Routine resite of peripheral intravenous devices every 3 days did not reduce complications compared with clinically indicated resite: a randomised controlled trial

Claire M Rickard
Damhnat Mccann
Jane Munnings
Matthew R Mcgrail
Routine resite of peripheral intravenous devices every 3 days did not reduce complications compared with clinically indicated resite: a randomised controlled trial
R E S E A R C H A R T I C L E Open Access
Background: Peripheral intravenous device (IVD) complications were traditionally thought to be reduced by limiting dwell time. Current recommendations are to resite IVDs by 96 hours with the exception of children and patients with poor veins. Recent evidence suggests routine resite is unnecessary, at least if devices are inserted by a specialised IV team. The aim of this study was to compare the impact of peripheral IVD 'routine resite' with 'removal on clinical indication' on IVD complications in a general hospital without an IV team.Methods: A randomised, controlled trial was conducted in a regional teaching hospital. After ethics approval, 362 patients (603 IVDs) were randomised to have IVDs replaced on clinical indication (185 patients) or routine change every 3 days (177 patients). IVDs were inserted and managed by the general hospital medical and nursing staff; there was no IV team. The primary endpoint was a composite of IVD complications: phlebitis, infiltration, occlusion, accidental removal, local infection, and device-related bloodstream infection. Results: IVD complication rates were 68 per 1,000 IVD days (clinically indicated) and 66 per 1,000 IVD days (routine replacement) (P = 0.86; HR 1.03; 95% CI, 0.74-1.43). Time to first complication per patient did not differ between groups (KM with log rank, P = 0.53). There were no local infections or IVD-related bloodstream infections in either group. IV therapy duration did not differ between groups (P = 0.22), but more (P = 0.004) IVDs were placed per patient in the routine replacement (mean, 1.8) than the clinical indication group (mean, 1.5), with significantly higher hospital costs per patient (P < 0.001). Conclusions: Resite on clinical indication would allow one in two patients to have a single cannula per course of IV treatment, as opposed to one in five patients managed with routine resite; overall complication rates appear similar. Clinically indicated resite would achieve savings in equipment, staff time and patient discomfort. There is growing evidence to support the extended use of peripheral IVDs with removal only on clinical indication.

Background

Peripheral intravenous device (IVD) insertion is the most commonly performed invasive procedure in hospitalised patients, with an estimated 150 million peripheral intravenous devices placed each year in North America alone [1]. IVDs are vital for delivery of hydration, medicines and nutrition but are not without complications. Serious adverse outcomes are fortunately rare, with IVD-related bloodstream infection reported in a recent meta-analysis of 110 studies to occur in 0.1% of devices and 0.5 per 1,000 device days [2]. IVD treatment is more frequently interrupted by phlebitis, an irritation of the vein characterised by pain, tenderness on palpation, erythema, warmth, swelling, induration or palpable cord (thrombosis) of the cannulated vein; diagnostic algorithms usually require two or more of these conditions [3][4][5]. Phlebitis is in almost all cases a biochemical reaction to the mechanical irritation by the presence of the IVD and associated infusate [3], although phlebitis symptoms such as erythema may be misperceived as indicative of an infection. In fact, there is not a high correlation between phlebitis and device infection, and the Centers for Disease Control (CDC) states that infection is rarely associated with peripheral, as opposed to central, venous devices [3,6,7]. Fluid infiltration or 'tissuing' of devices is another common IVD complication which may also reflect the inflammatory (phlebitic) response of the vein, rather than simple misplacement of the device tip [8].

Early cohort studies noted an association between increased device time in situ and phlebitis [9,10]. This association was responded to with policies for routine device removal. Recommended timelines for routine resite have been extended over the past three decades from 24, to 48, then to 72 hours. Currently, 72-to 96hour resite is recommended to reduce phlebitis by the CDC's 2002 Guidelines for the Prevention of Intravascular Device Infection, with the exemption that this is not required in children or those with poor veins [7]. Such policies cause increased workload in hospitals, where the task of removing and replacing well-functioning IVDs generally falls to busy nursing and junior medical staff. In addition, few patients welcome the prospect of additional venipuncture.

Despite the general clinical acceptance of routine IVD replacement as a phlebitis and infection prevention measure, it has not been supported by recent data. It may be that the risk of complications during the entire IVD treatment episode is similar, regardless of whether multiple short-dwell or fewer longer-dwell IVDs are used over this time. Three small (n = 47-60) randomised, controlled trials (RCTs) suggested routine replacement at 12, 24 or 48 hours may reduce phlebitis compared to resite on clinical indication, although a systematic review for the Swedish Council on Technology Assessment in Healthcare assessed these as low-to mediumquality studies providing 'limited scientific evidence' [11][12][13][14]. More recently, two well-conducted RCTs found no evidence of effect when comparing IVD replacement every 3 days with replacement only on clinical indication for medical and surgical inpatients [15,16]. The largest of these studies reported findings from 755 general medical and surgical patients with 1,428 IVDs and found a 5% difference in combined phlebitis and infiltration rates per patient (38% clinically indicated resite, 33% routine resite), suggesting a potential small clinical benefit of 3-day resite [15]. However, this difference was not statistically significant (RR 1.15; 95% CI, 0.95-1.40) and disappeared when overall cannulation time was considered (59.8/1,000 IVD days clinically indicated resite, 60.9/1,000 IVD days routine resite; RR 0.98; 95% CI 0.78-1.24) [15]. In addition, no clinically important or statistically significant differences were observed in the secondary endpoints of phlebitis, infiltration, occlusion, local infection or suspected bloodstream infection rates between study groups [15]. Another recent RCT in the 'hospital in the home' community setting also found no important clinical or statistically significant difference in phlebitis, occlusion or bloodstream infection rates in 316 patients when resite every 3 days was compared with clinically indicated resite [17]. A 2010 Cochrane Collaboration review concluded there was 'no conclusive evidence of benefit' of routine IVD resite and suggested organisations could consider adopting a resite on clinical indication policy [18]. There is growing evidence that routine IVD replacement may be ineffective, although caution has been urged in light of the large number (74% in both groups in the largest study to date) of reported devices inserted by a specialised IV team, a factor known to reduce complications [19].

Device insertion (and reinsertion) is unpleasant for patients, requires skilled and available clinical staff, and has associated costs for the health sector. If replacement only on clinical indication is safe and effective, this would have important benefits for patients and the health system. We report a RCT of 3-day routine IVD resite versus clinically indicated replacement in a medical-surgical hospital where IVDs were inserted by the general medical and nursing staff; the insitution did not have a specialised IV service.

Methods

Aim

The aim of the study was to compare the impact of 3day routine resite, with clinically indicated resite, on peripheral IVD complications.

Design

Open (nonblinded), parallel group RCT.

Ethics

The study was approved by the Tasmanian State Human Research Ethics Committee. Written informed consent was obtained prospectively from all participants.

