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Assessment of dose homogeneity in conformal interstitial breast brachytherapy with special respect to ICRU recommendations

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Abstract

Purpose

To present the results of dose homogeneity analysis for breast cancer patients treated with image-based conformal interstitial brachytherapy, and to investigate the usefulness of the ICRU recommendations.

Material and methods

Treatment plans of forty-nine patients who underwent partial breast irradiation with interstitial brachytherapy were analyzed. Quantitative parameters were used to characterize dose homogeneity. Dose non-uniformity ratio (DNR), dose homogeneity index (DHI), uniformity index (UI) and quality index (QI) were calculated. Furthermore, parameters recommended by the ICRU 58 such as minimum target dose (MTD), mean central dose (MCD), high dose volume, low dose volume and the spread between local minimum doses were determined. Correlations between the calculated homogeneity parameters and usefulness of the ICRU parameters in image-based brachytherapy were investigated.

Results

Catheters with mean number of 15 (range: 6-25) were implanted in median 4 (range: 3-6) planes. The volume of the PTV ranged from 15.5 cm3 to 176 cm3. The mean DNR was 0.32, the DHI 0.66, the UI 1.49 and the QI 1.94. Related to the prescribed dose, the MTD was 69% and the MCD 135%. The mean high dose volume was 8.1 cm3 (10%), while the low dose volume was 63.8 cm3 (96%). The spread between minimum doses in central plane ranged from −14% to +20%. Good correlation was found between the DNR and the DHI (R2=0.7874), and the DNR correlated well with the UI (R2=0.7615) also. No correlation was found between the ICRU parameters and any other volumetric parameters.

Conclusions

To characterize the dose uniformity in high-dose rate breast implants, DVH-related homogeneity parameters representing the full 3D dose distributions are mandatory to be used. In many respects the current recommendations of the ICRU Report 58 are already outdated, and it is well-timed to set up new recommendations, which are more feasible for image-guided conformal interstitial brachytherapy.

Keywords: breast cancer, homogeneity, dose-volume histogram, image-based brachytherapy

Purpose

In interstitial brachytherapy (BT), the non-homogeneous dose distribution around the radioactive sources is mainly determined by inverse square law. Other factors add only little modifications to this geometrical phenomenon. In the immediate proximity of the sources there are always regions of high dose, but with appropriate source distribution regions with low dose gradient can be attained, and in proper implants, the high dose volumes are relatively small. Historically, different parameters have been defined to characterize the dose homogeneity in BT [14]. The International Commission on Radiation Units and Measurements (ICRU) published the ICRU Report 58 in 1997 which deals with specification of dose homogeneity in interstitial BT [5]. For a reporting purpose it is recommended to use homogeneity parameters which have been validated in classical low dose rate (LDR) BT. However, in modern image-guided, dose optimized high-dose-rate (HDR) BT in which stepping-source remote afterloading equipment is used for irradiation, the practical applicability of these parameters is questionable. Boost dose after whole breast irradiation as well as accelerated partial breast irradiation (APBI) can be delivered with image-guided BT where conformal dose distribution can be achieved with optimized dose distribution [69]. Among the APBI techniques, the longest experience is present in multi-catheter based interstitial BT [79]. Now, follow-up data of up to 12 years are already available for HDR interstitial breast BT with comparable results to the WBI in terms of safety and efficacy [8]. In 2004, a European multicentre Phase III clinical trial was initiated by the Breast Cancer Working Group of the GEC-ESTRO to investigate the efficacy of the APBI [9]. Our institution actively participated in this study.

The purpose of this paper is to present the results of a detailed analysis on dose homogeneity of dose distributions in treatment plans made for our patients enrolled into the GEC-ESTRO trial and treated with interstitial BT. Furthermore, to investigate the suitability of the ICRU recommendations for dose uniformity in image-based conformal interstitial BT.

Material and methods

Dose plans of forty-nine patients were evaluated with respect to dose homogeneity. All patients were treated with microSelectron V2 HDR afterloader (Nucletron BV, Weenendaal, The Netherlands), and the used planning system was the Nucletron’s Plato Brachytherapy v.14.6. The details of our planning and implant techniques have been published elsewhere [10]. We used pre- and post-implant CT imaging for catheter placements and treatment planning. Following geometrical and graphical optimization, the dose was normalized to basal dose points and an isodose line was individually selected for dose prescription in order to obtain at least 90% of target volume coverage. The prescribed dose (PD) was 30.1 Gy delivered by 7×4.3 Gy, 2 fractions daily. Quantitative evaluation of dose plans was performed with dose volume histograms (DVHs). To characterize the homogeneity of dose distributions, the most common DVH based quality indices and parameters recommended by the ICRU were calculated. Descriptive statistics was calculated and correlation analysis between the indices and parameters was performed. Volumetric homogeneity parameters used for calculations were as follows:

Dose non-uniformity ratio (DNR)

The DNR is a simple and easy to interpret parameter for quantitative analysis of dose homogeneity in interstitial implants. The DNR is the ratio of the high dose volume to the reference dose volume [3]. The reference dose volume is the volume that receives dose equal or greater than PD, and the high dose volume is the volume that receives 1.5 times PD or more. The optimal dose distribution in terms of dose uniformity can be achieved at the minimum DNR value.

Dose homogeneity index (DHI)

The concept of DHI is similar to DNR, though different definitions exist in the literature [1113]. Sometimes it is used as a complementary parameter to the DNR (DHI = 1 – DNR). It can be calculated only for the implant geometry and can also be related to the volume of the PTV. In the latter case it is called relative homogeneity index (HI). In this paper we used the definition as follows: DHI=(V100 – V150)/V100. Where, V100 and V150 is the relative volume of the PTV in percent irradiated at least by the 100% and 150% of the PD, respectively.

Uniformity index (UI)

The UI is calculated from the “natural” volume dose histogram (NVDH) [1]. In the NVDH the “u” parameter is defined as –3/2 power of dose (D-3/2), and the volume (V) per unit “u” (dV/du) is plotted versus the “u” parameter. With this transformation the inverse square law is suppressed, and from this follows that for a point source the NVDH is a horizontal line. For a real implant, there is a peak on the graph which is graphical representation of the dose uniformity. The narrower the peak, the more uniform the dose distribution is. Evaluation of other peak parameters such as width, position and contained volume, in relation to treatment dose permits to define other quantitative volume-dose parameters such as UI and QI.

Per definition,

equation M1

 

where TD is the treatment dose (or PD) and HD (high dose) is dose value at dV/du that is half way between the (dV/du)max (peak dose, PkD) and the asymptotic value of dV/du as u → infinity (see HD definition in Fig. 1). The UI depends on the prescribed dose, thus it can be used to compare implants with the same dose prescription only.

Fig. 1

Natural volume-dose histogram for a breast implant. The arrows shows how the LD and HD are defined. The LD and HD is used to define the QI and UI, respectively

Quality index (QI)

The formula of QI is similar to UI, but instead of the treatment dose (TD) the low dose (LD) is used, where LD is dose value at dV/du that is half way between the (dV/du)max (peak dose, PkD) and the asymptotic value of dV/du as u → 0 (see LD definition in Fig. 1). Since treatment dose is excluded from the formula, QI is independent on the prescribed dose.

ICRU Report 58 recommendations

The ICRU recommends using the following parameters in interstitial BT: Minimum Target Dose (MTD) – minimum dose at the periphery of the clinical target volume, which in most cases practically coincides with the PTV, Mean Central Dose (MCD) – arithmetic mean of the local minimum doses between sources in the central plane (same as basal dose in the Paris system), High dose volume – volume encompassed by the isodose corresponding to 150% of the MCD, Low dose volume – volume within the clinical target volume encompassed by the isodose corresponding to 90% of the PD (corresponds to V90).

For high dose volume and low dose volume the maximum dimension of the volumes in the calculated planes should be reported. Dose uniformity parameters are the mean spread between the local minimum doses in the central plane (maximal±percentage deviations of the individual minimum doses from the MCD) and the MTD/ MCD (ratio of MTD and MCD).

Results

The median number of implanted catheters was 15 (range: 6-25) in a median of 4 (range: 3-6) planes. The mean volume irradiated by the PD was 78.8 cm3 (range: 23.2-209.5 cm3). The volume of the PTV ranged from 15.5 cm3 to 176 cm3 with a mean of 66.4 cm3. The volumetric dose homogeneity parameters are shown in Table 1. In 6 out of 49 cases (12%) the DNR value was higher than 0.35 which was the upper limit in the study. But, this was always accepted in order to obtain proper dose coverage. The dose homogeneity inside the PTV is characterized by 0.66 (range: 0.50-0.76) as a mean of the DHI.

Table 1

Volumetric homogeneity parameters for 49 HDR breast implants

Table 2 shows calculated parameters recommended by the ICRU. The wide range of the MTD (53-92%) indicates weakness of the use of this parameter in conformal BT. The average of the MCDs is 135%, which means that the mean isodose selected for dose prescription was 74% (range: 69-85%). The mean volume irradiated by 1.5 times MCD was 10% (range: 6-36%) which corresponds to absolute volume of 8.1 cm3 (range: 3.4-21.4 cm3). The mean low dose volume (96%) was close to 100% corresponding to 63.8 cm3. The mean deviation in local mean minimum doses from the MCD was 14% in negative and 20% in positive direction. The largest deviation was –25% and +61%. The minimum dose in the target related to the MCD (MTD/MCD) was quite low with 0.51 (range: 0.37-0.69) value.

Table 2

Homogeneity parameters recommended by the ICRU Report 58 for 49 breast implants

Figure 2 shows the correlation between DNR and DHI. Although, the former relates to the implant geometry and the latter to the PTV, the correlation is quite good (R2=0.7874). The UI also correlates with the DNR, which is presented in Fig. 3. No correlation (R2 <0.5) was found between the ICRU parameters (spread in individual minimum doses, MTD/MCD, low dose volume, high dose volume) and any other volumetric parameters (DNR, DHI, UI).

Fig. 2

Correlation between the DHI and DNR
Fig. 3

Correlation between the UI and DNR

Discussion

In interstitial BT, the classical Paris system has been successfully used clinically for different treatment sites for decades [14]. One of the advantages of the Paris system is that following its rules the resulting dose distribution will be always homogeneous. Although, it was originally based on LDR wire sources, its application is also possible with a HDR stepping source, when uniform dwell times are used [15]. In a previous study, comparing different dosimetry systems we found that the most homogeneous dose distributions occurred in the Paris dosimetry system and in the geometrical optimization [16]. For both systems the mean DNR was 0.25. The clinical availability of dose optimization algorithms and recent evolution of image-based brachytherapy have highlighted the limitations of the Paris system [17]. With 3D imaging, the exact definition of the PTV is possible, and this calls for tailoring the reference isodose surface to the PTV. However, good dose coverage sometimes can be achieved only with deterioration of dose homogeneity [16, 18].

At the time of publication of the ICRU Report 58, conformal interstitial BT was not widely available. This is well reflected by the recommended parameters which can be effectively used in projection-based classical implants. Use of an implant related parameters and point doses is recommended, and DVH is mentioned only as an additional representation of dose distributions. This is understandable, since at that time individual computerized treatment planning was not common. Without 3D volume calculation, the dimensions of the high dose volume in different planes have to be determined as per the recommendations. In current planning systems, however, calculation of the high dose volume can be easily performed from the DVH. In LDR BT or in HDR stepping source BT with uniform dwell times, the volume irradiated by 1.5 times MCD (high dose volume according to the ICRU) can be approximated by the dimensions measured in three planes, since the high dose region closely follows the catheters. But, in conformal BT the source dwell times can be very different due to optimization algorithms. From this follows, that the high dose volume will be irregular (bumpy) and its size can not be estimated with dimensions measured only in three planes. This is demonstrated in Fig. 4, where 3D representation of the high dose volume is shown in uniform and various dwell times in a two-plane breast implant. In the latter case, the dwell times were determined by optimization algorithm. It is evident from the images that knowing the dimensions in 2D planes only, can not be equivalent to calculation of the full 3D volume if the source dwell times are not uniform.

