# Corticosteroids and Cholelithiasis in Systemic  Lupus Erythematosus

Authors: T. M. Reshetnyak; G. V. Saparin; P. V. Ivannikov; V. I. Reshetnyak
Journal: Scholarly Research Exchange
 (2009)

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## Abstract

The aim. To evaluate the frequency
of gallstone formation and alteration of plasma lipid profiles in
SLE patients long treated with prednisolone. Material and methods.
Sixty patients with SLE were divided into 2 groups: (1) 38 SLE
patients without gallstones; (2) 22 SLE patients with gallstones.
Gallbladder ultrasonography was performed in all the patients, and
the serum lipid profile was determined. To identify the
composition and structure of gallstones obtained during
cholecystectomy, color cathodoluminescence scanning
electromicroscopy was used. Results. Gallstone disease was
detected in 22 (36.7%) patients of the 60 examinees and in 22
(68.8%) of the 32 SLE patients receiving prednisolone therapy;
whereas none of the 28 prednisolone-untreated patients was found
to have the disease (P=.001). There were the most significant
differences between the SLE patients with and without gallstones
in the duration of administration of prednisolone and in its mean
daily and mean monthly doses, and cumulative ones. Conclusion. Age
at the onset of the disease, the mean daily dose of
corticosteroids, and the duration of therapy with these agents are
the most likely factors predisposing to gallstone disease in SLE
patients. The CCL-SEM study identified predominantly the
protein-cholesterol structure of gallstones.


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## Body

## 1. Introduction

Systemic
lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by
immune dysregulation, which results in the production of autoantibodies,
activation of the complement system, and generation of immune complexes [1]. An intrinsic feature of the
disease is vascular injury manifesting itself as cutaneous vasculitis,
glomerulonephritis, cardiopulmonary, cerebrovascular and, less commonly,
gastrointestinal lesions.In several
past decades, the use of corticosteroids and cytotoxics has dramatically
improved the prognosis of the disease [2, 3]. Prednisone, prednisolone,
methylprednisolone, and hydrocortisone are the most commonly used
corticosteroids in the treatment of SLE. The long-term systemic use of
corticosteroids is one of the most important issues in the management of SLE
due to their potentially serious side effects. Long-term corticosteroid therapy
is well known to cause dyslipoproteinemia characterized by elevated plasma
total cholesterol (TCh), triglycerides (TGs), and low-density lipoprotein
cholesterol (LDL-Ch) [4, 5]. The major catabolic pathway for
cholesterol is its transformation into bile acids involving P450 cytochrome and subsequent bile excretion from the body. The elevated level of
total cholesterol may change a bile acid/cholesterol ratio and lead to the
formation of gallstones in patients with SLE.The aim of
this study was to evaluate the frequency of gallstone formation and alteration
of plasma lipid profiles in SLE patients treated long with prednisolone.


