Delayed apoptosis by neutrophils from COPD patients is associated with altered bak, bcl-xl, and mcl-1 mRNA expression

Jisong Zhang
†
Jian He
†
Jingwen Xia
Zhen Chen
Xiaodong Chen
Delayed apoptosis by neutrophils from COPD patients is associated with altered bak, bcl-xl, and mcl-1 mRNA expression
R E S E A R C H Open Access
Background: Delayed neutrophil apoptosis may be an important factor in the persistent inflammation associated with chronic obstructive pulmonary disease (COPD). Bcl-2 family proteins are important regulators of neutrophil apoptosis. We determined the mRNA levels of pro-apoptotic Bak and anti-aptototic Bcl-xl and Mcl-1 members of the Bcl-2 family in unstimulated peripheral blood neutrophils from patients with mild to moderate COPD and compared these to neutrophils from healthy controls. Methods: Neutrophils were isolated from peripheral blood samples of 47 COPD patients (smokers: N = 24) and 47 healthy controls (smokers: N = 24). Percentages of apoptotic cells were determined at 4, 24, and 36 h for unstimulated neutrophils cultured in vitro. Neutrophil mRNA expression of Bak, Bcl-xl, and Mcl-1 was determined by real-time polymerase chain reaction (PCR). FEV1 (% predicted) and FVC were determined by spirometry and correlations between mRNA levels and lung function parameters were determined.Results: The percentages of apoptotic cells among unstimulated neutrophils from COPD patients were significantly lower compared to cells from controls after 4, 24, and 36 h in culture; smoking history had only a minimal effect on these differences. Unstimulated neutrophils from COPD patients had significantly lower Bak mRNA expression and higher expressions of Bcl-xl and Mcl-1 mRNA than cells from healthy controls. Again, smoking history had only a minimal effect on these trends. Bak mRNA expression was significantly positively correlated with both % predicted FEV1 and the FEV1/FVC ratio, while Bcl-xl and Mcl-1 mRNA expressions were significantly negatively correlated with %predicted FEV1 and the FEV1/FVC ratio. Conclusions: The genes for pro-apoptotic Bak, and anti-apoptotic Bcl-xl and Mcl-1 may be important in regulating the delayed neutrophil apoptosis observed in COPD, which may contribute to COPD pathogenesis.Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/ vs/1605269445677066

Introduction

Chronic obstructive pulmonary disease (COPD) is characterized by irreversible airflow limitation [1]. COPD is also characterized by an abnormal inflammatory response to noxious gases, especially cigarette smoke, and these abnormal inflammatory responses exacerbate airflow obstruction [2]. An inflammatory pulmonary infiltrate comprised primarily of neutrophils is important in COPD pathogenesis. Delayed cellular apoptosis can prolong the life of neutrophils and can lead to their accumulation, resulting in a persistent inflammation in the lungs and airway, which is considered to be a critical step in COPD pathogenesis [3]. Aggregation of neutrophils in the lungs and airway is another characteristic of COPD [4][5][6].

Although a number of studies have confirmed the delayed apoptosis of neutrophils in the peripheral circulation, and in induced sputum and bronchoalveolar lavage fluid (BALF) samples during the development of COPD [7][8][9][10], the mechanisms underlying this phenomenon are not clear. One report found no relationship between serum or sputum cytokines and the rate of neutrophil apoptosis during COPD exacerbations [11]. However, Brown et al. found that neutrophil apoptosis was reduced in both stable COPD subjects and in healthy controls who were smokers, which might have been attributed to the activation of NFκB [12].

Some genes may increase the risk of COPD development in certain populations. Ning et al. found 6 genes whose expressions were different in mild COPD patients who were smokers and in smokers without lung injury [13]. Thus, we hypothesized that delayed neutrophil apoptosis was caused by differences in the expressions of genes involved in the apoptotic regulatory pathway.

Members of the Bcl-2 protein family are part of the mitochondrial intrinsic apoptotic pathway, and it has been suggested that regulation of neutrophil apoptosis primarily involves members of this family [14]. Human neutrophils express many members of the Bcl-2 family, including the pro-apoptotic proteins Bax, Bid, Bak, and Bad, and the anti-apoptotic proteins Mcl-1, A1, and, Bcl-xl [15,16].

We hypothesized that a reason for the delayed neutrophil apoptosis observed in COPD is the abnormal expression of Bcl-2 family members. For this study, we selected genes for the pro-apoptotic protein Bak and the anti-apoptotic proteins Bcl-xl and Mcl-1, as they have been considered to be critical genes in regulating neutrophil apoptotic pathways [14,15]. We examined the mRNA expressions of these genes in unstimulated peripheral blood neutrophils from patients with mild to moderate COPD and healthy volunteers to investigate the relationship between Bcl-2 family gene regulation, delayed neutrophil apoptosis, and COPD pathogenesis.

Methods

Subjects

All COPD patients in this study were in a stable stage and were recruited from the out-patient clinic of our hospital. These patients had mild-to-moderate COPD (total: n = 47; smokers: n = 24). The categorization of mild-to-moderate COPD was based on the Global Initiative on Chronic Obstructive Lung Disease (GOLD) criteria (http://www.goldcopd.org). Patients who were possibly asthmatic were excluded based on the criteria of the Global Initiative on Asthma (GINA; http://www. ginaasthma.org).