Fig. 4

3D representation of high dose volume according to the ICRU Report 58 in a breast implant planned A) without optimization, B) with geometrical and graphical optimization

In BT the dose inhomogeneity is unavoidable and it is particularly important in cases of breast implants, where all the breast tissue can be considered equally at risk for developing late side effects (e.g. fibrosis or fat necrosis). Wazer et al. [12] found a significant relationship between dose homogeneity and cosmetic outcome in interstitial LDR boost breast implants. With higher value of DHI they observed less late fibrosis. In another study from the same department, no clear statistical correlation between dose homogeneity and complication risk was found at sole HDR brachytherapy treatment for early-stage breast cancer [19]. In a study with LDR implants the probability of excellent cosmetic outcome linearly increased with DHI [20]. Vicini et al. [11] reported the DHI of 0.89–0.90 calculated for the implants of five patients. In the study of Das et al. [13] the DHI ranged from 0.46 to 0.85 with a mean of 0.73. Converting these values into DNR, the range will be from 0.15 to 0.54 with a mean of 0.27. Recently, a new dose volume uniformity index has been proposed where all volume elements irradiated by higher than the prescribed dose is taken into account [21].

The parameters for dose uniformity recommended by the ICRU relates to 2D dose distributions and point doses. Our results demonstrated that this simple representation of dose homogeneity did not correlate with volumetric parameters in HDR implants. The spread in individual minimum doses in the central plane may describe dose homogeneity in that plane, but the degree of homogeneity in the whole volume can be very different. In our study, there was no correlation between the deviations in the midpoint doses between the catheters in the central plane and volumetric parameters (DNR, DHI). The explanation for this is that in optimized dose plans the dose distributions in planes parallel to central plane can be unrelated to each other, not like in classic LDR implants or HDR implants with uniform source dwell times. Therefore, the central plane is no longer representative of the implant, as it was before. Nowadays, 3D assessments of dose distribution is mandatory with volumetric parameters to characterize the dose homogeneity.

Conclusions

In the era of image-guided conformal interstitial BT, the recommendations of the ICRU Report 58 seem to be outdated in many respects. The progress in imaging and dose optimization algorithms has recently made conformal BT as a routine procedure in many institutions. Considering this, it is mandatory to use DVH-related homogeneity parameters representing fully the 3D dose distributions. To decide which parameters have clinical significance requires more studies with clinical validation of their correlation with treatment outcome and side effects. In order to report the treatments in consistent way, new recommendations from international bodies and/or professional societies are highly awaited.

Acknowledgement

This paper is partially supported by the Sectoral Operational Programme Human Resources Development, financed from the European Social Fund and by the Romanian Government under the contract number POSDRU/89/1.5/S/60782.

References

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studentjournalofmedicine

Increased Carotid Intima-Media Thickness and Reduced Distensibility in Human Class III Obesity: Independent and Differential Influences of Adiposity and Blood Pressure on the Vasculature

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Guillermo López-Lluch, Editor

Abstract

Carotid intima-media-thickness (cIMT) and carotid distensibility (distensibility), structural and functional properties of carotid arteries respectively, are early markers, as well as strong predictors of cardiovascular disease (CVD). The characteristic of these two parameters in individuals with BMI>40.0 kg/m2 (Class III obesity), however, are largely unknown. The present study was designed to document cIMT and distensibility in this population and to relate these to other factors with established association with CVD in obesity. The study included 96 subjects (65 with BMI>40.0 kg/m2 and 31, age- and gender-matched, with BMI of 18.5 to 30.0 kg/m2). cIMT and distensibility were measured by non-invasive high resolution ultrasonography, circulatory CD133+/KDR+ angiogenic cells and endothelial microparticles (EMP) by flow cytometry, and plasma levels of adipokines, growth factors and cytokines by Luminex immunoassay kits. The study results demonstrated increased cIMT (0.62±0.11 mm vs. 0.54±0.08 mm, P = 0.0002) and reduced distensibility (22.52±10.79 10−3kpa−1 vs. 29.91±12.37 10−3kpa−1, P<0.05) in individuals with BMI>40.0 kg/m2. Both cIMT and distensibility were significantly associated with traditional CVD risk factors, adiposity/adipokines and inflammatory markers but had no association with circulating angiogenic cells. We also demonstrated, for the first time, elevated plasma EMP levels in individuals with BMI>40.0 kg/m2. In conclusion, cIMT is increased and distensibility reduced in Class III obesity with the changes predominantly related to conventional CVD risk factors present in this condition, demonstrating that both cIMT and distensibility remain as CVD markers in Class III obesity.

Introduction

Carotid intima-media-thickness (cIMT) and carotid distensibility (distensibility) represent structural and functional properties of carotid arteries respectively. Both increased cIMT, a noninvasive measure of subclinical atherosclerosis, and reduced distensibility, an indicator of regional artery stiffness, are independent predictors of future cardiovascular events [1], [2]. Importantly, a combined assessment of the two allows for a better analysis of the individual atherosclerotic burden and improved prediction of aortic atherosclerosis [3].

Increased cIMT or decreased distensibility has been linked to hypertension [4], diabetes mellitus [5] and obesity [6][10], determinant risk factors for cardiovascular disease (CVD) [11][14]. The occurrence of these three co-morbidities is linked with chronic low-grade inflammation. Furthermore insulin resistance present in obesity is believed to be a principal contributor to this link. The inter-relation between adipogenesis, inflammation, insulin resistance, hypertension and diabetes mellitus remains a current focus of obesity research. Nevertheless higher CVD incidences are evident in hypertensive and/or diabetic obese compared to non-obese counterparts. The prevalence of obesity is rising at an alarming rate worldwide. Moreover the prevalence of Class III obesity, defined as BMI≥40.0 kg/m2, is increasing at an even steeper rate [15], [16]. cIMT and distensibility in Class III obesity, however, are largely undocumented with only two papers providing both cIMT and distensibility data in people with BMI≥40.0 kg/m2 [17], [18]. Similarly, little information is available in Class III obesity on novel biomarkers of CVD, such as circulatory angiogenic cells [19] or endothelial microparticles [20].

The main objective of this study was therefore to document cIMT and distensibility in Class III obese subjects compared with a non-obese cohort, and to examine and compare traditional CVD risk factors (CVRF) and novel CVD biomarkers between the two populations. We hypothesized that cIMT and distensibility remain useful as CVD markers in the severely obese population despite technical difficulties that may be encountered and verified this by determining the association of cIMT and distensibility with other established CVRF in Class III obesity.

Materials and Methods

Ethics Statement

The study protocol was approved by the institutional ethics committee of Alfred Healthcare (#158/06), and informed written consent was obtained from each participant.

Study Population and Design

A total of 96 subjects (31 non-obese controls: BMI 18.5 to 30.0 kg/m2 and 65 class III obesity: BMI>40.0 kg/m2) were included in the study. Class III obesity subjects were recruited via the Obesity Research Groups at Monash University while the age- and gender-matched non obese were from the Baker IDI BioBank database. Exclusion criteria were known coronary artery disease, cardiac failure, vascular brain disease, peripheral obstructive artery disease, significant renal or hepatic dysfunction and pregnancy. Subjects with current or past history of multiple myeloma, blood dyscrasia or any form of leukemia or lymphoma were also excluded.

All individuals underwent a physical examination and had their medical histories recorded. In brief, participants were measured for height, weight, waist and hip circumferences and blood pressure. 30 ml of peripheral blood was drawn for routine blood tests following a 12 hr fast and also analyzed for levels of plasma adipokines, growth factors and cytokines, circulating angiogenic cells (CD133+/KDR+ PBMCs & Hill-CFU) and endothelial microparticles (EMP). Routine blood tests were performed by the Alfred Pathology Department including a full blood count, hsCRP, glucose and a lipid profile (HDL, LDL, total cholesterol and triglycerides). cIMT and distensibility were examined using non-invasive high resolution ultrasonography.

The measurement of cIMT and distensibility were compared between the two groups. The associations of cIMT or distensibility with traditional CVRF (age, gender, BP, glucose and lipids etc) and adiposity/adipokines (BMI, waist:hip, adiponectin and leptin) were examined, as were their respective associations with inflammatory markers and circulating angiogenic cells. Plasma levels of EMP were measured in randomly selected subpopulations including both males and females (Class III obese = 15 and non-obese = 16) to determine vascular inflammation and integrity.

Carotid Imaging and Measurement

The left and right common carotid arteries proximal to the carotid bifurcation were imaged through non-invasive high resolution ultrasonography using a Philips iE33 ultrasound system (Philips, Bothell, WA, USA) with a 11–3 MHz linear array transducer while the subject was at rest in a supine position. Briefly, the carotid arteries were imaged in longitudinal sections, 0–2 cm proximal to the carotid artery bifurcation, focusing on the far wall of the vessel. Two 10-second loops were captured for each of the left and right arteries and stored for offline analysis. cIMT was defined as the distance between the intima-lumen interface and the media-adventitia interface, and measured at end diastole (as determined from the simultaneous electrocardiogram recordings) over a 10 mm long portion of the vessel wall between 0–1 cm proximal to the carotid bulb. Diastolic and systolic diameters, for distensibility calculation, were determined as the smallest and largest diameter values during a cardiac cycle. An average of three measurements from consecutive cardiac cycles from each of the left and the right carotid artery was made, and the average of the left and right arteries was used for the final analysis. All measurements were conducted independently using an automatic edge detection system (Philips QLAB version 7.0) by two observers blinded to all participant information.

Carotid distensibility was calculated as (2Δd/ds)/ΔP in 10−3•kPa−1, where Δd is carotid internal diameter change between systole and diastole, ds is carotid systolic diameter and ΔP is pulse pressure [21].

Determination of Plasma Adipokines and Cytokines

Plasma samples were collected and stored at −80°C until use. Plasma levels of adiponectin, leptin, IL-10 and SDF-1 were measured using Luminex immunoassay kits (Millipore, USA) as per manufacturer’s instruction. Briefly, the appropriate adipokines or cytokine standards, plasma samples (25 µL), and fluorescent conjugated, antibody-immobilized beads were added to wells of a pre-wet filtered plate and then incubated in dark overnight at 4°C. The following day, the plate was washed twice with wash buffer and then incubated with secondary detection antibody for 1 hr, followed by subsequent incubation with streptavidin-PE for 30 min. After the plate was washed twice again with wash buffer, it was run on the Luminex system (BioRad) with the addition of sheath fluid. Concentrations of different analytes in the plasma samples were determined by using respective standard curves generated in the assays.

Measurement of Circulating Angiogenic Cells

Enumeration of peripheral blood CD133+/KDR+ PBMCs by flow cytometry

Peripheral blood mononuclear cells (PBMCs), isolated from fresh venous blood by ficoll-gradient centrifugation, were used to quantitate the number of CD133+/KDR+ PBMCs by flow cytometry. In brief, 100 µl of PBMCs (1.5×107/ml) was incubated with Fc-γ receptor blocking agent followed by 30 min incubation on ice with antibodies against human CD133 (PE-conjugated, Miltenyi Biotec, Germany) and VEGFR-2 (KDR) (APC-conjugated, R&D Systems, USA). PE- and APC-conjugated mouse IgG from the same manufacturers served as isotype controls. Following incubation, cells were washed with PBS and then fixed in 1% paraformaldehyde. Flow cytometry acquisition was performed on BD FACSCalibur™ using appropriate settings excluding debris and platelets as shown in Figure 1-A1 & Figure 1-B1. 106 events per sample were collected within the R1 monocyte gates. Cells positive for both CD133 and KDR (upper right quadrants of the FL2-FL4 plots as shown in Figure 1-A2 & Figure 1-B2) were characterized as angiogenic cells. Results were expressed as percentage of CD133+KDR+PBMCs/PBMCs. Analysis was carried out by a blinded randomized approach in regard to patient profiles using the FlowJo software (Tree Star Inc, USA).

Figure 1

Illustration of FACS gating analysis of angiogenic cells (AC133+/KDR+PBMCs).

Hill-CFU assay

The ability to clonally expand and generate colonies in an endothelial-specific medium is considered a key functional feature of angiogenic cells. The Hill-CFU assay was performed using the commercially available kit, EndoCultTM liquid medium Kit (Stem cells Technologies, USA) and used as per the manufacturer’s instructions. In brief, 5×106 ficoll-isolated PMNCs were resuspended with 2 ml EndoCult medium and plated in a well of fibronectin-coated 6-well plates (BD Biosciences, USA), which were incubated for two days at 37°C, 5% CO2 with ≥95% humidity. After two days the non-adherent cells were harvested, counted and plated as at a density of 1×106 cells per well onto a 24-well fibronectin-coated plate, which was then incubated at 37°C for another 3 days. Hill-CFUs, characterized by a central cellular cluster surrounded by emerging spindle-shaped cells were counted at day 5 in 24-well plates in a minimum of 3 wells per subject and the average count was recorded. Results were expressed as number of colony per well.