## 2. Matherial and Methods

### 2.1. Subjects

This was a
retrospective study covering 60 consecutive SLE patients (45 females and 15
males; Caucasians; mean age30.5±9.4 years) treated and followed up at the
Institute of Rheumatology, Russian Academy of Medical Sciences (Moscow, Russia)
with January 2003 to December 2006 years. All patients met four SLE diagnostic
criteria or more (American College of Rheumatology,
1982)  [6]. This study was approved by the Ethics Committee
of Institute of Rheumatology, Russian Academy
of Medical Sciences, and all the patients gave an informed consent. SLE was
first diagnosed in 28 patients and none of them had received corticosteroids.
Thirty two SLE patients had been treated with prednisolone. The average
statistical interval between the occurrence of the first sign of the disease
and the establishment of the diagnosis was one year (Table 1).Table 1
Baseline disease-related
features of the SLE patients recruited.
Variablen≥4 ACR criteria,n (%)60 (100)Sex (female/male)44/16Median age at the time of this study,n (range), years28 (18–44)Median age at the onset of disease,n (range), years20 (10–36)Median age at diagnosis,n (range),
years21 (15–29)Median disease duration,n (range),
years9.6 (1–21)Median (IQR) SLEDAI, score15 (10–22)Median (IQR) SLICCA damage index,
score1.1 (0.4–2.3)Current steroid therapy,n (%)32 (53.3)Median (IQR) current steroid
dosage, mg5.5 (0–15.5)Median (IQR) steroid therapy duration,
years5 (0–12)Median (IQR) monthly steroid dose,
mg/month12 (0–16)Cumulative steroid dose (range), g107.3 (0–583.7)Current antimalarial therapy,n (%)24 (40)Current immunosuppressive therapy,n (%)16 (26.7)Gallstone disease,n (%)22 (36.7)IQR: Interquartile range.The mean
monthly and cumulative dose of prednisolone and its treatment duration were recorded during a chart review.
The mean monthly dose of prednisolone was defined as its total sum in
milligrams divided by the total number of follow-up months, the cumulative dose
of the agent was as its total sum in grams taken during the same period, and
the duration of treatment with the drug was estimated as the time interval
(years) during which a patient had taken it (Table1).SLE activity
was quantified, by using the SLE disease activity index (SLEDAI)  [7].
None of patients had underlying conditions (nephrotic syndrome or renal insufficiency)
or concomitant diseases (diabetes mellitus) that could potentially cause
dyslipidemia. The study excluded pregnant women, 6-month nursing mothers, women
who had used oral contraceptives or estrogen-containing agents for 6 months
before the study, and patients who had used statins.The patients
were clinically interviewed and examined according to the standard protocol
comprising demographic characteristics, family history, and lifestyle (Table2). To evaluate the effect of corticosteroids on gallstone formation, all the
SLE patients were divided into 2 groups: (1) 22 patients with gallstones (a gallstone group); (2) 38 patients without
gallstones (a nongallstone group).Table 2
Comparisons
of baseline demographic characteristics, prednisolone therapy, SLE-related
features, and laboratory data in SLE patients with/without gallstones.
VariableSLE patients with gallstones
(n=22)SLE patients without gallstones (n=38)P-valueSex (female/male)17/527/11NS (χ2)Median age at the time of this
study (range), years33 (22–44)24 (18–40).0002Median age at the onset of the
disease (range), years23 (10–35)19 (8–36).015Median age at diagnosis (range),
years25 (18–35)21 (17–37)NSMedian disease duration (range),
years11.8 (8–15)1.8 (0–8.0).000001Median (IQR) SLEDAI, score7 (6–10)12 (10–20).000145Current steroid therapyMedian (IQR) current steroid
dosage, mg16.0 (10.0–18.5)2.1 (0–5.0).0001Median (IQR) steroid therapy
duration, years12 (8.0–15.0)0 (0–5.0).0001Median (IQR) monthly steroid dose,
mg/month16.1 (15.0–16.5)0 (0–8.0).0001Cumulative steroid dose (range), g848.0
(502.0–972.5)0 (0–65.7).0001Laboratory resultsTCh (range), mg/dL220.0
(172.0–251.0)177.0
(142.0–190.0).0004HDL-Ch (range), mg/dL46.0 (40.0–56.0)38.0 (34.0–42.0).008Ratio of TCh and HDL-Ch4.9 (4.1–5.5)4.3 (3.6–4.8).05LDL-Ch (range), mg/dL135.0
(103.0–173.0)111.2 (84.6–136.0).006ApoA1-HDL (range), mg/dL130.0
(115.0–151.0)112.0
(103.0–141.0).05ApoB-LDL (range), mg/dL108.0 (89.0–136.0)97.0 (77.0–119.0).005Fibrinogen (range), g/L3.8 (3.1–4.2)3.4 (2.6–4.2)NSTCh: Total cholesterol; HDL-Ch: HDL
cholesterol; LDL-Ch: LDL cholesterol; IQR: Interquartile range; NS: Nonsignificant.
All comparisons are Mann-WhitneyU-test unless where a chi-squared test is
indicated.


### 2.2. Laboratory Studies

#### 2.2.1. Routine Clinical Samples

All samples
were collected after a written informed consent had been given under
institutional review board-approved protocols in accordance with the Helsinki
Declaration. Hemoglobin, leukocyte and platelet counts, fasting plasma glucose,
serum creatinine, serum liver functional tests (bilirubin, cholesterol,
aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase,
total protein, albumin, and globulin levels), urine microscopy, DNA antibodies,
complement profile (C3, C4, C50), and
antinuclear antibodies were measured as a part of the routine laboratory assessment.


#### 2.2.2. Ultrasound Study

Ultrasonography was performed with a Combison 530 with 3.5 MHz convex (Kretz, Austria).
Gel was applied to the skin to provide an acoustic interface.