We also recruited age-matched healthy controls (total: n = 47; smokers: n = 24). For both COPD patients and healthy controls, smoking history was recorded in packyears. Exclusion criteria for all subjects were history of allergy, tuberculosis, neoplasm, asthma, thoracic or abdominal surgery, or other serious concomitant diseases. No subjects had had an infection of the respiratory tract for at least 3 months, and none were currently being treated with antibiotics or steroids. The study was approved by the ethics review committee of Huashan Hospital, Fudan University and was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. All patients gave written informed consent.

Lung function determinations

Lung function was determined in the Lung Function Laboratory according to standard protocols using a pneumotachograph (Jaeger MS Diffusion, Würzburg, Germany). A subject breathed smoothly several times, and then inspired forcibly until total lung capacity was achieved. Subsequently, the subject expired forcibly until reaching his/her residual capacity. Then, the subject inspired until total lung capacity was achieved followed by expiration. Results for the % predicted forced expiratory volume in one second (FEV1) and the forced vital capacity (FVC) were generated automatically.

COPD patients were asked to stop taking long-acting aminophylline 48 h or oral aminophylline 12 h before the examination and stopped using any inhaled β−agonist 6 h before the examination. If the FEV1/FVC was < 70%, patients were recommended to inhale salbutamol (Ventolin) (200 μg × 2 puffs), and spirometry was repeated 15 min later. An increase in FEV1/FVC of ≥ 12% and an absolute increase of FEV1 ≥ 200 ml were regarded as positive for bronchodilation.

Isolation of peripheral blood neutrophils

Neutrophils were isolated from peripheral blood samples collected in sterile heparinised tubes by a two-step procedure. First, erythrocytes were removed by sedimentation using dextran (T500, Pharmacia). Next, granulocytes were isolated using discontinuous Percoll gradient centrifugation [17]. We used 0.9% saline to prepare 60% and 70% (by volume) Percoll solutions (densities of 1.079 g/ml and 1.091 g/ml, respectively) from a stock solution of 1.130 g/ml (100% fine grade Percoll, Pharmacia). After centrifugation, the density of peripheral blood granulocytes, primarily neutrophils, in the Percoll solution was between 1.080 to 1.085 g/ml. The isolated cells were determined to be > 98% neutrophils by Giemsa staining.

Neutrophil apoptosis assay

Neutrophils were suspended to a concentration of 5 × 10 5 cells/ml in RPMI 1640 medium supplemented with 10% FCS and 100 U/ml of penicillin-streptomycin, and 200 μl of this suspension was pipetted into each well of a 36 well flat-bottom microtest plate. Unstimulated cells were cultured at 37°C in a 5% CO 2 atm for 4, 24, or 36 h, after which the percent of apoptotic cells was determined using previously described methods [18]. Briefly, after the indicated culture time, cells were stained with a FITC-Annexin V antibody (Abcam) followed by staining with propidium iodide (PI). After washing in PBS, cells were evaluated by flow cytometry using a FACScan (Becton-Dickinson, San Jose, CA). Forward and side scatter gates were set to evaluate neutrophils (granulocytes) only. The percent of cells that were double-positive for Annexin V and PI were recorded as percent apoptotic cells.

RNA extraction from neutrophils and reverse transcription (RT)-PCR

Total RNA was extracted from neutrophils with RNAi-soTMPlus (TaKaRa Biotechnology) and reverse transcribed using PrimeScriptTM RT reagent kits (TaKaRa Biotechnology). Reactions mixtures were 8 μL of total RNA, 4 μL of 5 × PrimeScriptTM Buffer, 1 μL of Prime-ScriptTMRT Enzyme Mix I, 1 μL of oligo dT primer, 1 μL of random primer, and 5 μL of RNase-free deionised H 2 O.

Real-time quantitative PCR

Neutrophils' expressions of Bak, Bcl-xl, and Mcl-1 mRNA were determined by real-time quantitative PCR [19]. Realtime PCR used an Applied Biosystems 7500 Fast Real-Time PCR System with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal control. The gene sequences for Bak, Bcl-xl, Mcl-1, and GAPDH were from the NCBI database (http://www.ncbi.nlm.nih.gov/). Primer Express Version 3.0 (Applied Biosystems, America) was used to design specific primers (details in Table 1). SYBR Green Master Mix (Applied Biosystems, America) was used for real-time PCR, with initial denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. An identical threshold cycle (Ct) was used for each gene of interest. Relative mRNA expression levels were determined according to the 2 -ΔΔCT method [20].

Statistical analysis

Results for subjects' demographic and clinical characteristics are given as means ± standard deviations (SD) for continuous variables and n (%) for categorical variables. Results for mRNA expression and apoptosis percentages are given as means ± standard errors of the mean (SEM).

For continuous variables, groups were compared using a two-sample t-test or a Mann-Whitney U test if data were not normally distributed. A Pearson chi-square test was used for categorical variables. Spearman correlation analysis was used to evaluate associations between mRNA expression levels and lung function parameters. Statistical significance was set at P < 0.05. Statistical analyses used SPSS 15.0 software (SPSS, Inc, Chicago, IL, USA).

Results

Study subject characteristics

A total of 94 subjects were enrolled in this study. The demographic and clinical characteristics of the healthy controls (n = 47) and COPD patients (n = 47) are shown in Table 2. There were more males than females (40 vs. 7 in each group), and the COPD patients were significantly older, on average, than the controls (61.3 ± 4.5 years vs. 58.8 ±4.6 years; P = 0.011). Slightly more than 50% of the subjects were smokers in both groups. Thirty (63.8%) COPD patients used inhaled shortacting bronchodilators as needed and, among these patients, 18 also used inhaled long-acting bronchodilators.