Isolation and Identification of Circulating EMPs

EMPs are defined as CD31+/CD41 particles sized between 0.1–1 µm in platelet-depleted plasma. They were determined by the analysis for the expression of surface antigens by flow cytometry. In brief, 500 µl of completely thawed plasma was centrifuged at 16000 g for 5 min at 4°C to deplete platelets or any cell debris. The top 450 µl of plasma was transferred into a fresh tube, which was centrifuged again at 16000 g for 30 min at 4°C. The top 250 µl plasma was carefully removed and the remaining 200 µl vortexed and used for FACS analysis. Following a 15 min incubation with 50 µl Fc-γ receptor blocking agent (Miltenyi Biotec, Germany) at room temperature to reduce non-specific binding, half of the treated plasma was incubated with antibodies against human CD31 (Alexa647-conjugated, BD Biosciences, USA) and CD41 (PE-conjugated, BD Biosciences, USA). The other half was incubated with Alexa647- and PE-conjugated mouse IgG from the same manufacturer served as isotype controls. At the end of 20 min incubation, 300 µl of double filtered 1% Formaldehyde/0.2% FBS/PBS (filtered through a 0.2 and then a 0.1 µm membrane filter before use) was added for fixation and 50 µl of diluted calibration beads (BD Biosciences, USA) was added for EMP calculation and size reference. Each sample and its corresponding control were counted on BD FACSCalibur™ (BD Biosciences, USA) for 5 min.

For flow cytometry counting, EMP gate (R2 as shown in Figure 2-B) was pre-defined using commercial beads sized at 0.1 and 1 µm (Sigma-Aldrich, USA). Only events included within this gate were further analysed for fluorescence signal as shown in Figure 2-D. For EMP enumeration, a formula was used based on the concentration of the added calibration beads [22], which discriminated themselves from the EMP population on the FSC-SSC cytogram (R1 as shown in Figure 2-B and Figure 2-C). All counting data were then processed with a blinded randomized approach using BD CellQuest Pro (BD Biosciences, USA). Results are presented as number of CD31+/CD41 EMP per µl of plasma.

Figure 2

Elevated levels of circulating CD31+/CD41 EMP in obesity.

Statistical Analysis

Logarithmic transformations were applied if appropriate to skewed data following histogram analyses and Kolmogorov-Smirnov test. Transformed data are expressed as geometric mean (95% CI) and non-transformed data are expressed as mean ± SD. Comparisons between the non-obese and Class III obese groups were performed by two tailed Student’s t-test. Bivariate correlation analysis of adiposity (BMI and waist:hip), carotid variables (cIMT and distensibility) and blood pressure (SBP) was performed to define each crude association with other variables measured. Multivariable linear regression models were then constructed with the use of important covariates concluded from correlation analysis (P<0.1), in a hierarchal fashion, to elucidate independent determinants of cIMT and distensibility. Model 1 was adjusted for traditional CVRF (age, SBP, BP-med, fasting glucose and triglycerides), while Model 2 for traditional CVRF and adiposity/adipokines (BMI, adiponectin and leptin), and Model 3 for traditional CVRF, adiposity/adipokines and inflammatory markers (hsCRP, IL10 and WBC). Multivariable analysis was also repeated with no BP adjustment to further assess reliability of independent association between SBP and distensibility, since distensibility is a derivative parameter related to pulse pressure. For the same purpose, multivariable analysis of distensibility was again carried out separately in non-hypertensive and hypertensive subjects. In addition, multivariable analysis of SBP was performed as well. All statistical analyses were performed with SPSS version 12.0 for Windows and a probability value P<0.05 was considered statistically significant.

Results

The demographic, anthropometric, clinic and laboratory characteristics of the 96 subjects included in the study are shown in Table 1. Except for age, gender and smoking history, the 31 non-obese and 65 Class III obese subjects presented as two distinctive phenotypes in respective to all parameters related to traditional CVRF, adiposity, and plasma levels of circulating adipokines as well as inflammatory markers. All traditional CVRF measured were significantly worse in the Class III obese group. The heavier atherosclerotic burden in the Class III obese subjects was demonstrated both through increased cIMT (P = 0.0002) and reduced distensibility (P<0.05) in comparison to their age- and gender-matched non-obese counterparts (Figure 3-A & Figure 3-B). Furthermore, significantly higher levels of circulating EMP (P = 0.02) indicated vascular inflammation and a compromised integrity of vascular endothelium in Class III obese subjects (Figure 2-A).

Figure 3

Increased cIMT (A) and reduced distensibility (B) in obesity.
Table 1

Anthropometric, clinical and biochemical characteristics.

As expected, significantly elevated plasma leptin and suppressed adiponectin, a typical adipokine phenotype of obesity, were shown in Class III obese subjects (Table 1), as was the inflammatory profile of much higher levels of plasma hsCRP but lower IL10. Both measures of adiposity, BMI and waist:hip, closely correlated to status of metabolic syndromes (blood pressure, glucose, type 2 diabetes, HDL and triglycerides) and inflammation (Table 2).

Table 2

Bivariate correlation between BMI, Waist:Hip, SBP, cIMT and CD with other covariates.

On the other hand, there was no difference in the number of circulatory angiogenic cells (CD133+KDR+PBMC) between obese and non-obese populations, despite significantly higher colony-forming capacities (Hill-CFU) in Class III obese subjects (Table 1). Plasma levels of SDF-1, a critical cytokine mobilizing angiogenic cells, were nearly doubled (P = 0.003) in subjects with BMI>40 kg/m2.

cIMT significantly correlated with age, BMI, waist:hip ratio, hsCRP, and status of the metabolic syndrome (Table 2). These correlations were also true for distensibility. As well, distensibility was also linked to BP-medication and plasma levels of leptin and adiponectin.

Subjects with hypertension were defined, in this study, for those who either presented with elevated blood pressure (≥140/90 mmHg) at the time of BP measurement or were taking antihypertensive medications (BP-med) or had a history of hypertension. Blood pressure was significantly elevated in Class III obese subjects, and multivariate regression analysis revealed that SBP was independently associated with BMI (β = 0.879, P<0.0001) and plasma levels of adiponectin (β = −0.304, P = 0.049) in this cohort. Multivariate regression analysis of cIMT and distensibility, therefore, was performed with and without BP adjustment (Table 3). The results showed that age and BMI were independently associated with cIMT regardless of BP adjustment (Table 3). In contrast, carotid distensibility was independently associated with age, BP-medication and plasma levels of adiponectin when adjusted for SBP, which was also associated with distensibility, but only with age and plasma adiponectin when not adjusted for BP. In further regression analysis for distensibility stratified by hypertensive status, age (non-hypertensive: β = −0.562, P<0.0001; hypertensive: β = −0.465, P = 0.007) and plasma adiponectin (non-hypertensive: β = 0.527, P = 0.001; hypertensive: β = 0.518, P = 0.023) were independently associated with distensibility in non-hypertensive and hypertensive groups, in addition to SBP in the hypertensive group (β = −0.465, P = 0.007).

Table 3

Multivariable linear Regression Analyses.

Discussion

The present study demonstrates that increased cIMT and reduced distensibility is observed in Class III obese subjects with no overt CVD conditions when compared to their age- and gender-matched non-obese counterparts. Changes in both cIMT and distensibility corresponded well with elevated traditional CVRF in this cohort. We also show that cIMT and distensibility are significantly associated with adiposity, adipokines and inflammatory markers, however, none had any connection with circulatory angiogenic cells.

Since obesity, and in particular abdominal obesity, is a major risk factor for CVD [23], tools to screen, monitor and predict CVD in this population can be very useful clinically. cIMT and distensibility, structural and functional parameters of carotid arteries, are early markers as well as strong predictors of CVD [1], [2]. While previous studies have demonstrated a strong association of increased cIMT and/or reduced distensibility with obesity [6][9], documentation of these two parameters of carotid artery in individuals with BMI>40 kg/m2 is lacking. Our finding that cIMT is increased and distensibility reduced in the Class III obese is consistent with the only other published studies that documented increased cIMT in 64 subjects with BMI of 42.3±4.3 kg/m2 [17], [18] and increased cIMT and decreased distensibility in 13 obese subjects with average BMI of 40.5±7 kg/m2 [18]. In addition, we show that cIMT is positively associated with age, adiposity, blood pressure, type-2 diabetes, hyperglycemia, dyslipidemia and hsCRP, while distensibility demonstrates negative associations with these covariates. These demonstrated associations verify that cIMT and distensiblity remain as CVD markers in Class III obesity.

Obesity is highly associated with the metabolic syndrome (MS), which is closely linked to cardiovascular morbidity and mortality [24]. Two of the most widely accepted MS criteria have been respectively promulgated by the World Health Organization (WHO) and the National Cholesterol Education Program (NCEP-ATP III). The principal distinction between the two is that that the NCEP-ATP III emphasizes CVD risks whereas the WHO focus on insulin resistance [25]. We used NCEP-ATP III criteria for this study. Based on the MS criteria defined by the NCEP-ATP III [25], about 86% of our Class III obese subjects had MS: dyslipidemia, hyperglycemia (diabetes), hypertension, or central obesity. This extremely high prevalence of MS was highly associated with BMI in our cohort as evidenced by the close association of BMI with various measures of MS status (Table 2). It is thus not surprising that BMI and/or plasma adiponectin was found to be independently linked to cIMT and distensibility, besides age which is a strong predictor of arterial remodeling [26], [27].

Hypertension and diabetes mellitus are common in obesity, which is also evident in this study. 60% and 56% of our Class III obese subjects were, respectively, hypertensive or diabetic in comparison to 20% and 7% in the non-obese group. Indeed in our obese subjects only 14% were free of both diabetes and hypertension. As both hypertension and diabetes are important in development of CVD, it is therefore difficult to apportion relative contributions to hypertension, diabetes or obesity per se. Further, artery stiffness and blood pressure are two closely related factors and distensibility is a derivative parameter of pulse pressure. Indeed, distensibility was revealed to independently associate with SBP and BP-medication besides age and plasma adiponectin when BP was adjusted in its multivariate regression analysis. The fact that an independent link between distensibility and plasma adiponectin stands regardless of the adjustment of BP demonstrates the essential role of adiposity in development of CVD suggesting the paramount importance of weight control in prevention and reduction of CVD. Obesity is also known to be characterized by a chronic systemic inflammatory state [28]. This was reflected by elevated plasma levels of CRP in our study. Plasma levels of CRP were strongly correlated with all important measures, cIMT, distensibility, BMI/waist:hip and as well as SBP, demonstrating a crucial role of inflammation in the development of obesity and obesity associated CVD. To further directly assess vascular inflammation and integrity, plasma endothelial microparticles (EMP) were examined and compared in randomly selected subpopulations including both genders from both groups. EMP are small membrane vesicles, 0.1–1 µm in diameter, which are released from the endothelium following endothelial cell activation or injury by a process of exocytotic budding of the plasma membrane [29] sometimes referred to as endothelial blebbing. Increased plasma EMP levels can be detected by FACS [30] and have been reported in various CVD conditions [29], [31]. In patients presenting a characterized endothelial dysfunction, levels of circulating EMP are inversely correlated with the amplitude of flow-mediated dilatation, independent of age and pressure [31], [32]. EMP has thus been suggested as a novel surrogate marker of endothelial injury, which precedes CVD, and hence a novel potential biomarker of CVD [20]. To our knowledge, this is the first report measuring EMPs in the Class III obese population and we show, for the first time, that circulating levels of EMP are significantly elevated in obesity.