Setting and sample

The study was undertaken at a large regional teaching hospital in Australia which functions as the tertiary referral centre for the northern half of the State of Tasmania. The hospital has more than 32,000 separations per annum, with a spectrum of medical and surgical specialties. Eligible patients were at least 18 years of age and scheduled or expected to have a peripheral IVD indwelling for at least 4 days, and they gave written informed consent. Exclusion criteria were immunosuppression, current bloodstream infection or an IVD already in situ for >48 hours. IVDs were inserted and cared for by the general nursing and medical staff; there was no special IV team or service.

Sample Size

Sample size calculations were performed using PASS 2008 (Version 8.0.8; Kaysville, UT) to detect a change in rates by 30% (from 36% to 25%, two-tailed α = 0.05, 90% power) on the basis of the complication rates of routinely resited IVs in a previous study [16]. Although this indicated that n = 378 per group (total 756) were required, the study was ceased early (total n = 606 IVs) because all investigators left the employment of the institution. Consequently, study power was reduced, but remained over 80% (required minimum n = 282 per group).

Recruitment

All adult patients admitted to the inpatient acute medical and surgical wards of the study hospital were screened by a full-time research nurse. This excluded paediatric, day-surgery, mental health, obstetric, critical care and dialysis units.

Study procedures

Patients were randomly assigned (computer generated) in a 1:1 allocation ratio to either the 'routine replacement' (control) or 'clinically indicated replacement' (intervention) group. Assignment was concealed until randomisation by use of a telephone service. A tag was placed on the insertion site indicating the study group. All devices for the patient were managed as per randomised group. The intervention group did not have their IVD resited unless clinically indicated. This decision was made by the treating clinician (not the investigators), who ordered IVD resite if the device failed or phlebitis occurred and ongoing IV treatment was required. The control group had a new device relocated to a different site by the general medical or nursing staff every 3 days. Control devices could also be removed at any stage by the clinical staff if they were not required or if complications occurred. Clinical nursing and medical staff undertook insertion and follow-up care of all IVDs as per the CDC Guidelines [7].

Laboratory staff undertaking microbiological culture assessments were blinded to the study group. Due to the nature of the intervention, patients, research, and clinical staff were unable to be blinded. However, the investigators had no involvement in assessing or documenting complications.

IVDs were assessed by the clinical nursing staff on each nursing shift for complications as part of standard clinical practice in the hospital. Times and dates of device insertion and removal were recorded along with the reason for device removal and any protocol deviations. A full-time research nurse collected data from the hospital chart and sought clarification from patients and clinical staff if necessary. Microbiological investigations (device tip, blood cultures and site cultures) were performed by the clinical staff on clinical suspicion of infection by the treating clinician. Demographic and clinical data were collected on age, sex, diagnosis at hospital admission, phlebitis risk based on Tagar et al.'s classification (low/medium/high risk) [20], past history of phlebitis, any comorbidities requiring active medical treatment (e.g., type 2 diabetes or congestive heart failure), haemoglobin, concurrent infection at other sites, antibiotic therapy, type of surgery, type of infusate and any additives (and their level of irritability), vein and skin quality assessment, size of device, insertion site, health professional inserting the device, and setting for insertion, presence of other vascular devices, wound drains and urinary catheters. Vein quality was assessed as good (vein easy to visualise and easy to palpate with tourniquet on), fair (not easily visible but can palpate with tourniquet), or poor (veins small, scarred or difficult to palpate with tourniquet; may require heat pack to aid vasodilation). Skin quality was assessed as good (healthy, well hydrated, elastic), fair (mildly dehydrated, reduced elasticity), or poor (papery, dehydrated, or reduced elasticity).

Analytic Approach

The primary outcome was a composite measure of any complication causing unplanned cannula removal prior to completion of IV treatment. The composite included phlebitis, infiltration, occlusion, accidental removal, local infection, and IV device-related bloodstream infection (IVD-BSI). These were also analysed individually as secondary endpoints. A composite measure was chosen due to the low rates of these conditions individually and to the assumption that they are comparable measures of 'infusion failure'; that is, the device can no longer be used to deliver treatment. This approach has been used in previous studies on the topic [15][16][17]. Phlebitis was defined as two or more of pain, erythema, purulence, streak formation, or a palpable venous cord [3]. Local infection IVD-BSI (bacteremia/fungemia with at least one positive blood culture obtained from a peripheral vein, clinical manifestations of infection, and no apparent source for the bloodstream infection (BSI) except the device with or without positive tip or entry site swab culture) were defined using CDC criteria [7]. Other secondary outcomes were time in situ (hours of catheterisation from insertion to removal, both per patient and per device) [7]; IVDs per patient (number of peripheral devices inserted to complete the course of treatment) [7]; costs (calculations based on 20 minutes nursing or medical insertion time at relevant rates [15], plus the cost of the required equipment (cannula, insertion pack including dressing and solution, gloves, saline, syringe, extension tubing and starter pack for all plus fluid bag, tubing and secondary tubing for medication administration for those patients requiring this) for insertions, nursing time and equipment to routinely remove IVDs that were otherwise functional, and the costs of treating any complications that occurred (e.g., IVD-BSI). Cost calculations were undertaken from the viewpoint of the hospital using negotiated wage costs and purchasing agreements for government hospitals in the State of Tasmania. Costs would be similar for other Australian hospitals.

All randomised patients were analysed by intention to treat. Each patient was randomised once and could have multiple IVDs, with subsequent IVD resites managed as per the randomised group. Relative incidence complication rates per 1,000 IVD days and 95% confidence intervals were calculated to summarise the impact of clinically indicated replacement relative to 3-day replacement. Kaplan-Meier survival curves were drawn to compare time to first IVD complication between patients in the two study groups. To assess for any potential impact of protocol violations, a per protocol analysis was also undertaken. All clinical and demographic variables were subjected to univariate testing against the primary endpoint to guide selection of possible covariates for the multivariable model. Cox proportional hazards regression modelling was used to examine the impact of age, gender, oncology status, number of comorbidities (nil, one, two, or more than two), IV gauge, site, vein quality, skin quality, oral antibiotics, IV antibiotics, wound drain, inserter occupation, initial versus subsequent IVDs, phlebitis in a preceeding IVD, haemoglobin level, parenteral nutrition, continuous versus intermittent infusion, patient risk category and study group on the outcome of time to complication events using an additive model [3,5,7,[20][21][22][23][24][25]. In addition, to adjust for any inherent correlations or codependencies in the failure times of IVDs (i.e., same patient multiple failure-time data) within the Cox model, we also used the Prentice-Williams-Peterson conditional risk-set method [26]. The Mann-Whitney test was used to compare various secondary outcomes between study groups. Cost differences were calculated using arithmetic means and the t-test [27]. P values <0.05 were considered significant. All statistical data were entered and analysed using SPSS (Version 15.0; Chicago, IL) and Stata (Version 8.2; College Station, TX).