Neutrophil apoptosis

Apoptosis was determined for unstimulated neutrophils in culture. Figure 1 shows the percentages of apoptotic peripheral blood neutrophils from COPD patients and healthy controls after different times in culture (4, 24, and 36 h.). Each subject group was also sub-divided into those with smoking and non-smoking histories. Compared to neutrophils from healthy controls, the percentages of COPD patients' apoptotic neutrophils were significantly lower after culture for 4, 24, and 36 h. A smoking history slightly increased the percent of COPD patients' apoptotic neutrophils, although the percent of apoptotic cells was still lower than for cells from controls who smoked. These results show that unstimulated neutrophils from COPD patients have significantly delayed apoptosis relative to neutrophils from healthy controls.

Neutrophil mRNA expression of apoptosis-related genes Figure 2 shows the relative expressions of Bak, Bcl-xl, and Mcl-1 mRNA's by unstimulated peripheral blood neutrophils that were analyzed using real-time PCR. In neutrophils from COPD patients, Bak mRNA expression was significantly reduced and the expressions of Bcl-xl

Lung function and neutrophil mRNA expression of apoptosis-related genes

As shown in Table 2, spirometric lung function parameters were significantly worse for COPD patients than for healthy controls, including % predicted FEV 1 and the FEV 1 /FVC ratio. We used Spearman correlation analysis to determine if these lung function parameters were associated with neutrophils' mRNA expression of apoptosisrelated genes. Figure 3 shows the correlations between the mRNA expression levels for Bak ( Figure 3A), Bcl-xl  Figure 1 Comparisons of percentages of apoptotic neutrophils from healthy controls and COPD patients. Peripheral blood neutrophils were isolated from healthy controls and COPD patients; these groups were sub-divided into those with and without smoking histories. As described in Methods, unstimulated peripheral blood neutrophils were cultured in vitro for 4, 24, or 36 h, after which the percent of apoptotic cells was determined. Results are means ± SEMs for: COPD/smokers (n = 24); COPD/non-smokers (n = 23); Healthy/smokers (n = 24); and Healthy/ non-smokers (n = 23). Group comparisons were by Mann-Whitney U test. * significant difference as compared with Healthy/non-smokers (p < 0.05); † significant difference as compared with Healthy/smokers (p < 0.05); { significant difference as compared with COPD/non-smokers (p < 0.05).

( Figure 3B), and Mcl-1 ( Figure 3C) and lung function. These correlation results were not affected by smoking status (data not shown); thus, these correlations are shown by COPD status only without stratifying according to smoking status. Bak mRNA levels were significantly positively correlated with lung function parameters, and Bcl-xl and Mcl-2 mRNA levels were significantly negatively correlated with lung function parameters.

Discussion

The aim of this study was to investigate whether changes in gene expressions for members of the Bcl-2 family of pro-and anti-apoptotic proteins were involved in the delayed neutrophil apoptosis seen in COPD. We found that, in peripheral blood neutrophils from subjects with stable mild to moderate COPD, mRNA expressions for two anti-apoptotic genes (Bcl-xl and Mcl-1) were upregulated and that for a pro-apoptotic gene (Bak) was downregulated ( Figure 2). Our findings showed that the abnormal expression of Bcl-2 family member genes was present in an early stage COPD and that this trend of abnormal gene expression was consistent with the delayed apoptosis seen in neutrophils from COPD patients (Figure 1). We also found that these mRNA expression levels were significantly correlated with lung function parameters (Figure 3). In our study, after 36 h in unstimulated culture, the apoptosis rate of peripheral blood neutrophils of COPD patients was significantly delayed relative to neutrophils from healthy controls. For peripheral blood neutrophils, others have reported decreased apoptosis during COPD exacerbations, but not in stable COPD [8,9]. Using cells from sputum samples, neutrophil apoptosis for COPD patients who were current smokers was reported to be only one-third of that seen in sputum samples from healthy non-smokers. 12 This difference in results between neutrophils from peripheral blood and sputum is presumably due to the additional effect on sputum neutrophils of the chronic inflammation present in the lungs of COPD patients, particularly those who smoke.

In our study, the differences between COPD patients and controls in neutrophil mRNA levels of Bcl-2 family genes were greater than the differences seen in the percentages of apoptotic neutrophils. The mRNA levels for the two antiapoptotic genes (Bcl-xl and Mcl-1) were twice as high as in controls, and the mRNA level of the pro-apoptotic gene (Bak) was 60% of that in controls' neutrophils.

The Bcl family contains pro-and anti-apoptotic proteins that are found in the outer membranes of mitochondria. The balance between the pro-and anti-apoptotic members of this family is the central factor in the opening and closing of permeability transition (PT) pores [21], which is a critical pathway for cytochrome c release from mitochondria [22] and causes apoptosis by activating caspase-9. The increase in anti-apoptotic mRNA expression and decrease in pro-apoptotic mRNA expression seen in our study could tip the balance toward neutrophil survival and decrease the apoptosis rate. and Healthy/non-smokers (n = 23). Groups were compared using two-sample t-test for Bak and Mcl-1 mRNA's, and a Mann-Whitney U test for Bcl-x1 mRNA due to its non-normal distribution. * significant difference as compared with Healthy/non-smokers (p < 0.05); † significant difference as compared with Healthy/smokers (p < 0.05). However, it is the relative abundance of Bcl-2 family proteins, rather than their mRNA expression, that would ultimately regulate apoptosis, and protein levels do not always correspond to mRNA levels. This was reportedly the case for Bcl-xL. Moulding et al. [15], who studied peripheral blood neutrophils from normal subjects, detected Bcl-xL mRNA in these cells, but could not detect the corresponding protein. They also found that blocking gene transcription with actinomycin decreased Mcl-1 mRNA and protein, but had virtually no effect on Bcl-xL mRNA levels. Therefore, Bcl-xL may not be involved in the control of apoptosis in human neutrophils.