In addition, we also examined plasma levels of angiogenic cells, an established cellular biomarker of CVD, and their associated cytokine SDF-1, a potent stimulator to mobilize angiogenic cells [33]. Decreased circulating levels of endothelial progenitor cells (EPC) have been used as an indicator of higher CVD risk [19]. We measured angiogenic cells in our study as CD133+/KDR+ peripheral blood mononuclear cells (PBMCs) by FACS. In comparison to EPC, commonly defined as CD34+/KDR+ PBMCs, CD133+/KDR+ PBMCs are earlier endothelial progenitors [34]. The role of EPC or angiogenic cells in obesity, and particularly in the Class III obese is largely unknown albeit there have been some studies which demonstrate low circulating levels of EPC in obesity with suggestions of the potential to predict CVD prevalence in this population [35], although contrary reports have also been observed [36]. In the current study, circulating levels of angiogenic cells were unchanged. This result might relate to the difference of angiogenic cells carrying different surface markers. We suggest, however, that the difference, at least partly, is the outcome of the contradicting processes occurring with inflammation suppressing the generation and mobilization of bone marrow-derived EPC [37] and highly activated adipogenesis promoting increased release of EPC from adipose tissue [38]. The latter is supported with the finding that SDF-1, secreted by adipose stromal cells [39], was significantly increased in the obese cohort. Intriguingly, the functional capacity of angiogenic cells measured as CFU-Hill colonies was significantly increased in the Class III obese group. The finding of a recent report by Hirschi et al. might explain this dichotomy. It reported that CFU-Hill colonies comprise primarily of monocytes and macrophages [40], and indeed what we are observing in the current study with increased CFU-Hill may simply reflect the activated inflammatory status in Class III obesity, in line with hsCRP. Our result of no correlation between cIMT or distensibility with angiogenic cells indicates that these cells may not be a cellular biomarker of CVD in Class III obesity.

It is a limitation of this study that only BMI or waist:hip ratio, but not direct measurement of body fat or body composition, was measured to reflect adiposity. Also neither insulin levels nor insulin resistance were evaluated. Further, this is a cross-sectional study, from which no casual relationship can be further explored. A future study in a cohort of subjects with BMI>40 kg/m2 with no overt CVD and also free of MS, esp. hypertension and diabetes mellitus, would be extremely useful to delineate the complexity of CVD pathophysiology in this population. Such individuals, however, are uncommon and thus difficult to identify.

In conclusion, we document in the current study that increased cIMT and reduced distensibility are present in Class III obesity. cIMT and distensibility correlate closely with traditional CVRF, adiposity and inflammatory markers, confirming the validity of these two important parameters in CVD detection in individuals with BMI>40 kg/m2. We also demonstrate, for the first time, elevated plasma EMP levels and unchanged circulatory CD133+/KDR+ angiogenic cells in Class III obesity.

Acknowledgments

We thank Ms Elizabeth Dewar and Ms Sofie Karapanagiotidis for their technical assistance on carotid imaging and measurement.

Funding Statement

This study was supported by a National Heart Foundation of Australia Project Grant (APP1012003) and in part by the Victorian Government’s Operational Infrastructure Support Program. MRS is a National Health and Medical Research Council of Australia (NHMRC) Career Development Fellow. AMD, JCD and JBD are NHMRC Fellows. Funding also provided by National Heart Foundation of Australia: http://www.heartfoundation.org.au/Pages/default.aspx, Victorian Government’s Operational Infrastructure Support Program: http://www.business.vic.gov.au/industries/science-technology-and-innovation/programs/medical-research-operational-infrastructure-program and National Health and Medical Research Council of Australia: http://www.nhmrc.gov.au/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Modulation of Intestinal Inflammation by Yeasts and Cell Wall Extracts: Strain Dependence and Unexpected Anti-Inflammatory Role of Glucan Fractions

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Jagadeesh Bayry, Editor

Abstract

Yeasts and their glycan components can have a beneficial or adverse effect on intestinal inflammation. Previous research has shown that the presence of Saccharomyces cerevisiae var. boulardii (Sb) reduces intestinal inflammation and colonization by Candida albicans. The aim of this study was to identify dietary yeasts, which have comparable effects to the anti-C. albicans and anti-inflammatory properties of Sb and to assess the capabilities of yeast cell wall components to modulate intestinal inflammation. Mice received a single oral challenge of C. albicans and were then given 1.5% dextran-sulphate-sodium (DSS) for 2 weeks followed by a 3-day restitution period. S. cerevisiae strains (Sb, Sc1 to Sc4), as well as mannoprotein (MP) and β-glucan crude fractions prepared from Sc2 and highly purified β-glucans prepared from C. albicans were used in this curative model, starting 3 days after C. albicans challenge. Mice were assessed for the clinical, histological and inflammatory responses related to DSS administration. Strain Sc1-1 gave the same level of protection against C. albicans as Sb when assessed by mortality, clinical scores, colonization levels, reduction of TNFα and increase in IL-10 transcription. When Sc1-1 was compared with the other S. cerevisiae strains, the preparation process had a strong influence on biological activity. Interestingly, some S. cerevisiae strains dramatically increased mortality and clinical scores. Strain Sc4 and MP fraction favoured C. albicans colonization and inflammation, whereas β-glucan fraction was protective against both. Surprisingly, purified β-glucans from C. albicans had the same protective effect. Thus, some yeasts appear to be strong modulators of intestinal inflammation. These effects are dependent on the strain, species, preparation process and cell wall fraction. It was striking that β-glucan fractions or pure β-glucans from C. albicans displayed the most potent anti-inflammatory effect in the DSS model.

Introduction

Probiotics are a popular alternative to antibiotics [1]. The positive effects of probiotics on humans and animals result either from a direct nutritional effect or a health effect, with probiotics acting as bioregulators of the intestinal microflora and reinforcing the host’s natural defences [2].

Saccharomyces cerevisiae var. boulardii (Sb) is described as a biotherapeutic agent in the clinical literature and is reported to be efficacious in the prevention of antibiotic-associated diarrhoea and colitis in humans [3], [4]. Orally administered Sb demonstrated clinical and experimental effectiveness in gastrointestinal diseases through modulation of host cell signalling pathways implicated in the pro-inflammatory response such as IL-1β and TNF-α. Sb exerts a trophic effect that restores intestinal homeostasis and activates expression of peroxisome proliferator–activated receptor-gamma which protects against gut inflammation [5].

It has recently been reported that Sb decreases inflammation and intestinal colonization by C. albicans in a BALB/c mouse model of colitis induced by dextran-sulphate-sodium (DSS) [6]. Interestingly, parallel studies indicated that Sb reduces C. albicans adhesion to human intestinal cell lines and decreases pro-inflammatory cytokine mRNA levels in response to C. albicans infection [7], [8].

Sb is generally administered as a lyophilized powder [9] and its use as a food additive has only been reported in a number of cases such as in the fermentation of raw vegetable materials [10] and incorporation into commercial yoghurts [11].Taxonomic studies indicate that Sb should be considered as a Saccharomyces cerevisiae strain [12], [13]. This leads to the question “do other strains of S. cerevisiae also possess probiotic properties?” [14].

In the present study, low doses of DSS were administered to mice for 2 weeks to induce colonic inflammation and promote the establishment of C. albicans colonization, followed by a 3-day restitution period. Either S. cerevisiae strains or glycan fractions were then administered daily by oral gavage for 2 weeks, starting 3 days after the C. albicans challenge, in order to assess their curative effects on both colonic inflammation and acceleration of colonic epithelial restoration. Using the DSS mouse model each dietary yeast was found to have its own effect on colitis and C. albicans colonization. The impact of orally administered glycan fractions extracted from S. cerevisiae that may reverse the adverse effects of DSS and C. albicans, and the biological activity of soluble β-glucan isolated from the C. albicans cell wall, were then investigated in this DSS mouse model.

Results

Comparison of the probiotic potential of S. cerevisiae var. boulardii and S. cerevisiae 1-1 strain

S. cerevisiae 1-1 strain (Sc1-1) was selected from our collection as having previously exhibited a probiotic effect. This yeast is prepared as an active dry yeast so that it can react quickly to its environment (Table 1). As Sc1-1 was comparable in vitro to Sb in terms of decreasing growth and germ tube formation by C. albicans (data not shown), the aim was to compare the Sc1-1 and Sb strains for their ability to reduce C. albicans colonization and intestinal inflammation.

Table 1

Yeast strains used in the investigation.

The model used is summarized in Fig. 1A and can be defined as “curative”. In this model, a single dose of C. albicans was administered to mice receiving DSS. Either the Sb or Sc1-1 strain was given 3 days later when C. albicans colonization was established. Low mortality was observed in mice that received DSS or DSS+C. albicans whereas none of the mice given the same regimen plus S. cerevisiae died (Fig. 1B).

Figure 1

Comparison of the probiotic potential of S. cerevisiae var. boulardii and S. cerevisiae 1-1 strain.

Concerning C. albicans colonization in mice that received DSS (Fig. 1C), the number of colony-forming units (CFUs) in stools gradually increased from the day of C. albicans administration. From S. cerevisiae administration on day 4 to the endpoint, a dramatic reduction in number of C. albicans CFUs was observed. No difference was observed between the two S. cerevisiae strains in their activity on C. albicans clearance (Fig. 1C).

The cumulated clinical and histological scores (Fig. 1D–E) were higher in mice that received DSS or DSS+C. albicans whereas they were reduced significantly by the administration of either Sb or Sc1-1 (Fig. 1D–E). Both strains were equally effective in reducing intestinal inflammation assessed by these parameters (Fig. 2).

Figure 2

Histological analysis of DSS-induced colitis in mice.

To analyze the possible mediators involved in the reduction of the inflammatory response to DSS and C. albicans in the colon following S. cerevisiae administration, we focused on levels of TNF-α and IL-10 mRNA as representative pro- and anti-inflammatory cytokines. As shown in Fig. 1F–G, administration of either Sb or Sc1-1 was associated with a significant reduction in TNF-α expression and increase in IL-10 production. No difference was observed between the two S. cerevisiae strains in their ability to redirect the inflammatory response.

Analysis of the anti-C. albicans and anti-inflammatory properties of other S. cerevisiae strains with different preparation processes

As both Sc1-1 and Sb strains displayed identical beneficial effects in this specific model the Sc1-1 strain was used as the reference strain for probiotic activity. In this part of the study, the influence of yeast preparation procedure and other yeast strains was investigated.

From an initial screening involving 10 strains or preparation procedures, five representative examples of strain activities were selected and compared to Sc1-1. These consisted of strains Sc1-2, Sc2, Sc3 and Sc4.

Sc1-1 showed important differences in mortality (Fig. 3A), ability to reduce C. albicans colonization (Fig. 3B) and a decrease in both histological and clinical scores (Fig. 3C–D). The Sc1-1 strain gave excellent results for all parameters, as did Sc3, which is used for its probiotic activities (Fig. 4) Interestingly, some of the beneficial effects induced by Sc1-1 were abolished when this strain was prepared as an instant dry yeast (Table 1). The most striking results were observed with the Sc4 strain, which did not display any particular effect on C. albicans colonization over 2 days compared to the other S. cerevisiae strains. However, the mice were extremely constipated, clinically inflamed and highly colonized with C. albicans starting from day 7. From this point on, we discontinued collecting faeces as the mice were becoming extremely ill. This yeast, which presents optimal growth at low temperatures, dramatically exacerbated both the clinical and histological scores and was associated with high mortality (around 80%). We also found that both C. albicans and Sc4 increased the staging of colitis in mice (Fig. 4). These high clinical and histological scores were associated with high numbers of C. albicans CFUs in different parts of the gut from moribund mice (data not shown).

Figure 3

Assessment of biological effect of S. cerevisiae 1-1 versus other S. cerevisiae strains.
Figure 4

Histological analysis of DSS-induced colitis in mice.

Finally, Sc2, another industrial strain used for the production of yeast proteins, displayed an “intermediate” behaviour with reduced beneficial effects and slight worsening of mortality and clinical scores.

Identification of the cell wall fractions supporting the beneficial and adverse effects on C. albicans colonization and inflammation

For this purpose, Sc2 was used as this strain can be easily induced to undergo autolysis in order to prepare cell wall extracts including mannoprotein (MP) and β-glucan fractions (Fig. 5).

Figure 5

Schematic diagram of glycan preparation from S. cerevisiae.

In contrast to living cells, none of the cell wall extracts induced mortality (Fig. 6A). However, the changes in body weight and clinical scores were worsened considerably by MP fractions while these parameters were ameliorated by β-glucan fractions (Fig. 6B, F). Notably, the MP fraction as well as the Sc2 strain induced an important loss in body weight up to day 9 that was correlated with its incapacity to control C. albicans colonization (Fig. 6C). By contrast, administration of β-glucan fraction maintained normal body weight, reduced inflammation scores and promoted C. albicans clearance (Fig. 6A–F). Although the body weight of mice receiving MP fractions started to increase from day 10, the clinical activity score was higher than that of mice receiving β-glucan fractions (Fig. 6E).