#### 2.2.3. Lipids

Blood was sampled after a 14-hour of fasting. On the day of sampling, plasma and
serum were immediately separated, centrifuged, and stored at −20°C before
testing the lipid profile. TCh,
TG, and HDL-Ch were measured, by employing commercially available assays (Biocon,
Germany) on an Airone-200 analyzer (Crony, Italy). HDL-Ch
was measured after precipitation of LDL and very low-density lipoprotein
(VLDL), by using sodium 12-tungstophosphate (1.1 mmol/L) and magnesium chloride
(45 mmol/L)  (a final concentration)  [8]. The level of LDL-Ch was
calculated using the Friedewald formula [9]. Apolipoprotein AI
(Apo-AI) and apolipoprotein B (Apo-B) were measured by radial immunodiffusion.
All assays were repeated.


#### 2.2.4. Composition and Structure of Gallstones

Color
cathodoluminescence scanning electron microscopy (CCL-SEM) was employed to
study the composition and structure of gallstones. A “Stereoscan MK-IIA” SEM
(Cambridge Instruments, UK) was equipped with a CCL-attachment (developed by
GVS, SKO, and PVI at the Department of
Physics, Moscow State University, Russia) and operated in the “real-color mode”
at an electron energy of under 20 keV and a beam current of 10 nA. As a result,
color images of the samples under examination were electron-induced luminescence
of major chemical gallstone components located onto the section surface. “Adobe
Photoshop” software was used to separate colors and to have intensity
histograms. The method and its possible fields of application were
characterized in detail in our earlier publications [10–12]. Ten
gallstones obtained from 4 SLE patients on 10- to 20-year prednisolone
treatment at cholecystectomy were selected for CCL-SEM evaluation of their
composition, microarchitectonics, and
microstructure. All samples of the gallstones were cut in half through
their centers.The standard
commercial unconjugated bilirubin (C33H36N4O6,
Merck, Germany), unesterified cholesterol (C22H46O Serva,
Germany), high molecular-weight protein, the prototype of a bile mucine
component (Serva, Germany) were used to obtain control CCL-SEM images of major
bile constituents. The color images of the control samples were as follows: the
unconjugated bilirubin luminiscented under electronic rays was colored
reddish-orange (Figure 1(a)), the crystals of unesterified cholesterol were
blue (Figure 1(b)), and high molecular weight protein was yellowish-green (Figure
1(c)). The color micrographs of gallstone samples were compared with those of
the control ones. The application of the computer program (software) and color
contrast by the CCL-SEM technique permitted the determination of cholesterol,
bilirubin, protein, and their relationships within the stone [12].CCL-SEM micrographs of bilirubin (a), cholesterol (b), high molecular weight protein (c) and their corresponding histograms obtained after color separation.
(a)(b)(c)


#### 2.2.5. Statistical Analysis

The results
for continuous variables were expressed as mean ± SD. Nonparametric tests were carried
out due to the skewed distribution of the Gaussian curve and the small number
of samples. Differences between groups were examined by the Mann-WhitneyU-test. Proportions were determined using the standard chi-square test (χ2) or the Fisher exact test.
The values of P<.05 were considered significant. Univariate analysis using the individual factors was
carried out for the association with gallstones. It was used to compare all
clinical and lipid profile variables between 2 groups (the gallstone group and
the nongallstone group). The variables found significant by univariate analysis
were considered to be predictors of gallstone formation in SLE patients. All
baseline variables that have found on univariate analysis significant at P<.05
were entered into a backward elimination variable selection procedure by the
multivariate logistic regression analysis. A multivariate logistic regression
analysis was used to assess the relationship between the predictor variables
and the presence of gallstones (present or not). The results were expressed as
odds ratio (95% confidence interval) (Table 3). The predictors with a P<.05 were considered as independent predictors of gallstone formation in SLE
patients. Discrimination analysis was used to detect the signs predicting the
development of gallstone disease in SLE patients.Table 3
Univariable association of predisposing factors to gallstone formations in the
study SLE patients.
VariableSLE patients withSLE patients withoutORLn ORSE ln OR95%  CIP-value (χ2)gallstone (n=22)gallstone (n=38)present,nnot,npresent,nnot,nDemographic characteristicsAge at the onset of the disease (≥25.1 years)Age at the time of this study (≥30.3 years)Prednisolone therapyPrednisolone therapy duration (≥7.0 months)Current steroid dosage (≥8.66 mg.)Minimum maintenance dosage of
prednisolone (≥10.2 mg)Maximum maintenance dosage of prednisolone (≥28.5 mg)Monthly steroid dose (≥10.4 mg)Cumulative steroid dose (≥498.5 mg)Pregnancy historyPregnancy in past historySLE-related featuresEnanthema of mucosal soft palateThrombocytopeniaHemolytic anemiaCentral nervous system lesionLupus nephritisIgG-anticardiolipin antibody (≤11.6)Antiphospholipid syndromeLaboratory resultsTCh (≥169.0 mg/dL)HDL-Ch (≤45.5 mg/dL)ApoB-LDL(≤74.0 mg/dL)LDL-Ch (≤136.0 mg/dL)ApoA1-HDL (≥116.5 mg/dL)Ratio of TCh and HDL-Ch (>4.5)Ratio of HDL-Ch and HDL-PhL (≥0.504)TCh: Total cholesterol; HDL-Ch: HDL
cholesterol; LDL-Ch: LDL cholesterol; HDL-PhL: HDL phospholipids.