Although gene transcription is important in determining mRNA levels, the mRNA half-life is also important. The half-life of the mRNA for Mcl-1, an anti-apoptotic protein, is relatively short and the half-life of the mRNA for Bak, a pro-apoptotic protein, is relatively long [15]. Thus, Mcl-1, whose mRNA levels can change relatively rapidly, might be more important than Bak in the short-term control of apoptosis. A relationship between decreased Mcl-1 mRNA expression and an increase in neutrophil apoptosis has previously been reported for patients with sepsis [23], but until now, research on COPD patients has been lacking.

We found only minimal differences in Bcl-2 family mRNA expression between healthy smokers and healthy non-smokers, and between smoking and non-smoking COPD patients. This suggests that the effects of smoking are not evident in neutrophils from peripheral blood as would be expected in cells from BALF, especially in patients with mild to moderate COPD like those included in the current study. An alternative explanation would be that Bcl-2 member expression is implicated in COPD pathogenesis, but not through a pathway that is affected by smoking. In addition, COPD is the result of the interactions between genes and the environment [24,25]. Genes other than Bcl-2 family members could be affected by these factors.

More studies are needed to investigate the relationships between mRNA and protein levels, and to determine whether the results of Bcl family members in normal neutrophils apply to COPD patients. An in-depth study is also needed on the neutrophil apoptosis pathways that are influenced by cigarette smoking and the complex effects of nicotine on neutrophil apoptosis in vivo.

Conclusions

We evaluated associations between Bcl-2 family members mRNA expressions and delayed apoptosis for unstimulated peripheral blood neutrophils from COPD patients. Relative to neutrophils from healthy controls, pro-apoptotic gene Bak mRNA was significantly downregulated in COPD patients' neutrophils, whereas anti-apoptotic gene Bcl-xl and Mcl-1 mRNA's were significantly upregulated. In addition, Bak mRNA was significantly positively correlated with %predicted FEV1 and the FEV1/FVC ratio, while Bcl-xl and Mcl-1 mRNA's were negatively correlated with these parameters. The trend for abnormal mRNA expression for members of the Bcl-2 family was consistent with the decline in lung function in COPD. Therefore, the most likely neutrophil apoptosis pathway in COPD patients may be the mitochondrial pathway, which could be a new target for future COPD research.

(See figure on previous page.) Figure 3 Correlations between mRNA levels of Bak, Bcl-xl, and Mcl-1 and lung function parameters. COPD patients are represented by solid circles and healthy controls by open circles (N = 47 for each group) Spearman correlation analysis was used to assess associations between variables. (A) Bak mRNA level was positively correlated with lung function. FEV1/FVC%: r = 0.72; P < 0.001; FEV1% reference: r = 0.70; P < 0.001. (B) Bcl-xl mRNA level was negatively correlated with lung function. FEV1/FVC%: r = -0.82; P < 0.001; FEV1% reference: r = -0.86; P < 0.001. (C) Mcl-1 mRNA level was negatively correlated with lung function. FEV1/FVC%: r = -0.79; P < 0.001; FEV1% reference: r = -0.79; P < 0.001.

Bak 5 0 -
0CCCAGGACACAGAGGAGGTTT-3 0 5 0 -GCCTCCTGTTCCTGCTGATG-3 0 SenseAntisense 65 bp NM001188 Bcl-xl 5 0 -TGCGTGGAAAGCGTAGACAA-3 0 5 0 -ATTCAGGTAAGTGGCCATCCAA-3 0 SenseAntisense 75 bp NM138578 Mcl-1 5 0 -AGGCTGGGATGGGTTTGTG-3 0 5 0 -CACATTCCTGATGCCACCTTCT-3 0 SenseAntisense 65 bp NM021960 GAPDH 5 0 -AACAGCCTCAAGATCATCAGCA-3 0 5 0 -CATGAGTCCTTCCACGATACCA-3 0 SenseAntisense 102 bp M33197and Mcl-1 mRNA's were significantly increased compared to neutrophils from controls. As with neutrophil apoptosis, a history of smoking had an effect on mRNA expression; however, these differences between mRNA expressions remained significantly different between COPD patients and controls regardless of smoking history. In COPD patients' neutrophils, mRNA expression for the two anti-apoptotic genes, Bcl-xl and Mcl-1, was almost twice that of controls (Bcl-xl:

Figure 2
2Expression of Bak, Bcl-xl, and Mcl-1 mRNA's in peripheral blood neutrophils. Real-time PCR was used to determine the expression of (A) Bak, (B) Bcl-xl, and (C) Mcl-1 mRNAs in peripheral blood neutrophils. The ΔΔCt value for each mRNA was normalized to the value of the GADPH housekeeping gene mRNA. Results are means ± SEMs for: COPD/smokers (n = 24); COPD/non-smokers (n = 23); Healthy/smokers (n = 24);

Figure 3 (
3See legend on next page.)

Table 1
1Nucleotide primer sequences used for real-time quantitative PCRTarget genes Nucleotide sequence
Orientation
Amplicon length Accession No.