Figure 6

Effect of glycan fractions derived from S. cerevisiae on Candida DSS-treated mice.

Activity of the homologous C. albicans oligoglucoside fraction in the DSS mouse model

As the glucoprotein fraction from S. cerevisiae unexpectedly displayed a protective effect in this model, it was decided to investigate both the structure and biological activity of the β-glucan fraction from C. albicans. The harsh whole cell extraction procedure leads to fraction-1, known as yeast ghosts (Fig. 7). Fraction-1 (F1) was analyzed by fluorescence microscopy with various fluorescent probes specific for cell wall glycans in comparison to zymosan, which is widely used for β-glucan immunological studies (Fig. 8). Both zymosan and F1 were labelled with monoclonal antibody (mAb) 2G8 specific for β-1,3 glucans and WGA which binds to chitin (Fig. 8). In contrast to zymosan, which was stained with both Concanavalin A (ConA) and GNL, no mannose residue signals were observed for F1. Immunofluorescent staining with antibodies to β-mannose, liable to be synthesized by C. albicans, was also negative with F1 (data not shown). Thus, yeast ghosts have no mannose residues in their cell wall (Fig. 8).

Figure 7

Schematic diagram of β-glucan preparation from C. albicans.
Figure 8

Immunofluorescence staining of C. albicans ghost cells and zymosan with various fluorescent probes specific for yeast cell wall glycans.

MALDI-MS analysis of the soluble fraction derived from F1 (F2) established that it consisted of a highly polydisperse hexose polymer consisting of 3–27 hexose residues (Fig. 9A). According to its reactivity with anti β-1,3 glucans, this component was susceptible to zymolyase digestion which produced a set of small water soluble fragments (2–5 Glc), as demonstrated by thin-layer chromatography (data not shown) and MALDI-MS analyses (Fig. 9B). After purification of this fraction by reverse phase and adsorption chromatography, its structure was established by NMR as a mixture of β1,3-substituted glucan oligomers with free reducing ends. Furthermore, these oligomers were shown to be partially substituted by a random single β1,6 glucopyranose residue (Fig. 9C).

Figure 9

Structural analysis of glycan fraction.

Mortality, weight loss, clinical activity, histological score and C. albicans colonization

After structure characterization of the β-oligoglucoside fraction (F2) extracted from C. albicans, the biological activity of F2 was tested in the DSS mouse model. After C. albicans challenge, mice were given F2 (1 mg/day) from day3 up to the endpoint (Fig. 1A). F2 administration significantly prevented mouse mortality due to either DSS or DSS+C. albicans (Fig. 10A). In contrast to DSS and DSS+C. albicans mice, which developed severe colitis and lost body weight, F2 administration reversed the adverse effect of colitis and the mice showed a significant amelioration of both the clinical inflammation score and body weight (Fig. 10B–D). Histological examination of colon sections from mice receiving either DSS or DSS+C. albicans showed important colonic inflammation which was associated with mucosal cell loss, crypt damage, mucosal ulceration and accompanying submucosal oedema (Fig. 10C–D). F2 administration significantly reduced colonic inflammation due to either DSS or DSS+C. albicans (Fig. 10C–D). C. albicans colonization in DSS-treated mice showed a steady increase as assessed by the number of CFUs in faeces which was consistent with the high load of C. albicans recovered from the stomach, ileum and colon of this group of mice at the endpoint of the experiments (Fig. 10E–F).

Figure 10

Effect of β-oligoglucoside fractions derived from C. albicans on Candida DSS-treated mice.

In contrast to the higher numbers of C. albicans CFUs recovered from different compartments of the gut in DSS-treated mice, oral administration of F2 decreased the number of C. albicans CFUs recovered from stools and all gut segments of DSS-treated mice (Fig. 10E–F). Furthermore, the reduction in clinical and histological scores was consistent with these low numbers of C. albicans CFUs in different parts of the gut (Fig. 11 and Table 2).

Figure 11

Summary of the effects of S. cerevisiae strains or glycan fractions on Candida DSS-treated mice.
Table 2

Effect of different strains and cell wall extracts on C. albicans colonization and inflammation in the curative C. albicans DSS model.

Discussion

Excessive use of antifungal agents has been implicated in the emergence of antifungal resistance in C. albicans and constitutes a serious clinical problem in hospitals by affecting the natural balance of the intestinal microflora in these individuals [15]. The non-pathogenic yeast, Sb, which is widely prescribed for the treatment of antibiotic-induced gastrointestinal disorders and Clostridium difficile-associated enteropathies, has been shown to be an alternative approach to counterbalance the equilibrium of the intestinal microflora and modulate the innate immune defence [16], [17]. Orally administered Sb was successful in both the treatment of inflammatory bowel disease (IBD) and the elimination of C. albicans colonization [7], [8], [18][21].

Recently, it has been shown that Sb decreases both C. albicans colonization and intestinal inflammation in a mouse model of DSS-induced colitis [6]. Following this study, S. cerevisiae strains, MP and β-glucan fractions were screened in a mouse model of DSS-induced colitis. As Sb is considered taxonomically to be a strain of S. cerevisiae [12], [13], strain Sc1-1 was compared to Sb in the DSS model. Sc1-1 is a gastro-resistant strain that reacts rapidly to its environment and is widely used in the food industry. Incidentally, it was observed that both Sc1-1 and Sb strains reduced C. albicans filamentation in vitro and C. albicans adhesion to plastic-plate wells (data not shown). In the present study, we did not chose a prophylactic but a curative model in which the animals develop colitis with histological features that are similar to those seen in patients with IBD before starting their treatment.

In this model low doses of DSS were used in order to establish C. albicans colonization, followed by S. cerevisiae or yeast extracts administration to assess their effects on the inflamed colon and colonic epithelium restitution.

Two weeks of DSS administration were scheduled to induce moderate colonic inflammation in mice, with low mortality rates. A recent study by Samonis et al. showed that mice receiving a high daily oral dose of C. albicans (around 108 CFU/day) for 2 weeks did not respond to Sb treatment [22]. In our model, a single inoculum of C. albicans was used and Candida colonization was maintained naturally in the mouse gastrointestinal tract by the DSS-induced colitis since a high C. albicans dose could dramatically hide the beneficial effect of Sb. In the present study, and similar to the Sb strain, Sc1-1 decreased both C. albicans colonization and intestinal inflammation in terms of clinical and histological score and mortality. Another notable finding was the acceleration of colonic epithelium restoration in mice treated with these dietary yeasts leading to the absence of submucosal oedema and epithelial erosion. Mechanistically, a recent report on intestinal inflammation showed that Sb secretes motogenic factors that enhance intestinal epithelial cell restitution [23].

Regarding the RT-PCR results, both Sb and Sc1-1 reduced the expression levels of pro-inflammatory cytokine TNF-α mRNA in the colonic mucosa with subsequent enhancement of IL-10 mRNA expression that inhibits intestinal injury [24]. Additionally, different pro-inflammatory cytokines were investigated in this set of experiments and were consistent with TNF-α expression. Further investigation is required to determine the role of Th17/Treg responses in different sets of experiments [25], [26]. A recent study in patients with IBD showed that Sb reduced TNF-α production and significantly inhibited T-cell proliferation induced by intestinal inflammation [19]. Generally, the biological activities of S. cerevisiae in gastrointestinal inflammatory conditions are mediated through modulation of host pro-inflammatory responses not only by the whole yeast, but also by secreted factors able to interfere with host signalling molecules that control inflammation at different levels such as NF-κB [27], [28]. Sb produces a soluble anti-inflammatory factor that inhibits NF-κB activation and attenuates pro-inflammatory signalling in host cells. In addition, Sb stimulates IL-10 secretion from intraepithelial lymphocytes infected by C. albicans and Escherichia coli [29]. As Sc1-1 was shown to be comparable to Sb and presents the same beneficial features against C. albicans and intestinal inflammation, Sc1-1 was considered as the reference strain in the DSS model. To assess if the observed anti-inflammatory properties were strain-dependent, other S. cerevisiae strains were selected deliberately for their high phenotypic diversity. The possible influence of yeast preparation process on anti-C. albicans activity was also studied. Surprisingly, some strains had a dramatic effect in the DSS mouse model and the process of yeast preparation also had an influence on the yeast’s biological properties [30]. Each strain selected in this study was well characterized in vitro in terms of cell growth, osmostress, fermentation, viability and metabolites. However, different factors could influence the biological activity of the strains when introduced by gavage in the DSS mouse model: (i) the resistance of the cell wall related to the yeast preparation process [30]; (ii) the viability of the strain in the stomach, ileum and colon; (iii) its interaction with the microflora and intestinal mucosa [31]; and (iv) its ability to produce soluble anti-inflammatory factors in the milieu triggering expression of mediators in the intestinal epithelium and cells of monocyte lineage present in the submucosae [27]. Altogether, each strain has its own unique properties and supports specific activities within the host. The in vitro findings, together with the results for all S. cerevisiae strains analyzed in this study, suggest that Sc1-1 has beneficial biological activities reversing all aspects of colitis, including histological damage, diarrhoea and mucosal levels of the pro-inflammatory mediator TNF-α.

The cell wall is an essential structural component of yeast cells playing a central role in the interaction of yeasts with their environment. Unfortunately, the biological activities of S. cerevisiae cell wall components are still unclear in terms of C. albicans colonization and intestinal inflammation. Two components (MP and β-glucans) produced industrially were explored in our experimental model. With MP fraction administration, C. albicans colonization was not consistent with intestinal inflammation parameters, suggesting that MP fractions have differential effects on C. albicans colonization and intestinal inflammation. In contrast to MP fractions, GP fraction administration decreased the number of C. albicans CFUs concomitantly to all intestinal inflammation parameters.

Both of these components are known to be potent immunological activators, but their mechanisms of action are different and controversial [32][34]. As an example, both MP and β-glucans act positively on tumour cells and several microbial infections [34], [35]. Conversely, administration of β-glucans derived from C. albicans has been shown to exacerbate arthritis in mice [36]. Structurally, MP have extensive N-and O-linked mannosylation which serve as ligands for galectin-3 (Gal-3), mannose receptor and DC-SIGN on macrophages and dendritic cells [37]. Different MP express β-Man epitopes, which have been identified as the principal ligand for Gal-3 [38]. In a previous study using the DSS model with C. albicans, Gal-3 knock-out mice were less affected by intestinal inflammation and C. albicans colonization than wild-type animals [39]. Recently, it was shown that C. glabrata deficient in β-Man was less virulent in DSS-treated mice as revealed by low clinical and histological scores and reduction of C. glabrata colonization [40]. β-glucans have affinities towards different receptors such as CD11b/CD18 [41], located on neutrophils, or Dectin-1 on macrophages [42]. This results in β-glucan activation of cytokine production and in turn activation of adaptive immunity. Thus, β-glucans attenuate the impact of colitis compared to MP [43].

As our results also showed a beneficial effect of β-glucans on inflammation/colonization, insoluble ghost yeast cells derived from C. albicans containing β-glucans were prepared and compared to zymosan which is widely used in β-glucan studies where many investigators refer to it as β-glucan [44], [45]. Zymosan stimulates the production and activity of pro-inflammatory cytokines [45]. Additionally, when chemically characterized zymosan containing only β-(1–3)-glucans was added to macrophage cells, the production of IL-10, reactive oxygen species (ROS) and TNF-α increased in a dose-dependent way [46]. Bonifazi et al. demonstrated the capacity of zymosan to activate both inflammatory and tolerogenic dendritic cells (DCs) leading to the triggering of both Th17 and Treg cells in vivo [47]. Our observations showed that zymosan contains both mannans and β-glucans exposed together on the cell wall surface in comparison to C. albicans ghosts that contain only β-glucans. This evidence prevented us from further studies on zymosan. Different observations showed that the biological activities of soluble β-glucans differ from those of cell-associated β-glucans [32], [48], [49]. Ishibashi et al. showed that insoluble cell wall β-glucans induced intensive inflammatory and immunomodulating activities compared to soluble β-glucans [49]. Following the β-glucan analysis, the chemical structure of the soluble β-glucan fraction derived from C. albicans ghosts was characterized and its biological activities were tested in the DSS mouse model. Interestingly, orally administered β-glucans from C. albicans decreased intestinal inflammation and C. albicans colonization.Several reports show that β-glucan enhances the immune response and improves the clearance of pathogenic bacteria in animal models [50][52]; this supports our findings that smaller oligoglucosides derived from C. albicans showed beneficial activities against C. albicans and these results are comparable to β-glucans derived from S. cerevisiae. However, it may also be hypothesized that these individual oligoglucosides could block receptors such as dectin-1 and CD11b/CD18 and prevent multivalent binding necessary for strong triggering of the inflammatory responses [53]. Besides the importance of yeast molecules sensing for immune response, a third player may also possibly act in the general interplay. This is the mouse microbiota. Oligosaccharides are well known prebiotics active on the intestinal flora [54], [55], and although such a role has not been investigated for C. albicans derived oligoglucosides it cannot be ruled out. Altogether, these results demonstrate that oligoglucosides behave differently from the original C. albicans whole yeast cells in the DSS mouse model.