Results

Sample

Over a 10-month period, 1,954 patients were screened for eligibility. Of these, 788 were eligible, with 362 (46%) recruited into the study. The most frequent exclusion criterion was altered mental state that precluded consideration of consent as assessed by the research nurse. Altered mental state was generally related to older medical patients and the immediate postoperative phase for surgical patients. Reasons for exclusion are shown in Figure 1. The 362 patients were randomised into either the routine change group (n = 177 participants, 323 devices) or the clinically indicated replacement group (n = 185 participants, 280 devices). In total 50,173 IVD hours were studied (routine change group 23,288 hours, clinically indicated group 26,885 hours). More patients in the routine change group had an active infection (53% vs. 44%) and were receiving IV antibiotics (73% vs. 64%). However, as shown in Tables 1 and 2, the two groups were generally comparable at baseline for patient-and cannula-related factors.

Effect of intervention on primary outcome

Outcome data were available for all patients. Table 3 shows the rates of primary and secondary outcomes. Differences in complication rates between groups were not significantly different (routine replacement 66.0 per 1,000 IVD days; clinical replacement 67.8 per 1,000 IVD days; HR 1.03; 95% CI, 0.74-1.43; P = 0.86). As shown in Figure 2, the time to first complication per patient was also not significantly different between groups (Kaplan Meier [KM] with log rank P = 0.53). On crude rate per IVD, the catheters replaced on clinical indication had higher complication rates (110/280 or 39% vs. 91/323 or 28%; P = 0.004). However, total complication rates per patient (to deliver the course of IV therapy) were not significantly different (P = 0.39) between clinically indicated (76/185, 41%) and routine resite patients (64/177, 36%).

Patient-and device-related variables considered in the multivariable model were older age, number of comorbidities (nil, one, two or more than two), smaller cannula size, poor skin or vein integrity, IV antibiotics, insertion by medical staff and study group. None of these were found to be statistically significant. The final Cox proportional hazards model after adjusting for time found study group was not a significant factor (HR 1.02; 95% CI, 0.77-1.36; P = 0.89). Variance-adjustment testing for potential multiple-failures per patient (cannula data) found no inconsistency in significant results compared to the main Cox model.

Protocol compliance

Compliance with the study intervention was achieved in 78% (251 of 323) of routinely replaced IVDs removed at Day 3 and 100% (280 of 280) of IVDs resited on clinical indication. Noncompliance in the routine resite group was generally related to high staff workloads on that day or to staff's perception that the patient was soon to be discharged or have therapy completed, and so the IVD remained in situ beyond Day 3. A per protocol analysis was performed including only those patients in the routine replacement group whose IVDs were all actually removed by 96 hours (n = 251). This found no significant difference (KM with log rank P = 0.16) in the rate of complications between groups (routine replacement 92 per 1,000 IVD days vs. clinically indicated 68 per 1,000 IVD days).

Effect of intervention on secondary outcomes

There was no statistically significant difference in group outcomes for any phlebitis (P = 0.34), infiltration (P = 0.57), occlusion (P = 0.75), or accidental removal (P = 0.43). No cases of local infection or IVD-related bloodstream infection occurred in either group.

For overall IVD therapy (total cannulation time per patient for all devices), clinically indicated devices had a median therapy of 120 hours (quartiles 86.5 and 172.5 hours), and those replaced routinely had median therapy of 113 hours (quartiles 72 and 172 hours) (P = 0.21). For individual IVD dwell times, the clinically indicated group devices had a median dwell time of 85 hours (quartiles 51 and 121 hours), and those replaced routinely had a median dwell time of 71 hours (quartiles 48 and 90 hours) (P < 0.001). The maximum IVD dwell time was 1,023 hours (43 days) in the clinical replacement group, and this cannula was still working well on removal for completion of therapy.

The overall number of IVDs per patient was significantly less (P = 0.004) for those replaced on clinical indication (mean 1.5, SD 0.8, median 1, quartiles 1 and 2) than for those routinely replaced (mean 1.8, SD 1.1, median 1, quartiles 1 and 2). A total of 22% of patients in the routinely replaced group had three or more IVDs compared with 9% in the clinical indication group. A total of 82 (28%) IVDs in the routine replacement group were resited after 3 days despite functioning well and ongoing treatment being required. (The remainder removed at this time were no longer required and so not resited, or infusion failure had already occurred.) Mean hospital costs per patient for the course of IV therapy were significantly higher (P < 0.001) for those

Discussion

The finding that 3-day routine resite was not an effective intervention was consistent across the intention-totreat primary analysis and the per protocol analysis. There remained no effect when events were expressed per patient or per 1,000 IVD days. Neither composite nor individual complication rates differed between groups, and there were no cases of local or devicerelated bloodstream infection. It appears safe and practical to leave IVDs in situ as long as they are functioning well and are still needed for clinical treatments.

All IVDs will fail eventually, but this study shows that artificially shortening the lifespan of individual catheters does not reduce the overall complication rates over the course of therapy. Our results indicate that the average duration of IV therapy is 5-6 days and that many catheters can remain complication-free for this period. If catheters are not routinely resited, the median dwell time would remain within the 72-96 hours recommended by the CDC, but about 10% would remain in situ for longer (in this study up to 43 days with no complications). Our data show that a policy of resite on clinical indication would mean that one of every two patients would need a single cannula to receive treatment, whereas a 3-day change policy would result in only one in five patients having this scenario, with the rest requiring multiple cannulations and therefore additional pain and inconvenience.

The results are consistent with the findings of recent RCTs in both hospitals and the community that have found no benefit in routinely resiting IVDs every 3 days [15][16][17]. In these studies, many cannulae were inserted by an expert IVD team [15][16][17], which may have minimised complications in both groups [19]. Our study confirms and extends these findings into the general medical/surgical setting without an IV team where IVDs were inserted by a variety of nursing and medical staff. Data from this study were included in a 2010 Cochrane Collaboration systematic review and meta-analysis on the topic [18]. This review included six trials (n = 3,455) and reported no clinically important or statistically significant difference in catheter-related bloodstream infection or phlebitis between IVDs that were routinely resited (at 48-96 hours) or resited on clinical indication, yet there were significantly lower costs in the group resited on clinical indication [18].

The belief that routine resite of IVDs will prevent complications appears to stem from early observational  studies that noted longer-dwelling IVDs had more complications than shorter-dwelling IVDs [9,10]. This is intuitively true, given, for example, that an IVD in situ for 6 days has twice the time exposure of risk than an IVD in situ for 3 days. However, this does not prove that two sequentially inserted IVDs (in the same patient), both used for 3 days, have a combined lower risk over time than the 6-day dwell device. Indeed, this and other recent trials strongly suggest that the risk for the patient over the 6-day period is similar. Well-designed cohort studies with modern catheter materials suggest that the daily risk of phlebitis is relatively stable after the first 24 or 48 hours [3,21,[28][29][30][31]. The peak in phlebitis between 24 and 48 hours is likely associated with the time taken by the body to mount a biological response after the instigation of therapy; those most likely to develop phlebitis will do so at this time.