Table 2
2Subjects' demographic and clinical characteristics Results are means ± SDs for †,} continuous variables and n (%) for { categorical variables. †{} Groups were compared using † two-sample t-test or } Mann-Whitney U test if data were not normally distributed.Variables
Healthy controls (n = 47)
COPD patients (n = 47)
P-value

Age (years)  †
58.8 ± 4.6
61.3 ± 4.5
0.011 *

Sex {
1.000

Males
40 (85.1%)
40 (85.1%)

Females
7 (14.9%)
7 (14.9%)

Smokers {
24 (51.1%)
24 (51.1%)
1.000

Smoking history (pack/year) }
31.9 ± 7.4
31.7 ± 6.0
0.755

FEV 1 /FVC %  †, }
86.6 ± 4.5
56.8 ± 5.3
<.001 *

FEV 1 ,% Reference  †, }
103.1 ± 9.5
59.4 ± 6.3
<.001 *

ISAB as needed {
0 (0)
30 (63.8)
-

ILAB regularly {
0 (0)
18 (38.3)
-

Abbreviations: COPD: chronic obstructive pulmonary disease; ISAB: inhaled short-acting bronchodilators; ILAB: inhaled long-acting bronchodilators.
 †,{,} { Pearson Chi-square test was used for
categorical variables.
* P < 0.05, significantly different between groups.

http://www.diagnosticpathology.org/content/7/1/65
AcknowledgementsThis study was supported by a grant from the Science and Technology Commission of Shanghai (No 074119508).Competing interestsThe author(s) declare that they have no competing interests.
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Delayed apoptosis by neutrophils from COPD patients is associated with altered bak, bcl-xl, and mcl-1 mRNA expression. Zhang, Diagnostic Pathology. 65Cite this article asCite this article as: Zhang et al.: Delayed apoptosis by neutrophils from COPD patients is associated with altered bak, bcl-xl, and mcl-1 mRNA expression. Diagnostic Pathology 2012 7:65.


THE POSSIBLE INFLUENCE OF RISK MANAGEMENT, FORECASTING, AND PERSONNEL TRAINING IN PHYSICAL ASSET MANAGEMENT

J S Schoeman
P J Vlok firstname.lastname@example.com
Department of Industrial Engineering
Stellenbosch University

Department of Industrial Engineering
Stellenbosch University
South Africa

THE POSSIBLE INFLUENCE OF RISK MANAGEMENT, FORECASTING, AND PERSONNEL TRAINING IN PHYSICAL ASSET MANAGEMENT
EB192D329E35B76859639096CA5C3792
The work presented in this paper highlights the possible effects of risk management, forecasting, and personnel training in the physical asset management environment.Each section is taken separately, and an argument is formed as to why it is important and what portion of the physical asset management five-year budget each should receive.This paper considers the long-term and short-term benefits in its conclusion that risk management should receive 60 per cent of the budget, personnel training 25 per cent, and forecasting the final 15 per cent.OPSOMMINGHierdie studie beklemtoon die moontlike effekte wat risiko bestuur, voorspelling van voorvalle, en die opleiding van personeel kan hê binne die fisiese bate bestuur omgewing.Die afdelings word elk apart bespreek, en 'n argument word gevorm om te sê waarom die afdeling belangrik is en watter bedrag van 'n maatskappy se vyf-jaar begroting elk moet ontvang.Die finale besluit was dat risiko 'n totaal van 60 persent van die begroting moet ontvang omdat dit so 'n breë afdeling is, die opleiding van personeel vereis 25 persent, en die voorspelling van gebeure die laaste 15 persent.AA

INTRODUCTION

Most, if not all, companies with physical assets that they use to create value have a limited budget to manage these assets.This paper takes a closer look at the physical asset management (PAM) environment, and advises the reader on how to divide the organisation's PAM budget into three critical areas.

Asset management (AM) is essential to ensuring equipment availability, quality products, on-time deliveries, and plant safety [1].AM is much more than just the maintenance of equipment; as mentioned in Hastings [17], it encompasses the following:

• identifying what assets are needed, • identifying funding requirements, • acquiring assets, • providing logistic and maintenance support systems for assets, and • disposing of or renewing assets.

This must all be done while still ensuring that the desired objectives are met and that the strategies used are in line with the business objectives [17].In more mature organisations, PAM has proven itself to be an investment and not just an expense [8].

There has been a great demand for PAM within organisations; and this realisation led to the creation of the Publicly Available Specification 55 (PAS 55) document.PAS 55 was created and published in 2004 by the British Standards Institute to provide a framework to manage mainly physical assets, although it can be applied to all asset types.The scope of those considered to be physical assets by PAS 55 [20] are shown in Figure 1: this gives the reader an idea of all the different areas to which PAS 55 can be applied.The document was created with asset-intensive organisations in mind.This framework is a continuous process, and the different processes can be grouped and described in the Deming Cycle structure (plan, do, check, and act): this helps to structure the AM system [20].AM is an on-going process, and manages the assets over their entire life cycle -something that many organisations do not understand.

The PAS 55 document proved so successful that an ISO standard (ISO 55000) was created and is currently in the final stages of being published.This will be an international standard for PAM, largely based on the PAS 55 document [2].Both documents are all about reducing and managing risk associated with the physical assets in an organisation.Thus risk management is a vital area within the PAM environment -although the entire PAM process can also be seen as risk management.In order to achieve AM excellence, risk management is one strategy used to identify and execute AM activities.Risk management alone has many supporting tools (activities executed to enforce risk management) that can be applied, but that are sometimes so specific that special expertise and their own budgets are needed.