In summary, Sc1-1 was found to be comparable to Sb and had beneficial biological activities against C. albicans and intestinal inflammation. Clinical trials are currently being conducted with Sc1-1 and promising results have been seen in patients with IBD. In the second part of this study, we focused on cell wall components involved in direct contact with the host and demonstrated that, in contrast to MP, β-glucan fractions from either S. cerevisiae or C. albicans have a more potent anti-inflammatory effect against colonic colitis induced by DSS in mice. In conclusion, this study generated some progress in deciphering the nature of the yeast molecular components differentially favouring inflammation and/or C. albicans clearance. Future studies will include experiments on oligosaccharide administration to mice in order to determine how these glycans stimulate the growth of beneficial bacteria in the gut and boost the immune system providing therapeutic perspectives for digestive disorders and life-threatening fungal infections of endogenous origin.

Materials and Methods

Yeast strains

The yeast strains used in this study are shown in Table 1.

Preparation of β-glucan fractions from yeasts

The composition in dry matter of spray-dried S. cerevisiae Sc2 cell wall fractions is shown in Table 3 and the preparation procedure for MP and β-glucan fractions from the cell wall of the same strain is shown in Fig. 1. The fractionation and digestion procedure for extraction of the β-glucan fraction from C. albicans is summarized in Fig. 2. Briefly, the cell pellet of C. albicans (50 g wet weight) was incubated twice in 200 ml of 1 M NaOH at 70°C for 30 min. After washing with distilled water, the supernatant was removed and the pellet was oxidized with 100 mM NaIO4 (Sigma-Aldrich, France) at room temperature for 24 h in the dark [56]. After completion of the reaction, excess periodate was destroyed by adding ethylene glycol. After washing several times with water, the pellet was reduced with 1 M NaBH4 (Sigma Aldrich, France) at room temperature. The reaction was terminated by lowering the pH to 5 by the addition of acetic acid. After washing several times with water, the insoluble fraction was then lyophilized to produce fraction-1. Fraction-1 was treated with zymolyase 20T (0.2 mg/mL, Immuno™; ICN Biomedicals Inc.) at 37°C for 3 h. Zymolyase inactivation was performed at 70°C for 5 min. After centrifugation, the supernatant was dialyzed against distilled water. The dialyzed solution was loaded onto a Sep-Pak C18 column (Alltech) equilibrated with 0.1% TFA (trifluoroacetic acid). Eluate-1 was evaporated and the resulting oligoglucosaccharides were dissolved in distilled water and further purified on a carbograph column (Alltech carbograph SPE column). Eluate-2 from the carbograph column was lyophilized to produce fraction-2 (F2). Fluorescence microscopy was performed to assess surface oligomannose expression on fraction-1 in comparison to zymosan (Sigma-Aldrich). Fraction-1 and zymosan suspensions deposited on slides were incubated with either monoclonal antibody (mAb) 2G8 specific for β-1,3 glucans [57], [58], or wheat germ agglutinin(WGA), which binds to chitin [59], or Concanavalin A (ConA) or Galanthus nivalis lectin (GNL) or DAPI, as described previously [6], [40]. For animal experimentation, fraction-2 was suspended in water and divided into 200 µL aliquots (each aliquot of 200 µL contained 1 mg of β-glucans).

Table 3

Composition in dry matter of spray-dried Sc2 cell wall fractions.

Structural analysis of β-glucans

NMR experiments were performed at 300 K using a Bruker AvanceII 900 MHz spectrometer equipped with a 5 mm triple-resonance cryoprobe. Prior to NMR spectroscopic analyses in deuterium, oligosaccharides were repeatedly exchanged in 2H2O (99.97% 2H, Euriso-top; Saint-Aubin, France) with intermediate freeze-drying and finally dissolved in 2H2O and transferred into Shigemi (Allison Park, USA) tubes. Chemical shifts (ppm) were calibrated taking the methyl group from internal acetone at δ1H 2.225 and δ13C 31.55 ppm. MALDI-TOF mass spectra were acquired on a Voyager Elite DE-STR mass spectrometer (Perspective Biosystems, Framingham, MA). Prior to analysis, samples were prepared by mixing 1 µL of oligosaccharide solution (1–5 pmol) with 1 µL of 2,5 dihydroxybenzoic acid matrix solution (10 mg/mL in CH3OH/H2O, 50[ratio]50, vol/vol) directly on the target. Between 50 and 100 scans were averaged for each spectrum.

Animals

Six- to 8-week-old female BALB/c mice were used. All mice were maintained by Charles River Laboratories (France). Four sets of experiments were performed independently and each experiment was divided into control groups (eight mice/cage), including assessment of the effect of DSS alone, and experimental groups (10 mice/cage).

Ethics statement

All mouse experiments were performed according to protocols approved by the Subcommittee on Research Animal Care of the Regional Hospital Centre of Lille, France, and in accordance with the European legal and institutional guidelines (86/609/CEE) for the care and use of laboratory animals.

Inoculum preparation and induction of colitis

Each animal was inoculated on day 1 by oral gavage with 200 µL of phosphate-buffered saline (PBS) containing 107 live C. albicans cells. Mice were given 1.5% DSS (MW 36–50 kDa; MP Biomedicals, LLC, Germany) in drinking water from day 1 to day 14 to induce intestinal inflammation. Three days after C. albicans oral challenge, mice were administered by oral gavage with a single-daily dose of either 107 lyophilized S. cerevisiae strains or 1 mg of β-glucan fraction for 2 weeks. Lyophilized S. cerevisiae strains were rehydrated for 30 min in PBS at 37°C before administering to the mice [60]. The presence of yeasts in the intestinal tract was followed daily by performing plate counts of faeces (approximately 0.1 g/sample) collected from each animal [39]. The faecal samples were suspended in 1 mL saline, ground in a glass tissue homogenizer and plated onto Candi-Select medium (Bio-Rad Laboratories, Marnes la Coquette, France). This chromogenic medium is designed for the isolation of yeasts from clinical specimens and is intended to differentiate medically important yeast species depending on the colour of the colonies [61]. Colonies of C. albicans were counted after 48 h incubation at 37°C. The results were noted as colony forming units (CFUs)/µg of faeces.

Presence of C. albicans colonization in the gastrointestinal tract

To check for C. albicans colonization, the animals were sacrificed and the gastrointestinal tract was removed and separated into the stomach, ileum and colon. The tissues were cut longitudinally. After removal of intestinal contents, the tissues were washed several times in PBS to minimize surface contamination from organisms present in the lumen [62]. Serial dilutions of homogenates were performed. The results were noted as C. albicans CFUs/mg of tissue.

Assessment of clinical parameters

The mortality rate of DSS-treated mice was determined daily and a colon biopsy was taken immediately after death for histological analysis. Total body weight was measured daily. The data are expressed as mean percent change from starting body weight. Daily clinical activity score ranging from 0 to 8 was calculated as described elsewhere [39], [63].

Determination of histological score

Rings of the transverse part of the colon were fixed overnight in 4% paraformaldehyde-acid and embedded in paraffin for histological analysis. Cross-sections (4 µm thick) were stained with haematoxylin-eosin (Sigma-Aldrich, France). Histological scores were evaluated by two independent, blinded investigators who observed two sections per mouse at magnifications of ×10 and ×100. The scores were determined in accordance with Siegmund et al. [63] and the sections were evaluated for the following two subscores: (i) a score for the presence and confluence of inflammatory cells, including neutrophils, in the lamina propria and submucosa or transmural extension; and (ii) a score for epithelial damage, focal lymphoepithelial lesions, mucosal erosion and/or ulceration and extension to the bowel wall. The two subscores were added together and the combined histological score ranged from 0 (no changes) to 6 (extensive cell infiltration and tissue damage).

Real-time mRNA quantification

Total RNA was isolated from colon samples using a NucleoSpin RNA II kit (Macherey-Nagel, France) following the manufacturer’s instructions, with 20–50 units of DNase I (RNase-free) at 37°C for 30 min to avoid contamination with genomic DNA. RNA quantification was performed by spectrophotometry (Nanodrop; Nyxor Biotech, France). Reverse transcription of mRNA was carried out in a final volume of 26 µL from 1 µg total RNA using 300 U M-MLV reverse transcriptase (Invitrogen, France) according to the manufacturer’s instructions with 500 ng oligo(dT) 12–18 and 50 U ribonuclease inhibitor (RNase-Out, Promega). PCR was performed using an ABI 7000 prism sequence detection system (Applied Biosystems, France) with SYBR green (Applied Biosystems, France). Amplification was carried out in a total volume of 25 µL containing 0.5 µL of each primer [6], [39] and 1 µL of cDNA prepared as described above. SYBR green dye intensity was analyzed using Abiprism 7000 SDS software (Applera Corp.). All results were normalized to the housekeeping gene β-actin.

Statistical analysis

Data are expressed as the mean ± SE of five mice in each group. All comparisons were analyzed by the Mann-Whitney U test. Statistical analyses were performed using the StatView™ 4.5 statistical program (SAS Institute Inc., Meylan, France). Differences were considered significant when the P value was <0.05.

Acknowledgments

The authors thank Nadine FRANÇOIS, Caroline DUBUQUOY, Emilie GANTIER, and Edmone ERDUAL for their excellent technical assistance and Val HOPWOOD for editing the manuscript.

Funding Statement

This work was funded by the program LEVACI issued from the French Government research plan FUI 5th AAP (DGE-Lille University contract number 082906131, European funds FEDER and local funds from the Région Nord-Pas de Calais, Lille Métropole Communauté Urbaine). LEVACI partners belong to research clusters Nutrition-Santé-Longévité and Végépolys. This work was also funded by the FP7 Health 260338 “ALLFUN” project “Fungi in the setting of inflammation, allergy and auto-immune diseases: translating basic science into clinical practices.” The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Herpes Zoster Vaccine Effectiveness against Incident Herpes Zoster and Post-herpetic Neuralgia in an Older US Population: A Cohort Study

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Stephan Harbarth, Academic Editor
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Abstract

Background

Herpes zoster is common and has serious consequences, notably post-herpetic neuralgia (PHN). Vaccine efficacy against incident zoster and PHN has been demonstrated in clinical trials, but effectiveness has not been studied in unselected general populations unrestricted by region, full health insurance coverage, or immune status. Our objective was to assess zoster vaccine effectiveness (VE) against incident zoster and PHN in a general population-based setting.

Methods and Findings

A cohort study of 766,330 fully eligible individuals aged ≥65 years was undertaken in a 5% random sample of Medicare who received and did not receive zoster vaccination between 1st January 2007 and 31st December 2009.

Incidence rates and hazard ratios for zoster and PHN were determined in vaccinated and unvaccinated individuals. Analyses were adjusted for age, gender, race, low income, immunosuppression, and important comorbidities associated with zoster, and then stratified by immunosuppression status. Adjusted hazard ratios were estimated using time-updated Cox proportional hazards models.

Vaccine uptake was low (3.9%) particularly among black people (0.3%) and those with evidence of low income (0.6%). 13,112 US Medicare beneficiaries developed incident zoster; the overall zoster incidence rate was 10.0 (9.8–10.2) per 1,000 person-years in the unvaccinated group and 5.4 (95% CI 4.6–6.4) per 1,000 person-years in vaccinees, giving an adjusted VE against incident zoster of 0.48 (95% CI 0.39–0.56). In immunosuppressed individuals, VE against zoster was 0.37 (95% CI 0.06–0.58). VE against PHN was 0.59 (95% CI 0.21–0.79).