The results support the extension of the use of peripheral IVDs beyond the 72-96 hours currently recommended by the CDC [7]. There is incongruity in the  CDC recommendations; they recommend not to routinely resite IVDs in children or in those with limited venous access. If it is safe in these populations, it is unclear why it would be necessary to routinely resite adults or those with better veins. Higher-risk cannulae such as central venous devices are no longer recommended by the CDC for routine replacement, because trials showed this was not of benefit [7]. Our study also confirms that the CDC guidelines are not always complied with; one fifth of IVDs in the routine change group were not replaced by this time. However, the per protocol analysis showed that the intervention remained ineffective even for those who truly had their IVDs resited every 3 days.

Limitations of the study included a 9% higher frequency of IV antibiotics and concurrent infection in the routine resite group. This may have put the group at higher risk due to vein irritation, or conversely it protected against bacterial entry. Neither variable was significant in the multivariable model. The unblinded study design was unavoidable, but also a limitation. Our use of clear outcome measures, a full-time research nurse and laboratory staff blinded to culture assessments should have reduced the risk for potential bias. Resource constraints prematurely ended recruitment, thus reducing the anticipated power of the study from 90% to 80%.

Routine IVD resite involves pain for patients, staff procedural time, equipment costs and environmental waste. Contemporary evidence suggests the current policy for routine resite by 72-96 hours is ineffective and should be replaced with a 'resite on clinical indication' policy. It remains imperative that clinical staff monitor IVDs closely and that a daily review of the need for continuing therapy be made, with cessation as soon as possible; the only no-risk IVD is no IVD. Of the 4.3 million acute hospital admissions in Australia each year (excluding day cases), over half have IV treatment [15,32]. Conservatively, if even 2 million courses of IV therapy were managed with clinically indicated rather than routine resite, this would save the unnecessary insertion of approximately 660,000 IVDs and free 280,000 hours of staff insertion time. Assuming our costs are similar to those in other Australian hospitals, a change to resite on clinical indication would save approximately AUD$24 million nationally each year.

Conclusions

Although larger, multisite trials are required, evidence to date suggests that routine resite of peripheral IVDs increases patient discomfort and healthcare costs, but does not reduce IVD complications as has traditionally been thought.

Figure 1
1Participant routine resite (mean $55.42, SD $35.26) compared with resite on clinical indication (mean $43.35, SD $26.78).

Figure 2
2Kaplan-Meier survival curve of time to first intravenous device complication per patient (log rank, P = 0.53).

Table 1
1Baseline characteristics of study participantsVariable
3-Day Routine Change Group
(n = 177)

Clinically Indicated Change Group
(n = 185)

Sex -Male, n (%)
96 (54%)
103 (56%)

Age, mean (SD)
65.1 (17.3)
62.7 (15.4)
Reason for admission, n (%)
• Gastrointestinal
46 (26%)
52 (28%)
• Respiratory
37 (21%)
34 (18%)
• Oncology
21 (12%)
21 (11%)
• Orthopaedic
16 (9%)
22 (12%)
• Cardiac
10 (6%)
10 (5%)
• Neurological
9 (5%)
10 (5%)
• Vascular
7 (4%)
7 (4%)
• Renal
16 (9%)
9 (5%)
• Other
15 (8%)
20 (11%)

Number of comorbidities, n (%)
• None
6 (3.4%)
10 (5.4%)
• 1
22 (12.4%)
21 (11.4%)
• 2
44 (24.9%)
54 (29.2%)
• >2
105 (59.3%)
100 (54.1%)

Type of surgery, n (%)
• Nil
124 (70%)
132 (71%)
• Gastrointestinal
22 (12%)
22 (12%)
• Orthopaedic
8 (5%)
15 (8%)
• Other
23 (13%)
16 (9%)

Most recent Hb -Mean (SD)
124.8 (23.2)
126.2 (21.9)

Table 2
2Baseline infusion-related characteristics of study devicesVariable
3-Day Routine Change Group (n = 323),
n (%)

Clinically Indicated Change Group (n = 280),
n (%)

IV cannula gauge
• 22
43 (13%)
46 (16%)
• 20
241 (75%)
201 (72%)
• 18
27 (12%)
30 (11%)
• Other
2 (1%)
3 (1%)

Vein assessment
• Good
143 (44.3%)
107 (38.2%)
• Fair
160 (49.5%)
144 (51.4%)
• Poor
20 (6.2%)
29 (10.4%)

Skin integrity
• Good
138 (43%)
104 (37%)
• Fair
178 (55%)
164 (59%)
• Poor
7 (2%)
12 (4%)

Past history of phlebitis
2 (0.6%)
4 (1.4%)

Insertion site
• Hand
206 (64%)
187 (67%)
• Forearm
59 (18%)
49 (18%)
• Cubital fossa
51 (16%)
38 (14%)
• Other
7 (2%)
6 (2%)

Receiving infusate
241 (75%)
215 (77%)

pH of infusate -Mean (SD)
6.0 (0.5)
6.0 (0.4)

Receiving oral antibiotics
24 (7%)
24 (9%)

Receiving IV antibiotics
236 (73%)
176 (63%)

pH of IV antibiotics -Mean (SD)
6.9 (1.1)
6.9 (1.3)

Receiving other IV meds
190 (59%)
179 (64%)

pH of other IV meds -Mean (SD)
5.6 (2.6)
6.0 (2.8)
Wound drain
50 (16%)
40 (14%)

Urinary catheter
55 (17%)
41 (15%)

Other vascular device
26 (8%)
24 (9%)

Inserted by
• Junior doctor
232 (72%)
207 (74%)
• Registered Nurse
67 (21%)
57 (20%)
• Senior doctor
24 (7%)
16 (6%)

Where inserted
• Ward
216 (67%)
188 (67%)
• Emergency department
74 (23%)
70 (25%)
• Other
33 (10%)
22 (8%)

Current infection (site)
• Respiratory
97 (30%)
49 (18%)
• Urinary
24 (7%)
19 (7%)
• Wound
16 (5%)
15 (5%)
• Other
29 (9%)
37 (13%)
• >1 site
5 (2%)
4 (1%)

N/A (none)
152 (47%)
156 (56%)

Risk of phlebitis (Tagar scale)
• Low
174 (54%)
144 (51%)
• Medium
149 (46%)
136 (49%)
• High
0
0

Rickard et al. BMC Medicine 2010, 8:53
http://www.biomedcentral.com/1741-7015/8/53

Table 3
3Effect of intervention on primary and secondary endpointsMean difference $12.07 (95%CI $5.57, $18.56), p < 0.001Outcomes
3-Day Routine Change
Group
(n = 177)

Clinically Indicated Change Group
(n = 185)