One of these tools is the forecasting of events: it plays a large role in the modern PAM environment [3].The residual life of equipment is calculated to try to prevent costly failures from occurring.This makes use of condition monitoring (CM) to capture the data needed to make the predictions [9].Historical data then becomes the foundation of the future predictions, and so plays an important role.Even in the modern age it still happens that small organisations cannot afford the CM equipment, or the measuring equipment does not fit on to the equipment that needs to be measured.This then causes a lack of historical data, preventing the engineers from making predictions.Determining when a piece of equipment will break is a very important part of PAM, and can save organisations a lot of money.

Figure 1: Scope of PAS 55 [20]

The support infrastructure of the AM system is a large part of the system, and so it cannot be ignored.The correct training for personnel is essential to get the maximum efficiency from the PAM strategy.People and their leadership are just as important as the AM system itself, and training of personnel is a key factor to ensure that the required knowledge and skills are present [17].

The next section discusses risk management, its importance, and where it fits into PAS 55.The same is then done in Sections 3 and 4 to stress the importance of the different fields and to justify why they should receive a portion of an organisation's PAM budget.Section 5 presents the main reasons why it is a good idea that these specific areas should be funded by the PAM budget of an organisation.The last section then wraps up the discussions and presents what portions of the budget should be assigned to each area.

RISK MANAGEMENT

The decisions that are made on a day-to-day basis are based on the interpretation of the data that is currently available to the one deciding.It is known that data is full of uncertainty because of human error, biased readings, equipment error, etc. Unfortunately uncertainty is part of our everyday lives.Risk is defined in ISO 31000 as "the effect of uncertainty on objectives", whether these effects are positive or negative.The idea of risk management is not to eliminate risk, since this is impossible, but rather to decide which risks are worth taking and which ones we want to avoid completely [5,14].Risk management as described in Hubbard [4] is the identification, assessment, and prioritisation of risks, followed by coordinated and economical application of resources to minimise, monitor, and control the probability and/or impact of unfortunate events.Risk can be seen as the product of the probability of an event occurring and the consequences of that event.Hastings [17] states that ranking the potential events can then help to identify, assess, and prioritise the risks involved.Risk management is a very broad field, and it should be carefully monitored and managed.

The risks faced in PAM include environmental or health and safety risks; intangible losses such as damage to the brand name or to the organisation's reputation can also be a result of some of the risks [17].It is important to manage the risks in PAM.It is a moral obligation to try to protect the people we work with, the environment we live in, and the future of the organisation.As with financial investments, different risks are considered when we want to do a long-term or a short-term assessment [5].Complying with the PAS 55 document also requires risk management: the document explicitly states in just over 71 per cent of its sections that some form of risk management should be done.

When managing physical assets, the keyword is reliability: equipment with a higher reliability means lower risk [6,7].The reliability of equipment has been proven to relate directly to the safety of workers, environmental incidents, and cost effectiveness [7].The relationship differs between industry types, but it is assumed that the effects only differ in magnitude and that there is never an inverse effect [6].The correct maintenance strategies will increase the reliability of equipment in a plant; and with increased reliability comes a higher use of assets.Moore's study [7] showed that fewer environmental incidents occurred as the number of assets used increased, and the number of injuries decreased as the number of preventative and predictive work orders increased.The same study also revealed that the injury rate decreased as the maintenance schedule compliance increased [7].This study indicates that risk can be reduced by applying AM activities in the appropriate areas.

There is no doubt that correctly managing risks will have a beneficial impact on the AM system of an organisation.Part of the risk management is selecting the correct AM activities and applying them.Forecasting of events and personnel training are also discussed in the context of risk management, but because they can have such a large impact in the PAM environment, and because they require so much attention, they are discussed separately.

A different study -carried out by the Aberdeen Group in the manufacturing industrycategorised organisations into three different groups: the best-in-class, the industry average, and the laggards.Shah and Littlefield [14] used two key performance indicators (KPIs) to separate the organisations: 'percentage unscheduled asset down time' and 'percentage production compliance'.The top 20 per cent of organisations are considered to be the 'best-in-class', while the bottom 30 per cent are the 'laggards' and the remaining 50 per cent are the 'industry average'.On average the best-in-class had 11 per cent lower asset down time and 14 per cent more products and processes in compliance [14].They achieved their best-in-class results by applying risk management strategies, such as creating or improving visibility into the top risks, providing real-time visibility into operations, and educating employees on how to manage operational risks [14].With this study, the relative value of risk management is seen and found to be rather substantial.

The strategies applied by the best-in-class organisations are another indication that the training of personnel should be looked at separately because it is capable of having a large financial benefit to the organisation.

Considering the evidence of the effects of a mature risk management system, it is worthwhile investing money into this field over a period of time.Many organisations see a five-year budget as short-term; this will most likely be the minimum consideration time when wanting to develop a more mature and therefore more effective and efficient risk management system.Having the PAM system of an organisation act as a financial benefit requires commitment and hard work, as explained in Fogel [8].

FORECASTING

Forecasting of events is a useful tool because it can be used to reduce unexpected asset down time, reduce the risk of costly and/or dangerous failures, and prevent redundant maintenance activities [9].The condition of certain machines and their components affects the quality of the products produced; therefore it is important to know the condition of the machines at any particular time.Forecasting of events is a tool used to support the risk management process, because it can help to determine the possible time of an unwanted event, allowing predictive maintenance to be done.Forecasting also helps to avoid unnecessary preventative maintenance actions, saving money that would otherwise be spent on replacing healthy equipment.Considering this, it can be seen that if implemented correctly, the forecasting of unwanted events can have a favourable impact on an organisation's balance sheet and reputation.Forecasting requires the aid of CM, which measures the current health of the equipment and provides analysts and engineers with the data needed to make predictions [9].

