (p65) subunit. NAC is an antioxidant thiol that raises the intracellular pool of glutathione the major redox regulating mechanism in the A549 epithelial cell line \[[@B45]\]. Interestingly, it has been reported that NAC can have differential effects on NF-κB binding activity suggesting only a subset of NF-κB binding complexes are redox sensitive \[[@B58]\]. Moreover, it has been shown that NAC inhibits Rel A/p50 heterodimers but not p50 homodimers and can inhibit NF-κB binding to only certain variant κB elements which presumably interact with distinct redox sensitive NF-κB binding complexes \[[@B59]\]. These observations are consistent with our findings that indicate NAC predominantly inhibits Rel A homodimers since that was the major NF-κB subunit induced by RSV infection of A549 cells.

Consistent with RSV and TNFα inducing functionally distinct NF-κB binding complexes, AP-1 was recently shown to be the preferential cooperative partner with NF-κB in RSV-induced IL-8 expression \[[@B46]\], whereas in TNFα-induced expression NF-κB cooperates preferentially with NF-IL-6 \[[@B18],[@B19],[@B22]\]. Apparently, the stimulus-specific gene regulation of chemokines is complex involving differential cooperativity of redox sensitive and redox resistant NF-κB complexes with other transcription factors to form unique higher order transcription complexes.

Conclusions
===========

Taken together, our data indicate that RSV, in contrast to the cytokine TNFα, activate NF-κB and chemokine gene expression through a redox sensitive NF-κB signaling pathway involving predominately Rel A.

Competing interests
===================

None declared.

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

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Acknowledgments
===============

We thank Mark Peeples and Larry Thomas for reading the manuscript prior to submission. This work was in partial fulfillment of the doctoral degree of Laura R. Carpenter and was supported by a National Institutes of Health grant AR45835 and grants from the American Lung Association (Blowitz-Ridgeway Foundation).