RR (95% CI),
P Value

Primary:

IVD complications per patient, n (%)
64 (36%)
76 (41%)
RR 1.14 (0.88, 1.47),
p = 0.39

IVD complications per 1000 IVD days
66.0
(95% CI 49.8, 82.1)

67.8
(95% CI 52.6, 83.1)

HR 1.03 (0.74, 1.43),
p = 0.86

Secondary:

Phlebitis, n (%)
12 (7%)
18 (10%)
RR 1.44 (0.71, 2.89), p = 0.34
Infiltration, n (%)
53 (30%)
61 (33%)
RR 1.10 (0.81, 1.49), p = 0.57

Occlusion, n (%)
5 (3%)
4 (2%)
RR 0.77 (0.21, 2.80), p = 0.75

Accidental removal, n (%)
11 (6%)
16 (9%)
RR 1.39 (0.66, 2.92), p = 0.43

Local infection
0
0
-

IVD-related BSI, n (%)
0
0
-

IVD costs per patient,
AUD$ mean (SD)

$55.42 ($35.26)
$43.35
($26.78)

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Rickard et al. BMC Medicine 2010, 8:53
AcknowledgementsThe authors wish to thank the participants and the nursing and medical staff at Launceston General Hospital who supported the study, in particular the Department of Medicine. The authors gratefully acknowledge the support of a competitive project grant from the Clifford Craig Medical Research Trust, Launceston, Tasmania. The funding body was not involved in the design, undertaking or publication of the study. Further financial support of the study came from the Launceston General Hospital, University of Tasmania, Monash University and Griffith University. We thank the BMC reviewers for their constructive comments on the manuscript.Authors' contributions CMR designed the study, applied for funding and drafted the manuscript. DM participated in study design and manuscript preparation, and managed the study. JM recruited patients, ensured protocol integrity and collected data. MRM undertook statistical analyses and assisted with drafting the manuscript. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.
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Enhanced interleukin-1β production of PBMCs from patients with gout after stimulation with Toll-like receptor-2 ligands and urate crystals

Eleni E Mylona
Maria Mouktaroudi
Tania O Crisan
Stamatoula Makri
Aikaterini Pistiki
Marianna Georgitsi
Athina Savva
Mihai G Netea
Jos Wm Van Der Meer
Evangelos J Giamarellos-Bourboulis
Leo Ab Joosten
Enhanced interleukin-1β production of PBMCs from patients with gout after stimulation with Toll-like receptor-2 ligands and urate crystals
R E S E A R C H A R T I C L E Open Access
Introduction: Monosodium urate monohydrate (MSU) crystals synergize with various toll-like receptor (TLR) ligands to induce cytokine production via activation of the NOD-like receptor (NLR) family, pyrin domain-containing 3 (NLPR3) inflammasome. This has been demonstrated in vitro using human cell lines or monocytes of healthy volunteers. In the present study, we have investigated the effect of MSU crystals and of their combination with TLR ligands in peripheral blood mononuclear cells (PBMC) of patients with gout. Methods: PBMCs from 18 patients with primary gout and 12 healthy donors were exposed to MSU crystals in the presence or absence of saturated fatty acid C18:0 (free fatty acid, TLR2 ligand), palmitoyl-3-cystein (Pam 3 Cys, TLR1/2 ligand) and fibroblast stimulating factor-1 (FSL-1, TLR 2/6 ligand). Production of IL-1β, IL-6, IL-8, IL-17 and tumor necrosis factor alpha (TNFα) was determined by ELISA. mRNA transcripts of IL-1β were measured by real-time PCR.Results: MSU crystals alone failed to induce IL-1β, IL-6 or TNFα in both patients and control groups, but a stronger synergy between MSU/Pam 3 Cys and MSU/C18:0 for the induction of IL-1β was found in patients with gout compared to healthy controls. IL-6, but not IL-8, followed the kinetics of IL-1β. No production of the neutrophilrecruiting IL-17 was detectable after stimulation of the patients' PBMCs with MSU in both the presence or absence of TLR ligands. No change of gene transcripts of IL-1β after stimulation with MSU and Pam 3 Cys or with MSU and C18:0 was found. A positive correlation was found between synergy in IL-1β production from PBMCs of patients between C18:0 and MSU crystals, as well as the annual number of attacks of acute gouty arthritis (r s : +0.649, P: 0.022). Conclusions: The synergy between MSU crystals and TLR-2 ligands is more prominent in patients with gout than in controls. This is likely mediated by the enhanced maturation of pro-IL-1β into IL-1β.

Introduction

Gout is a crystal-induced inflammatory disease induced by the deposition of crystals of monosodium urate monohydrate (MSU) in the joints and in the synovial membranes [1]. The arthritis is mediated by pro-inflammatory cytokines, produced by activated innate immune cells [2]. Among these cytokines, IL-1β seems to play a pivotal role [3]. This was proven in clinical studies, in which selective blockade of IL-1β effectively suppressed pain and inflammation in patients with gout that was refractory to other treatments [4][5][6].

Synthesis of bioactive IL-1β is induced in two steps: in the first step, gene expression leads to synthesis of inactive pro-IL-1β; in the second step, pro-IL-1β is cleaved by the protease caspase-1 to yield mature IL-1β [7]. For activation of caspase-1, intracellular molecular platforms called inflammasomes have to be assembled [8]. MSU crystals have been reported to activate the MOD-like receptor (NLR) family, pyrin domain-containing 3 (NLPR3) inflammasome and induce IL-1β production via caspase-1dependent processing [9,10]. However, purified MSU crystals induce moderate amounts of IL-1β by themselves [11,12]; they require co-stimulation with toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS) [11] or free fatty acids (FFA) [12].

This necessity of co-stimulation is in agreement with the clinical wisdom that, in the context of chronically elevated uric acid concentrations, attacks of gout are preceded by infections or excess intake of alcohol or dietary fat.

The in vitro studies performed until now have either studied human monocyte cell lines [9] or primary mononuclear cells of healthy volunteers [9,11,12] but the IL-1β production capacity of primary cells of patients with gout has not been investigated. In this study, we investigated whether primary cells [peripheral blood mononuclear cells (PBMCs)] of patients with gout also need this dual stimulation for cytokine production. The role of IL-1β stimulation in gout was investigated in an effort to decipher if the pathophysiological phenomena of attacks of acute gouty arthritis are related to the excess production of IL-1β or of other cytokines mediating chemotaxis of neutrophils at the inflamed synovium, namely IL-18 and IL-17.