Earlier methods of maintenance, which were 'usage-based' (also known as preventative maintenance) and 'run-to-failure', did not consider the immediate condition of the equipment.Condition-based maintenance, also known as 'predictive maintenance', takes into consideration the current health of the equipment and uses the CM data to make predictions as to how much longer the equipment is likely to survive [10].This remaining life of the equipment is known as the 'residual life', and different mathematical models have been developed to predict the residual life of certain equipment with considerable accuracy [10].By knowing more or less when a piece of equipment is going to fail, the risk to the operators and to the production schedule can be calculated and analysed, helping to decide which actions should be taken.Here both functional breakdowns and physical breakdowns are considered to be failures.

Forecasting goes hand-in-hand with CM and data analysis, since it needs both fields.Expert opinion also plays a role in forecasting events: the best forecasting methods combine quantitative analysis with the opinion of experts in a certain field to make the prediction [11].If you are able to predict, for example, when the gearbox of just one haul truck running in a mine is going to break, it could save the mine the unplanned asset down time and also save the possible risk of an accident caused by the failure of the gearbox somewhere on-site.The relative value of the forecasting of events can differ greatly, depending on the equipment to which they are applied.Correct predictions can save an organisation the cost of something as small as a single bearing, up to something as big as the turbines used by ESKOM to generate South Africa's electricity.

Since forecasting requires data from CM and impacts on the maintenance method used, it overlaps different sections in the PAS 55 document [20].Forecasting affects Section 4.3. of PAS 55 [20] because it forms part of the AM strategy used, and it will affect the plans to maintain the equipment.Forecasting also falls within Section 4.4 because it acts as enabler and control measure for monitoring and controlling the equipment.Section 4.5 is about the implementation of plans, and the forecasting of events is an on-going process that should be conducted once all the measurements have been taken by the CM equipment [20].

Forecasting is also relevant in Section 4.6 in PAS 55 [20], because without the CM equipment, no predictions could be done: forecasting therefore plays a role in complying with PAS 55.The forecasting field is still relatively immature, on average, when looking at industry; the majority of organisations are only now starting to invest in the CM equipment, as this technology for making predictions is becoming increasingly cheaper [12].

PERSONNEL TRAINING

All personnel, from the operator to the technician responsible for the CM of equipment and the technician responsible for maintenance, must have the proper training and skills to perform their tasks.They should also have the ability to know when a piece of equipment, on which they are not expert, is malfunctioning.This is difficult to enforce because most people today only focus on carrying out their specific task.They do not worry about anything that is not specifically stated on their job card, since they cannot be held accountable for anything beyond that.To comply with the specifications of PAS 55 [20], the organisation that wants the AM tasks done must ensure that the employees responsible for the AM activities have the appropriate education, training, and experience for the job.This must also be done if the AM activities are outsourced, as stated in Section 4.4.3 of PAS 55 [20].

Mobley [13] argues that, in order to achieve AM excellence, a culture change must take place at the corporate level.Most of the people at that level still have the perception of predictive technology as only being a tool to prevent failures and to help with maintenance [13]: this must change.If this change takes place, the training of personnel responsible for the equipment's reliability will become a high priority [13].In order to foster this new culture among lower-level employees, all of the employees that work with the physical assets must be trained and educated about the possible results of the different failures, and be made aware of different situations.Telling employees that they are accountable gives them a sense of responsibility, and makes them feel that they have more of a purpose than just doing their own bit each day.The training of the employees must be recorded to enable the organisation to prove their compliance with the PAS 55 document [20].The impact of training is not only to comply with PAS 55, but also to produce a more effective workforce that is better prepared to react in situations they have never faced before.

Two strategies are used to improve the maintenance methods of organisations: reliabilitycentred maintenance (RCM) and total productive maintenance (TPM).Mobley [13] states that TPM is based on five pillars, and stresses that the most important of these pillars is the education and training of personnel.Training is also one of the key requirements in RCM in order to be successful [16].When carrying out tasks such as a criticality analysis, RCM expects that the person is well-informed about the task and is able to execute such a process.Operators not only have to know how to operate the equipment in the correct manner, but they should also be able to do routine inspections.The supervisors must also be able to provide proper supervision while knowing what is going on under their noses [13].

It is difficult to assign a quantitative value to the knowledge and skills of employees because it is not possible to know exactly how much their knowledge and skills are worth to the organisation.It is well-known, however, that the effect of expert knowledge and skills in organisations is beneficial.

To comply with the PAS 55 standard, the training of personnel in many different areas is mandatory.The employees responsible for the predictive maintenance must be able to carry out proper predictions, but training is required to ensure that these employees know how to use the required methods.Training is therefore an important supporting pillar of AM; and since risk management is such a broad area, training is required in many risk management fields.As stated in Campbell et al. [16], the training of the necessary personnel must be supported by management.Many organisations have realised this, which has meant that this field is now relatively mature and well-developed.Organisations offer their employees the opportunity to do more training and develop new skills, to benefit not only themselves but the organisation as well.