Conclusions

Vaccine uptake was low with variation in specific patient groups. In a general population cohort of older individuals, zoster vaccination was associated with reduction in incident zoster, including among those with immunosuppression. Importantly, this study demonstrates that zoster vaccination is associated with a reduction in PHN.

Please see later in the article for the Editors’ Summary

Introduction

Herpes zoster is a significant public health problem affecting 1 million individuals in the US per year and associated with important sequelae [1],[2]. Herpes zoster occurs following reactivation of latent varicella zoster virus (VZV) infection and presents with a painful vesicular rash, which frequently in older individuals leads to prolonged pain, post-herpetic neuralgia (PHN), with a major impact on quality of life [2]. Vaccine efficacy has been shown in trials [3],[4]; in a selected insured population [5]; and among people with any of five specific immune-mediated diseases [6] but not among an unselected population in a clinical setting. Zhang et al. demonstrated that despite Advisory Committee for Immunization Practices (ACIP) recommendations, individuals with immunosuppression received the live herpes zoster vaccine in clinical practice [7]. The lack of adherence to ACIP recommendations on vaccination is not entirely surprising given that individuals with immunosuppression are not only at increased risk of incident herpes zoster but also at significantly increased risk of herpes zoster complications, in particular prolonged, severe PHN [8],[9]. Previous research has suggested that the varicella vaccine may be efficacious and safe in people with immunosuppressive disorders [10][12]. Similar evidence about vaccine effectiveness (VE) is lacking in relation to the zoster vaccine in individuals with serious immune suppression, beyond effectiveness among those with the selected immune-mediated disorders examined to date.

Important outstanding research questions with great relevance to policy include VE in unselected population-based elderly US populations; this includes effectiveness against PHN, which has not been assessed in routine practice. The report by Zhang et al. also highlights the additional importance of studying further VE in those with immunosuppression [6]. This is the first study to the best of our knowledge to assess the effectiveness of herpes zoster vaccine against both incident herpes zoster and PHN in an unselected older population including those with immunosuppression.

Methods

Ethics

Ethics approval was obtained from Centers for Medicare & Medicaid Services (CMS) (data use agreement 21520) and the Ethics committee of the London School of Hygiene and Tropical Medicine. Any data cell containing fewer than 11 beneficiaries have not been shown as per the CMS Data Use Agreement.

Data Source

Medicare is a US administrative claims program mainly for individuals aged >65 y covering 15% of the US population. There are 44 million beneficiaries, of which more than half the individuals are aged 65–75 y.[13] This study was based on the 5% random Medicare Standard Analytic Files (SAF) including Denominator, Inpatient hospital discharge records (MedPAR), Physician/Supplier (Carrier) and Outpatient files from January 1st 2007 to December 31st 2009 obtained from the CMS.

Study Population

Study participants were aged 65 y or greater with at least 12 mo continuous enrolment in Medicare parts A (which covers inpatient care) and B (physician services and facility charges) and at least 6 mo continuous enrolment in part D (drug benefits) of Medicare. The start of follow-up was the first date an individual fulfilled all the eligibility criteria with an additional 12-mo baseline pre-study observation period added to ensure observation of incident rather than prevalent zoster. End of follow-up was defined as the earliest of end of eligibility, date of death, development of herpes zoster, or the end of the study period. Individuals enrolled in health maintenance organizations or Medicare Advantage plans were excluded from the study as their records are not processed by CMS, hence information on clinical events is not available. Individuals with episodes of herpes zoster in the first year pre-study observation period were excluded from analysis to exclude prevalent cases. Additionally, individuals who received the herpes zoster vaccine during the baseline pre-study observation period were excluded from analysis (Figure 1).

Figure 1

Flow chart of analysis cohort.

Exposure

Herpes zoster vaccine was identified based on the presence of Current Procedural Terminology (CPT) code 90736. Additionally, specific National Drug Codes (NDCs) for herpes zoster vaccine were identified. A definite administration date was considered present if a CPT code 90741 or Healthcare Common Procedural Coding system (HCPCS) code G0377 was present within 7 d of vaccine purchase; otherwise the date of recording of the NDC code for herpes zoster vaccine was considered to be the administration date.

Outcomes

Incident herpes zoster cases were identified as those with both the presence of a specific International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) diagnostic code for herpes zoster, excluding those with specific ICD-9-CM codes for PHN, and the use of antivirals, including acyclovir, famciclovir, or valacyclovir, within 7 d either before or after the diagnostic code for herpes zoster [6]. Cases were identified from outpatient, inpatient, or health care provider (carrier) files. The approach of using both the presence of diagnostic codes and receipt of antivirals was proposed by Zhang et al. to increase the positive predictive value of a herpes zoster diagnosis in an administrative data source [6]. Incident herpes zoster was defined as an episode of herpes zoster without any evidence of herpes zoster or PHN for at least 1 y previously. A sensitivity analysis was undertaken defining incident herpes zoster as the presence of an ICD-9 code for herpes zoster irrespective of receipt of antiviral therapy. PHN was identified using a modification of the method proposed by Klompas et al. for administrative sources [14]. On the basis of this method, PHN was identified as those with a first episode of zoster with a further zoster diagnostic code after 90 d with a relevant prescription for analgesia, anticonvulsant, or antidepressant therapy on the same day as the recorded consultation. The presence of codes for non-specific neuralgia or for neurological complications of zoster after 90 d was also consistent with PHN. The PHN analysis was repeated after 30 d using the same diagnostic criteria.

Covariates

Current age was determined using date of birth identified from the Medicare beneficiary file; age was categorized into 5-y age bands as a time-varying covariate. Age at the start of the study was also modelled as a continuous variable in a sensitivity analysis. Race was identified from the Medicare SAF Denominator files, derived from the Social Security Administration’s master beneficiary record (designated via self-report) and categorized into white, black, and other (not including those with missing data on race). It is critical to study race in any study of zoster epidemiology as incidence rates in individuals with black skin are significantly lower than in white individuals [15]. “State buy -in” at any point during follow-up was assessed as a marker for low income. “State buy-in” reflects that the state pays Medicare premiums for an individual who is eligible due to low income. It can be assumed that people with state buy-in have resources that are less than twice that of the Supplemental Security Income threshold; hence “state buy-in” can be used a proxy for low income. We also determined the proportions of individuals receiving herpes zoster vaccination and developing incident herpes zoster by state and used these data to define quintiles of states defined by each of these two variables.

Immunosuppression status was identified as a time-varying covariate. Once individuals developed leukaemia, lymphoma, or HIV, as determined by the presence of two diagnostic ICD-9-CM codes on different days within outpatient, inpatient, or provider files, they were defined as being immunosuppressed from that point forward. These specific disorders were selected as these are specific ACIP contraindications for the zoster vaccine [7].

Immunosuppressive medications were identified from the part D drug files and an individual was defined as being immunosuppressed for 6 mo following the prescription of any immunosuppressive medication. If a patient received a further script during that period, they remained immunosuppressed. Other comorbidities including immune-mediated disorders or others previously identified as being associated with increased risks of zoster such as chronic obstructive pulmonary disease (COPD), diabetes, and systemic lupus erythematosis (SLE) were identified from medical records by the presence of two ICD-9-CM codes on different days in the outpatient or carrier (provider) files or one or more codes from inpatient records. Individuals with autoimmune disorders such as SLE were considered immunocompetent unless they received immunosuppressive therapy; these disorders are not ACIP contraindications to vaccination.

Statistical Analysis

Characteristics associated with receipt of the herpes zoster vaccine were explored by examining proportions of person-years of follow-up contributed by demographic and clinical attributes. Incidence rates for herpes zoster and PHN overall and by population characteristics were determined by identifying the number of events divided by person-years of follow-up. Cox regression was used to derive hazard ratios for herpes zoster and PHN in the vaccinated compared with the unvaccinated, adjusting for relevant confounders identified from the previous literature [3],[15], including age, gender, race, low income, immunosuppression, and comorbidities associated with herpes zoster, with age and immunosuppression being included in the analysis as time-varying covariates. Other comorbidities including COPD were treated as binary variables. State quintiles of vaccination and zoster incidence were added to the model to determine if they were confounders. Checks for collinearity were undertaken by assessing variance inflation factors for independent variables. Interaction terms were explored for associations between vaccination and age group and gender. VE was calculated as (1 – the adjusted hazard ratio). Stratified analysis of VE by immune status was adjusted for demographic characteristics with a sensitivity analysis adjusting for other comorbidities. A further sensitivity analysis was undertaken assessing VE against PHN using logistic regression amongst those with zoster with at least 6 mo of follow-up following zoster. All analyses were undertaken using STATA (version 11.0).

Results

Vaccination Rates

Of the 766,330 eligible participants, 29,785 (3.9% of people; 2.1% of person-time) had herpes zoster vaccination during the study period. Vaccination rates were lower in the oldest age group (1.5% in those aged 80 y or greater), in black individuals (0.3% compared to 2.4% in white individuals), and lower in those with evidence of low income (Table 1)—0.6% in those with evidence of low income were vaccinated as compared to 2.6% in individuals with no evidence of low income. 140,925 individuals were immunosuppressed at some point during follow-up and 4,469 of these individuals were immunosuppressed at the time of herpes zoster vaccination.

Table 1

Person-years by vaccination status and characteristics.

Herpes zoster Incidence Rates

Incidence rates for herpes zoster using the antiviral definition were higher in older age groups, in women, in those with any immunosuppression (adjusted hazard ratio 1.80 [95% CI 1.70–1.90]) and in those with specified immune-mediated disorders, including inflammatory bowel disease and SLE, and other disorders such as chronic kidney disease and COPD (Table 2). Lower incidence rates were seen in people who reported being black (adjusted hazard ratio 0.51 ([95% CI 0.47–0.56]) and those with any evidence of low income (adjusted hazard ratio 0.86 [95% CI 0.82–0.90]).

Table 2

Incidence rates for herpes zoster by disease definition and patient characteristics.

Herpes zoster Vaccine Effectiveness

Overall, 154 vaccinees experienced incident herpes zoster episodes (defined using the specific antiviral definition) during 28,291 person-years of follow-up compared to 12,958 events in 1,291,829 person-years of follow-up in those not vaccinated, giving an incidence rate of herpes zoster in vaccinees of 5.4 (95% CI 4.6–6.4) per 1,000 person-years compared to 10.0 (95% CI 9.8–10.2) per 1,000 person-years in those not vaccinated. All variance inflation factors were less than 1.2, suggesting collinearity was not a major issue, and no significant interactions were detected. The overall vaccine effectiveness (VE) for herpes zoster in vaccinees adjusted for age, gender, race, immunosuppression, low income, and comorbidity was 0.48 (95% CI 0.39–0.56) (Table 3). Incorporating age at the start of the study as a continuous variable did not change study findings: adjusted VE, 0.48 (95% CI 0.40–0.56). The median time to vaccine failure was 168 d. In immunocompromised vaccinees, there were 24 events in 1,981 person-years of follow-up, giving an adjusted VE of 0.37 (95% CI 0.06–0.58). Adjusting for state quintiles of either proportions receiving herpes zoster vaccination or proportions with incident herpes zoster did not modify study findings (adjusted VE 0.48 [95% CI 0.30–0.56] and 0.48 [95% CI 0.30–0.56, respectively]). Proportions vaccinated per state varied from 0.05% to 11.02% (Figure 2) and proportions of individuals developing incident herpes zoster during follow-up per state varied from 0% to 7%. At 90 d or greater following zoster, the adjusted VE was 0.59 (95% CI 0.21–0.79) for PHN in vaccinees compared to those not vaccinated, after adjusting for age, gender, race, and other comorbidities (numbers suppressed to remain compliant with CMS’s small-sized cell privacy policy). At 30 d or longer following zoster, 16 vaccinees developed PHN during 71,457 person-years of follow-up compared to 1,665 events during 2,563,404 person-years of follow-up in those not vaccinated, giving an adjusted VE of 0.62 (95% CI 0.37–0.77) for PHN, after adjusting for age, gender, race, immunosuppression status, and other comorbidities (Table 4). Results of the logistic regression analysis amongst those with zoster showed materially similar estimates of protection against PHN, albeit with wider confidence intervals (adjusted VE 0.64 [95% CI 0.11–0.85]). Lower VE against incident herpes zoster and PHN was seen when using the general rather than the specific disease definition.

Figure 2

Percentage vaccinated by state.
Table 3

Zoster vaccine effectiveness against incident herpes zoster by characteristics and disease definition.
Table 4

Zoster vaccine effectiveness against PHN by characteristics and disease definition.