Materials and methods

Patients

Eighteen patients with gout (13 men, five women, aged 63.5 ± 13.6 years, Table 1) and 12 healthy volunteers (6 men, 6 women, aged 41.17 ± 14.41 years) were asked to donate blood. The study protocol received approval from the Ethics Committee of ATTIKON University Hospital. Patients were enrolled after written informed consent. Inclusion criteria were: a) diagnosis of primary gout; b) history of at least two acute gouty attacks; and c) blood sampling in interictal periods. Patients taking any anti-inflammatory medication in the previous 15 days were excluded from the study. Primary gout and an acute attack of gout were defined according to the criteria outlined by the American Rheumatism Association [13]. After informed consent, 30 ml of venous blood was collected under sterile conditions. Preparation of monosodium urate monohydrate (MSU) crystals and of ultrapure C18:0 MSU crystals were prepared according to the method described by Seegmiller et al. [14]. Briefly, a solution of 0.03 M of MSU at a volume of 200 ml was prepared after diluting 1.0 g of uric acid (Sigma Co, St Louis, MO, USA) in 200 ml of sterile water containing 24 g of NaOH. The pH was adjusted to 7.2 after addition of HCl and the solution became pyrogen-free after incubation for six hours at 120°C. Concentrations of LPS were below 0.01 ng/ml as measured by the kinetic LAL QCL-1000 assay (BioWhittaker-Walkersville, Maryland. USA). The solution was left to cool at room temperature and stored at 4°C. Produced crystals were 5 to 25 μm long ( Figure 1). On each day of the experiment, a small amount of crystals was weighted under sterile conditions for application.

A 200 mM stock solution of ultrapure C18:0 fatty acids (Sigma Co) at a volume of 5 ml ethanol 100% was prepared. The stock was warmed at 37°C in a water Although this concentration appears high, it is consistent with the concentrations that exist in the joint as reflected indirectly by the circulating concentrations of free fatty acids of patients before and after total hip arthroplasty [15].

Stimulation of cytokine production

PBMCs were isolated after gradient centrifugation of heparinized whole blood over FicolHypaque (Biochrom, Berlin, Germany All experiments were run in duplicate with cells of at least one healthy donor per day of experiment. After 24 hours of incubation, plates were centrifuged and supernatants were collected and stored at -80°C until the cytokine assays were performed. For assessment of IL-17 production, the culture period was five days with fetal bovine serum (FBS) added to the culture medium at a final concentration of 10%. PBMCs used for this analysis consist of lymphocytes and monocytes in an almost 3:1 ratio. Ideally, to measure cytokines released from the monocyte fraction of PBMCs, further monocyte separation should be performed. This generates some danger for ex vivo stimulation of monocytes by the separation media providing erroneous falsepositive results. However, stimulation of PBMCs for 24 hours provides cytokines coming from the monocyte fraction as already considered in previous publications of our group [16].

Cytokine measurement

Human TNFα, IL-1β, IL-6, IL-8 and IL-17 in supernatants were measured by ELISA (R&D systems, Minneapolis, MN, USA) according to the instructions of the manufacturer. The lowest limits of detection were 40 pg/ml for TNFα, 20 pg/ml for IL-1β, 16 pg/ml for IL-6, 165 pg/ml for IL-8, and 80 pg/ml for IL-17.

Quantitative PCR for mRNA expression of IL-1b

PBMCs were cultured with the stimuli as mentioned above and after four hours of incubation at 37°C in 5% CO 2 the plates were centrifuged, the cell pellets were lysed with 400 μl of Trizol (AppliChem GmbH, Darmstadt, Germany) and kept at -80°C until extraction of RNA.

RNA was extracted with chloroform and gradient centrifugation for 15 minutes at 4°C and 12,000 g followed by treatment for 30 minutes at 37°C with 0.04 U/μl of DNAase (New England BioLabs, Ipswich, MA, USA). RNA was detected after 3% agarose gel electrophoresis and ethidium bromide staining. A total of 1.5 μg of RNA (Pharmacia Biotech photometer) was used for the production of cDNA using 0.4 mM of dNTPs (New England Bio-Labs), 1 U of RNA-sin (New England BioLabs), 10 mM dithiothreitol (DTT) (AppliChem GmbH) and 5x of the reverse transcriptase buffer in a Sensoquest thermal cycler LabCycler Gradient using appropriate blanks (Eppedorf, Hamburg, Germay). After an initial incubation step of 10 minutes at 60°C, 1 μU of reverse transcriptase (New England BioLabs) was added followed by three cycles: 10 minutes at 25°C, 50 minutes at 42°C and 15 minutes at 70°C. cDNA was kept at -80°C until assayed Expression of mRNA was tested by the iCycler system (BioRad, Philadelphia, PA, USA) using 1μl of cDNA, 0.1 mg/ml of sense and antisense primers, 3mM of MgCl 2 (New England BioLabs), 0.25 mM of dNTPs (New England BioLabs), 10x buffer and 1 mM of Taq polymerase with SYBR-Gr as a fluorochrome in each tube. Primer sequences were: for IL-1β sense 5'-CAG CTA CGA ATC TCC GAC CAC-3' and antisense 5'-GGC AGG GAA CCA GCA TCT TC-3', and for β 2microglobulin sense 5'-ATG AGT ATG CCT GCC GTG TG-3' and antisense 5'-CCA AAT GCG GCA TCT TCA AAC-3'. After an initial denaturation step for 10 minutes at 95°C, 34 cycles were performed. Each cycle consisted of: denaturation for 30 seconds at 95°C, annealing for 30 seconds at 72°C and elongation for 30 seconds at 95°C. Amplification was followed by a melting curve, appropriate blanks were applied. The PCR product was identified after 3% agarose gel electrophoresis and ethidium bromide staining. Quantitative results were expressed as defined by the Pfaffl equation [17], using the efficiency of a standard curve created with known cDNA.

Statistical analysis

Data on cytokines are expressed as medians ± 95% confidence intervals (CI); those of gene transcripts by their mean ± SE. Comparisons between groups were done using the Mann-Whitney U test. Folds of change of cytokine production after treatment with a combination of stimuli compared with the most active single stimulus were calculated; comparisons between patients and healthy volunteers were done by the Mann-Whitney U test. Any more than a 30% increase was considered as significant synergy. Comparison between groups was done by the Fischer's exact test.

Correlations between folds of changes of cytokine production and of the annual number of attacks of acute gouty arthritis of patients were determined according to Spearman. P values less than 0.05 were considered significant.

Results

Cytokine production after exposure to MSU and TLR ligands

We stimulated PBMCs of patients with gout and of healthy donors with either the TLR2/6 ligand FSL-1 or TLR1/2 ligand Pam 3 Cys (Figure 2). We found that production of IL-1β did not significantly differ between patients and controls, although IL-1β production in cells of gout patients showed a wide variation and a tendency to higher production when exposed to Pam 3 Cys (Figure 2A).

Exposure of PBMCs to ultrapure MSU crystals did not induce IL-1β production in either of the two groups ( Figure 3A). Co-incubation of MSU with FSL-1 did not change the release of cytokines achieved by single FSL-1 ( Figure 3). When PBMCs of gout patients were incubated with MSU crystals together with Pam3Cys, production of IL-1β was significantly increased compared to Pam3Cys alone ( Figure 4). IL-6, IL-8 or TNFα released by PBMCs was not significantly different between gout patients and controls for any of the stimuli, single or in combination (Figures 2, 3 and 4).