DISCUSSION

In an asset-intensive organisation -for example, a mining company -Shah and Littlefield [14] found that an 11 per cent decrease in asset down time [14] could mean a saving of millions of dollars each month.For manufacturing companies, a 14 per cent increase in their products' compliance could save a lot of money, depending on the size of the company.These are just two examples of what can happen when risk is managed correctly.

In the PAM environment, managing risk means maintaining equipment correctly, which can increase their reliability, and acquiring the correct assets.The higher reliability of the equipment used can have a positive effect on the risks involved.Therefore it is crucial to have the correct maintenance strategy and implement the tactics correctly.To be able to comply with the PAS 55 [20] framework, risk management must be applied from the start: when setting up the PAM strategy, risk identification and assessment must be done.PAS 55 [20] also requires that a methodology is laid out beforehand for how risks will be managed.As mentioned earlier, risk management is directly related to more than 71 per cent of the sections in the PAS 55 framework.So it is an important field within AM, and cannot be ignored.Since training and forecasting are tools that support risk management, it would make sense that the bulk of the budget is allocated to risk management; even if more money is needed in one of the two other areas, it can be awarded to that field through the risk management budget.

The driving factors that caused AM to develop further over the last 40 years were, for the most part, plant/equipment availability, lower maintenance costs, equipment reliability, minimising environmental effects, and human safety [15].Forecasting of unwanted events is the basis of predictive maintenance.By applying this maintenance method, a positive effect on all of the driving factors can be achieved [16].Preventing casualties and/or environmental incidents from occurring at any organisation can save them money and boost their reputation [20].Environmental incidents can have a quick and drastic effect on the balance sheet of an organisation, as exemplified in the BP oil spill in the Gulf of Mexico.A good reputation increases share prices and improves the marketability to the public, and is more valuable than often realised [20].More reliable equipment lowers the risk associated with both human casualties and environmental damage.The prediction of equipment's residual life means that money can be saved on maintenance actions and spare parts, since replacements will only be made when needed.A higher reliability and better prediction of failures can yield a higher availability of assets; this enables planned shut-downs, allowing provision to be made for times when the assets are not operating.Forecasting of events is relevant for several areas described in PAS 55 [20]; and it is an important tool that can help an organisation to comply with the PAS 55 document and assist the risk management process.

In order to be able to do a risk analysis, create a maintenance strategy, make predictions, and even to operate the necessary equipment, special knowledge and skills are required.To support the AM framework, the training of employees is essential.Proper training will decrease the number of times that certain jobs have to be repeated, or decrease the number of recurrent and/or specific breakdowns [16].According to Campbell et al. [16], achieving maintenance excellence requires the combination of four main elements: leadership and people, methods and processes, materials and physical plant, and the systems and technologies used.Training the personnel and ensuring that they have the necessary skills and knowledge falls under the element of leadership and people.According to Hastings [17], training plays an important role in change management, and change is equal to 'uncertainty', which also means 'risk' [18].Training therefore also helps to manage the risks involved by equipping employees with the tools to make better-informed decisions.Dhillon [19] argues that personnel with inadequate training are also more prone to being injured or to injuring others: by providing proper training to personnel, the risk of injury is reduced and the effectiveness of the work force is increased.

CONCLUSION

This conclusion is based on the authors' interpretation, and is therefore subjective.The specific choices in this paper are not provided in the literature; but the literature did provide the foundation for the decisions discussed below.

The training of personnel and the forecasting of unwanted events can both be seen as tools to manage the risks involved with PAM.Risk management includes many other actions that may require changes at the managerial level.Therefore risk management should receive the largest portion of the organisation's budget.Since risk management includes those actions mentioned in Section 1, something like acquiring a new physical asset can easily drain the organisation's finances if not properly planned.Since risk management is directly related to nearly 75 per cent of all the sections in the PAS 55 [20] document, it seems fair to dedicate 60 cents of each South African Rand in the PAM budget to risk management.Doing this over a course of five years -and ensuring that the risk management is being implemented correctly -will ensure a mature and effective AM system in the long run.

The second largest portion of the budget should go towards the training of personnel, because the proper training of employees has a directly positive impact on the risks involved with physical assets.With the correct training, the health risk, environmental risk, and production risk of an organisation can be lowered: and avoiding these risks can have a substantially positive impact on the organisation's balance sheet.It is ideal to train all the people involved in the PAM system, but this is not always possible.Even if only the operators received training to make them more aware of equipment health, this would benefit the organisation.Training can consume precious time; and although this makes it pricey, the returns on it make it worthwhile.PAS 55 [20] states that continual improvement of an organisation's AM system is necessary.Since the improvement of an AM system is seldom cheap, 25 per cent of the budget should be made available for the training of various employees: this will cause the organisation to benefit from their knowledge gained, immediately and in the future.Training supports risk management and forecasting because both of those areas require special training to allow the employees to conduct the activities.It is important to note, however, that these are not the only areas that would benefit from training.

Forecasting is important; but because the decision whether or not to use CM will fall within the risk management section, the money to pay for the equipment will have to come from the risk management budget.The forecasting section will be for the analysis of the collected data and the study of new failure methods or signals.Software packages are available, but as more and more equipment is being fitted with CM equipment, new failure signals are being discovered.There is thus a lot of opportunity for further research.The remaining 15 per cent of the budget should therefore go towards the forecasting section for analysis of the data and further development in the field.If continual improvement occurs over the five years of the budget period, great positive financial implications will be realised.By preventing just one unexpected failure of a critical piece of equipment, an organisation can save the money it invested in forecasting, especially if the failure could have caused a fatality or an environmental risk.

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