Discussion

This is the first population-based study, to the best of our knowledge, to demonstrate the effectiveness of herpes zoster vaccination against PHN in a routine population setting. Study findings are consistent with efficacy data from the Shingles Prevention Study (SPS) randomised controlled trial (RCT) [3]. This work complements the findings of previous observational studies that have shown effectiveness of herpes zoster vaccination in immunocompetent insured individuals in southern California and in older individuals with selected immune-mediated diseases, as this study is the first study to the best of our knowledge to determine effectiveness against incident herpes zoster in a population-based cohort of older individuals across the US, not restricted by geographic region, immune status, or insurance status [5],[6].

Low uptake of herpes zoster vaccination (4%) was seen overall with variations in uptake by age, race, and low income levels. Overall VE of 48% was demonstrated against incident herpes zoster, 62% against PHN after 30 d and 59% against PHN after 90 d. In immunosuppressed individuals, VE against incident herpes zoster was 37%.

In the SPS RCT, Oxman et al. demonstrated herpes zoster vaccine efficacy against incident herpes zoster (51%) and PHN (67%) in 38,546 immunocompetent individuals (19,270 of whom were vaccinated) aged 60 y or greater with no history of herpes zoster [3]. Subsequent to this study, Schmader et al. performed an RCT in 22,439 immunocompetent individuals aged 50–59 y in North America and Europe, demonstrating vaccine efficacy of 69.8% for incident herpes zoster; efficacy against PHN was not determined [4]. Our findings are closer to those of the SPS study with similar estimates and confidence intervals for VE against incident zoster, which likely relates to the older age of participants in our study population. Our findings for VE against PHN were similar to those observed in the SPS trial [3]. RCTs typically have excellent internal validity, but post-licensure observational studies are necessary to inform generalisability of research findings.

Our study also confirms the results of the study by Tseng et al., which demonstrated the effectiveness of the herpes zoster vaccine against herpes zoster incidence in 75,761 immunocompetent vaccinees aged 60 y or greater matched (1[ratio]3) to unvaccinated members; all study participants were fully insured individuals in Kaiser Permanente Southern California [5]. The authors reported VE of 55% against incident herpes zoster, which is very similar to our estimates, despite differences in the study population. Zhang et al. assessed herpes zoster VE in 463,541 Medicare beneficiaries aged 60 y or greater with a restricted range of immune-mediated diseases and 18,683 vaccinees and found an adjusted hazard ratio of 0.61 (95% CI 0.52–0.71) overall [6]. The lower VE in this population likely reflects their underlying immunosuppression. The authors did not observe an increase in risk of herpes zoster following vaccination in those with immune-mediated disease, nor did they detect any cases of herpes zoster in individuals vaccinated while on biologic therapy.

Medicare is an administrative data source so some misclassification of exposures and outcomes is possible. However, this misclassification is likely to be random leading to the possibility of bias towards the null. Despite taking steps to guard against misclassification of the PHN by using the method proposed by Klompas et al. [14] for administrative data sources, the incidence of PHN in this study is lower than in the SPS [3]—1.38 per 1,000 person-years in the placebo group compared to 0.65 per 1,000 in the unvaccinated in our study—which might suggest some misclassification. In a recent study combining administrative data with medical record review, 3.9% of those with zoster developed PHN, which is lower than the 6.7% of people observed in this study [16]. There is a possibility that we underestimated herpes zoster vaccine uptake if individuals paid for their own vaccination; however, given that all of these individuals have part D Medicare (drug benefit) coverage, individual payment is unlikely because of the cost of the vaccine (US$159 for a single dose, not including administration costs). As these are observational data, the exposure—herpes zoster vaccination—was not randomly allocated. Our study demonstrates that vaccine uptake was not random and was likely to have been influenced by the demographic characteristics of beneficiaries. As data on exposures and outcomes were not collected for research purposes, there are unmeasured potential confounders including smoking and obesity, which are not routinely recorded in an administrative data source, despite the availability of diagnostic codes. Previous studies have not suggested that either of these covariates are major risk factors for the development of incident herpes zoster or PHN and, therefore, they are unlikely to confound the associations [15]. The study period was relatively short; the first 12 mo following eligibility was excluded to enable study of incident rather than prevalent herpes zoster. This limited duration results in the inability to study long-term vaccine effects but does not impact the study of VE. Additionally, while VE was assessed in individuals with immunosuppression, assessment of adverse effects or vaccine safety was not the hypothesis under study. The number of vaccinated immunosuppressed individuals in the study was modest resulting in a lack of precision of the estimate of VE in this group.

Our study is a large population-based cohort; the size of the cohort gives sufficient statistical power to study VE against herpes zoster and PHN and results are estimated relatively precisely; our findings would be unlikely if there was no effect in the population. In addition, Medicare beneficiaries are reasonably representative of the general US elderly population, with 98% of Americans aged 65 y or greater being enrolled in Medicare in 2009, increasing the generalisability of our findings [13]. Medicare datasets have high quality data available on demographic details of beneficiaries and clinical encounters, including prescription data. A strict definition for herpes zoster was used and therefore misclassification of incident herpes zoster is not likely, although it is not possible to completely exclude misclassification [16],[17]. The higher VE when using the specific definition could reflect some misclassification of zoster using the general definition. Alternatively, those with zoster who did not receive antivirals might include a large proportion with very mild disease; in the SPS, the zoster vaccine was shown to have higher efficacy against zoster with appreciable acute morbidity than against any zoster [3]. If those not receiving antivirals had milder incident zoster, this could lead to over-estimation of VE while providing reasonable estimates for significant zoster episodes. In this study VE was determined after adjusting for a wide range of confounders, including demographic details, immunosuppression, and immune-mediated diseases; despite the large size of this dataset, in some instances adjusting for confounding led to wide confidence intervals, for example when assessing VE in immunocompromised vaccinees.

Conclusions

Herpes zoster vaccination was associated with a significant reduction in incident herpes zoster and PHN in routine clinical use. This study also supports effectiveness of the vaccine against incident herpes zoster in immunosuppressed individuals, although the number of immunosuppressed individuals was small, resulting in lack of precision in the estimate. Given that these individuals are at greatest risk of both herpes zoster and complications, this may have important implications for policy. The findings are relevant beyond US medical practice, being of major importance to the many countries, including the UK, that are actively considering introducing the zoster vaccine into routine practice in the near future.

Despite strong evidence supporting its effectiveness, clinical use remains disappointingly low with particularly low vaccination rates in particular patient groups. This study shows that herpes zoster vaccination is associated with a reduction in PHN in routine clinical use. As PHN is the major complication of herpes zoster and is associated with highly significant morbidity and adverse impacts on quality of life, substantial efforts are needed to increase vaccine use in routine care of elderly individuals.

Acknowledgments

We thank Krishnan Bhaskaran for advice on statistical analysis and Ole Hoffstad for assistance creating Figure 2.

Abbreviations

ACIP
Advisory Committee for Immunization Practices
CMS
Centers for Medicare & Medicaid Services
COPD
chronic obstructive pulmonary disease
PHN
post-herpetic neuralgia
RCT
randomised controlled trial
SLE
systemic lupus erythematosis
SPS
Shingles Prevention Study
VE
vaccine effectiveness

Funding Statement

This research was funded by an NIHR Clinician Scientist award from the National Institute for Health Research, UK Department of Health, awarded to SML. The funders of the study had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the UK Department of Health.

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studentjournalofmedicine

A strange case of ingrown toenail treated with phenol

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Abstract

We experienced a strange case of ingrown toenail, which had developed as a huge mass and enveloped the nail of the left first toe. The patient had self-treated his ingrown toenail for a period of one year with an ointment available over the counter. However, the granulation tissue on both sides of the nail had increased gradually and advanced over the nail plate in the medial direction. Finally, the granulation tissue on both sides had adhered to the nail and epithelial cells advanced over the granulation tissue completely. During surgery, the epithelized granulation tissue was excised at the bilateral terminal base point, and the posterior nail fold and the nail matrix were cauterized completely with phenol. Eighteen months after the operation there was no recurrence of the ingrown toenail.

INTRODUCTION

The ingrown toenail is a common problem which occurs mostly in the first toe and causes a high amount of morbidity in affected patients. In this report we document a strange case of ingrown toenail of the first toe, which had developed as a huge mass and enveloped the nail. We describe here the cauterization of the nail matrix with phenol after surgical resection of the tumor, a treatment that had good results.

CASE REPORT

A 56-year-old man came to our office complaining of pain, offensive smelling discharge and disappearance of the nail of the right great toe. Physical examination showed epithelized mass on the nail plate and fistulation from the proximal nail fold to the tip of the toe, epithelization did not occur on the nail plate side of the mass (Figure 1). The clinical diagnosis of this tumor was epithelized granulation tissue caused by ingrown toenail, however squamous cell carcinoma was not denied completely.

Figure 1

The first toe of a 56-year-old man. Photograph shows epithelized granulation tissue on the nail plate and fistulation from the proximal nail fold to the tip of the toe

The operative treatment involved anesthetizing the toe with a digital nerve block using 1% lignocaine. The tumor mass was excised at the bilateral terminal base point, and was sent to pathology for an immediate diagnosis. The pathological finding was granulation tissue with partial scar formation, and the external surface of the tumor was covered with epithelial cells. Next, the nail plate was incised longitudinally from top to the root at a width of approximately 5mm. The posterior nail fold and the nail matrix were cauterized completely with an 88% phenol-immersed cotton-tipped applicator for five minutes. The excessive skin and soft tissue of the tip of the toe were excised and trimmed (Figure 2).

Figure 2

Bilateral chemical matricectomy of the matrix was done with phenol after resection of the tumor

Eighteen months after the operation, there is no recurrence of the ingrown toenail (Figure 3).

Figure 3

Eighteen months after the operation there was no recurrence of the ingrown toenail

DISCUSSION

Ingrown toenail deformity is a common nail pathology that causes intractable pain and discomfort, hindering normal walking and markedly decreasing the quality of life of patients. Ingrown toenails could be a cause of granulation tissue of the lateral nail fold of the finger or toe (1). However, an ingrown toenail creating such a huge mass on the nail plate, as in this case, is very rare. In the literature, to our knowledge, there have been no reports of cases similar to our patient.

In the case presented here, the patient had treated his ingrown toenail himself for one year with an ointment purchased over the counter before he came to our office. However the granulation tissue of both sides of the nail had increased gradually and advanced on the nail plate in the medial direction. Finally, the granulation tissue from both sides adhered and epithelial cells advanced over the granulation tissue completely. We believe such pathogenesis of this tumor is very uncommon.

Conservative and surgical forms of therapy for ingrown nails have been used, however, there is no common or unique form of treatment (26). In recent years, matrix phenolization of the nail bed has been used increasingly, and has been reported to give less discomfort and lower recurrence rates (7,8). The procedure of phenolization is easy to perform and does not require specialized equipment (9). In our reported case, after resection of the epithelized granuloma, both sides of the posterior nail fold and the nail matrix were cauterized completely with phenol. After this simple treatment, there was no recurrence of the ingrown toenail. The only disadvantage of the treatment was the smallness of the toenail, however, the patient did not complain of this aesthetic disadvantage due to the advantages of freedom from pain and the bad odour.

REFERENCES

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2. Moriue T, Yoneda K, Moriue J, et al. A simple therapeutic strategy with super elastic wire for ingrown toenails. Dermatol Surg. 2008; 1729–1732. [PubMed]
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5. Nazari S. A simple and practical method in treatment of ingrown nails: splinting by flexible tube. J Eur Acad Dermatol Venereol. 2006; 20: 1302–1306. [PubMed]
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7. Di Chiaccihio N, Belda W, Jr, Di Chiacchio NG, et al. Nail matrix phenolization for treatment of ingrowing nail: technique report and recurrence rate of 267 surgeries. Dermatol Surg. 2010; 36: 534–537. [PubMed]
8. Tatlican S, Yamangokturk B, Eren C, et al. Comparison of phenol applications of different durations for the cauterization of germinal matrix: an efficicacy and safety study. Acta Orthop Traumatol Turc. 2009; 43: 298–302. [PubMed]
9. Kimata Y, Uetake M, Tsukada S, et al. Follow-up study of patients treated for ingrown nails with the nail matrix phenolization method. Plast Reconstr Surg. 1995; 95: 719–724. [PubMed]

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