Cytokine induction by MSU and saturated fatty acids

Recently, we demonstrated that the addition of C18:0 fatty acid augments IL-1β production of PBMCs exposed to MSU [12]. In the present study, we show that PBMCs from gout patients produce more IL-1β than controls when incubated with C18:0 and MSU ( Figure 5A). However, we noted that exposure of PBMCs to C18.0 alone also stimulated IL-1β production, most likely due to a different preparation of C18.0 stimuli than in previous studies (containing a low final concentration of 0.55% ethanol). The production of IL-6 by MSU/C18:0 was in line with IL-1β production in cells isolated from gout patients, with a trend towards higher cytokine production in gout patients than in healthy controls. Release of TNFα and of IL-8 after costimulation with C18:0 and MSU did not differ between patients and healthy controls.

Undoubtedly, the wide variation in cytokine production raises questions about the robustness of the finding. In order to make the result robust four types of analysis were performed: a) statistical comparisons reported in Figures 5A and 5B were performed using non-parametric tests; b) qualitative analysis disclosed that synergy of FFA C18:0 with MSU crystals for the production of IL-1β and IL-6 was found in 15 of 18 patients versus 2 of 12 controls, P = 0.001); c) fold-changes of IL-1β and of IL-6 production after stimulation of PBMCs of patients with the combination of C18:0 and MSU crystals compared to C18:0 alone were significantly greater compared to respective changes of healthy controls ( Figure 6); and d) a positive correlation was found between the change of IL-1β production after stimulation of PBMCs of patients with the combination of C18:0 and MSU crystals compared to C18:0 alone and the annual number of attacks of acute gouty arthritis of these patients (P = 0.022, Figure 7). Significant correlations were not found between changes of IL-1β by the combination of Pam3Cys and MSU compared to Pam3Cys alone and the annual number of attacks of acute gouty arthritis (data not shown, r s : +0.596, P = 0289).

To exclude the possibility that part of the explanation for the synergy between C18:0 and MSU is due to the younger age of healthy controls, correlation analysis was done between the age of both healthy donors and patients and the fold change of IL-1β production by the combination compared with C18:0 alone. No statistically significant results were found (r s : +0.170, P = 0.428).

Lack of IL-17 production by PBMCs after MSU and TLR2 ligands

A hallmark of acute gout is the influx of neutrophils into the synovium and joint fluid [18]. In order to define over-production in IL-1β release as the major culprit for attacks of gout, production of other cytokines mediating chemotaxis of neutrophils, notably IL-8 and IL-17, should not be affected by co-incubation with C18:0 and MSU. The cytokine IL-17 induces neutrophil-attracting chemokines and granulopoietic cytokines, and recruits neutrophils to the site of inflammation [19]. PBMCs of both gout patients and healthy controls exhibited no detectable production of IL-17 after stimulation with MSU crystals for five days, either in the presence or absence of FSL-1, Pam3Cys or C18:0 fatty acids (data not shown).

Effect on gene transcription

The synergy between TLR-2 ligands and MSU crystals might lead either to enhanced expression of the IL-1β gene or to increased cleavage of pro-IL-1β by caspase-1. mRNA transcripts of IL-1β in the PBMCs of gout patients did not have any difference after addition of MSU crystals to FFA C18:0, compared with healthy donors ( Figure 8A). Similar findings were observed after stimulation with MSU and Pam3Cys ( Figure 8B).

Discussion

In the present paper, we report that PBMCs of gout patients who have experienced attacks of arthritis produce more IL-1β than PBMCs of healthy volunteers when the cells are exposed to a combination of TLR2 ligands and MSU crystals. In addition, the IL-1β response in the presence of C18:0 fatty acids together with MSU is augmented in the majority of the gout patients.

The responses in the gout patients show a much wider variation than those in healthy volunteers. These variations may be partly explained by the variations of the annual attacks of acute gouty arthritis in the patient group since synergy for IL-1β production is pronounced for patients with frequent attacks of acute gouty arthritis. We found that the synergism between MSU and FFA C18:0 leading to production of IL-1β is not due to enhanced gene expression. The most likely explanation is increased cleavage of pro-IL-1β to IL-1β in patients with gout. Enhanced cleavage may be mediated through the NLRP3 inflammasome, since MSU is one agonist for the NLRP3 inflammasome. Our finding that this synergy was also present for IL-6 (which is at least in part induced by endogenous bioactive IL-1) underscored the activation of the inflammasome. However, it should not be neglected that other proteinases, namely, neutrophil elastase and cathepsin G, may mediate cleavage of pro- IL-1β to IL-1β [20] but this is unlikely in the present study since they are present at very low levels in the PBMC preparations.

Our findings suggest that the NLRP3 inflammasome may be activated more readily in patients with gout than in healthy controls. The synergistic effects for the production of IL-1β were mainly found between MSU and Pam3Cys and between MSU and FFA C18:0, but not between MSU and FSL-1, providing evidence that MSU synergizes with TLR2/1 agonists but not with TLR2/6.

Several groups have shown that MSU crystals function as inflammasome activators [9][10][11]. Initial reports suggested that urate crystals were able to induce IL-1β on their own. When we reported that MSU crystals need the presence of a second stimulus such as LPS [11] or FFA C18:0 [12], it turned out that others had also used LPS as an inducer of pro-IL-1β transcripts [13]. This would fit with everyday clinical practice: while hyperuricemia is a prerequisite for gout, its presence does not always lead to disease and a second stimulus such as infection, excessive alcohol intake or a fatty meal is needed for an attack to occur. The data in the literature on IL-1β production in gout were obtained by the use of either primary mononuclear cells of healthy volunteers [9,11,12] or human monocytic cell lines (THP-1) cells [10]; this is the first study using cells of gout patients.

The infiltration of the synovium by neutrophils is considered to be a feature of the acute inflammation in gout [1]. The recent literature indicates that IL-17 is a potent mediator of neutrophil influx, by expansion of the neutrophil lineage through induction of Granulocye-colony stimulating factor (G-CSF) as well as by recruitment of neutrophils through the induction of chemokines [19]. IL-17 is mainly released by a distinct subset of T helper    cells, called Th17, which derive from the naïve CD4 + T-cells [21]. We measured IL-17 in order to find out whether acute gouty inflammation is under the control of IL-17. The lack of production of IL-17, observed in the present study, supports the view that neutrophil influx in gouty inflammation is probably due to the release of other neutrophil-recruiting cytokines, such as IL-1β. In fact, Th17 cells are generated after days and this is not compatible with the rapid occurrence of inflammation in gout.

Despite our inability to find a direct relationship between uric acid concentrations in serum and the degree to which the inflammasome is activated, hyperuricemia still may be the explanation for the increased activation of the inflammasome in patients with gout. At the molecular level uric acid, in its soluble form, has been reported to have a role in regulating redox homeostasis by forming radicals in reaction with other oxidants [22]. Moreover, it has been suggested that reactive oxygen species (ROS) may activate the NLRP3 inflammasome [23]. However, based on the findings of a previous study by our group, ROS are not involved in inflammasome activation, since PBMCs from ROS deficient patients produce significantly more IL-1β after exposure to MSU crystals than the cells from healthy individuals [24].

Our findings were generated with circulating PBMCs of patients. As such, it may be speculated that the over-inflammatory state produced in gout is systemic and not limited to joints. This speculation is consistent with the wide variation of co-morbidities recognized in these patients [1].

Conclusions

The present study provides evidence that the PBMCs of patients with gout display a more prominent synergistic production of IL-1β after stimulation with MSU crystals and TLR-2/1 ligands, compared to healthy controls. This synergy may be mediated by enhanced maturation of pro-IL-1β into IL-1β.

Figure 1
1Polarized microscopy of the prepared crystals of monosodium urate.

Figure 2
2PBMCs from gout patients produce more IL-1band IL-6 compared to healthy controls when exposed to TLR ligands. Release of IL-1β(A), IL-6 (B), IL-8 (C) and TNFα (D) by PBMCs of patients with gout and healthy donors after their stimulation with growth medium (RPMI), palmitoyl-3-cystein [Pam 3 Cys] (TLR2/1 agonist) or fibroblast stimulation ligand-1 [FLS-1] (TLR2/6 ligand). Asterisks denote outliers and circles denote extremes. Gout patients n = 18 and healthy controls n = 12. PBMC, peripheral blood mononuclear cells; TLR, toll-like receptor.

Figure 3
3Lack of synergy between MSU and FSL-1 in the induction of IL-1b. Release of IL-1β (A), IL-6 (B) IL-8 (C) and TNFα (D)by peripheral blood mononuclear cells of patients with gout and healthy donors after having been left untreated (RPMI) or stimulated by monosodium urate monohydrate (MSU) crystals alone or in combination with fibroblast stimulation ligand-1 [FLS-1]. Asterisks denote outliers and circles denote extremes. P values indicate significant differences between respective values of patients and healthy controls by Mann-Whitney U-test. Gout patients n = 18 and healthy controls n = 12.

Figure 4
4Synergy between MSU and Pam 3 Cys in the induction of IL-1b was greater in patients than controls. Release of IL-1β (A), IL-6 (B), IL-8 (C) and TNFα (D) by peripheral blood mononuclear cells of patients with gout and healthy donors after having been left untreated (RPMI) or stimulated by monosodium urate monohydrate (MSU) crystals alone or in combination with palmitoyl-3-cystein [Pam 3 Cys]. Asterisks denote outliers and circles denote extremes. P values indicate significant differences between respective values of patients and healthy controls by Mann-Whitney U-test. Gout patients n = 18 and healthy controls n = 12.

Figure 5
5Patients displayed greater synergy between MSU and C18:0 in the induction of IL-1b than healthy donors. Release of IL-1β (A), IL-6 (B), IL-8 (C) and TNFα (D)by peripheral blood mononuclear cells of patients with gout and healthy volunteers after stimulation with medium alone (RPMI), monosodium urate monohydrate (MSU) crystals and saturated fatty acids C18:0 alone or in combination with MSU. Asterisks denote outliers and circles denote extremes. P values indicate significant differences between respective values of patients and healthy controls by Mann-Whitney U test. Gout patients n = 18 and healthy controls n = 12.

Figure 6
6Changes of production of cytokines from PBMCs of gout patients and of healthy volunteers after stimulation with FFA C18:0 and MSU. x fold refers to changes compared with the most active single stimulus. P values indicate significant differences between respective values of patients and healthy controls by Mann-Whitney U-test. Gout patients n = 18 and healthy controls n = 12. FFA, free fatty acids; MSU, monosodium urate monohydrate; PBMC, peripheral blood mononuclear cells.

Figure 7
7Correlation between synergy for IL-1b production and the annual number of attacks of acute gouty arthritis. The x fold change of IL-1β produced by the combination of free fatty acids C18:0 and monosodium urate (MSU) compared with C18:0 alone is shown.

Table 1
1Clinical characteristics of 18 patients with gout enrolled in the study.No
Age (years)
Co-morbidities
Number of affected joints
Number of annual attacks
Therapy of attacks

1
75
DM2, VH, COPD
2
2
Colchicine

2
80
DM2, VH, CHD
4
5
Colchicine

3
62
DM2, VH, obesity
1
1
Colchicine

4
80
DM2, COPD, CHD
1
1
Colchicine

5
45
VH, obesity
1
1
Colchicine

6
70
VH
1
1
Colchicine
7
67
DM2, VH, CHD
7
5
NSAIDs

8
65
VH, obesity
1
1
Colchicine

9
62
VH
1
1
Colchicine

10
55
VH, obesity
2
1
Colchicine

11
80
DM2, VH
2
2
NSAIDs

12
32
Obesity
4
6
NSAIDs

13
70
VH
1
1
Colchicine

14
70
DM2, VH
3
2
Colchicine
15
68
Obesity
1
3
Colchicine

16
52
DM2, CRD
1
10
Colchicine

17
69
CRD
5
4
Colchicine

18
46
CRD
1
5
Colchicine

CHD, coronary heart disease; COPD, chronic obstructive pulmonary disease; CRD, chronic renal disease; DM2, diabetes mellitus type 2; NSAIDs, non-steroidal anti-
inflammatory drugs; VH, vascular hypertension.

Mylona et al. Arthritis Research & Therapy 2012, 14:R158 http://arthritis-research.com/content/14/4/R158
Mylona et al. Arthritis Research & Therapy 2012, 14:R158 http://arthritis-research.com/content/14/4/R158 Page 8 of 10
Mylona et al. Arthritis Research & Therapy 2012, 14:R158 http://arthritis-research.com/content/14/4/R158 Page 10 of 10
AcknowledgementsMGN was supported by a Vici grant of the Netherlands Organization for Scientific ResearchAuthor detailsAuthors' contributions EM and MM participated in the acquisition of data, analysis and interpretation and the preparation of the manuscript. TOC, SM, AP, MG and AS contributed to the acquisition of data. EG-B, MN, JvdM and LJ participated in study design, analysis and interpretation of data and manuscript preparation. All authors approved the final version of the article.Competing interestsThe authors declare that they have no competing interests.
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Enhanced interleukin-1β production of PBMCs from patients with gout after stimulation with Toll-like receptor-2 ligands and urate crystals. Mylona, Arthritis Research & Therapy. 14158Cite this article asCite this article as: Mylona et al.: Enhanced interleukin-1β production of PBMCs from patients with gout after stimulation with Toll-like receptor- 2 ligands and urate crystals. Arthritis Research & Therapy 2012 14:R158.