## 2.1. Subjects

This was a
retrospective study covering 60 consecutive SLE patients (45 females and 15
males; Caucasians; mean age30.5±9.4 years) treated and followed up at the
Institute of Rheumatology, Russian Academy of Medical Sciences (Moscow, Russia)
with January 2003 to December 2006 years. All patients met four SLE diagnostic
criteria or more (American College of Rheumatology,
1982)  [6]. This study was approved by the Ethics Committee
of Institute of Rheumatology, Russian Academy
of Medical Sciences, and all the patients gave an informed consent. SLE was
first diagnosed in 28 patients and none of them had received corticosteroids.
Thirty two SLE patients had been treated with prednisolone. The average
statistical interval between the occurrence of the first sign of the disease
and the establishment of the diagnosis was one year (Table 1).Table 1
Baseline disease-related
features of the SLE patients recruited.
Variablen≥4 ACR criteria,n (%)60 (100)Sex (female/male)44/16Median age at the time of this study,n (range), years28 (18–44)Median age at the onset of disease,n (range), years20 (10–36)Median age at diagnosis,n (range),
years21 (15–29)Median disease duration,n (range),
years9.6 (1–21)Median (IQR) SLEDAI, score15 (10–22)Median (IQR) SLICCA damage index,
score1.1 (0.4–2.3)Current steroid therapy,n (%)32 (53.3)Median (IQR) current steroid
dosage, mg5.5 (0–15.5)Median (IQR) steroid therapy duration,
years5 (0–12)Median (IQR) monthly steroid dose,
mg/month12 (0–16)Cumulative steroid dose (range), g107.3 (0–583.7)Current antimalarial therapy,n (%)24 (40)Current immunosuppressive therapy,n (%)16 (26.7)Gallstone disease,n (%)22 (36.7)IQR: Interquartile range.The mean
monthly and cumulative dose of prednisolone and its treatment duration were recorded during a chart review.
The mean monthly dose of prednisolone was defined as its total sum in
milligrams divided by the total number of follow-up months, the cumulative dose
of the agent was as its total sum in grams taken during the same period, and
the duration of treatment with the drug was estimated as the time interval
(years) during which a patient had taken it (Table1).SLE activity
was quantified, by using the SLE disease activity index (SLEDAI)  [7].
None of patients had underlying conditions (nephrotic syndrome or renal insufficiency)
or concomitant diseases (diabetes mellitus) that could potentially cause
dyslipidemia. The study excluded pregnant women, 6-month nursing mothers, women
who had used oral contraceptives or estrogen-containing agents for 6 months
before the study, and patients who had used statins.The patients
were clinically interviewed and examined according to the standard protocol
comprising demographic characteristics, family history, and lifestyle (Table2). To evaluate the effect of corticosteroids on gallstone formation, all the
SLE patients were divided into 2 groups: (1) 22 patients with gallstones (a gallstone group); (2) 38 patients without
gallstones (a nongallstone group).Table 2
Comparisons
of baseline demographic characteristics, prednisolone therapy, SLE-related
features, and laboratory data in SLE patients with/without gallstones.
VariableSLE patients with gallstones
(n=22)SLE patients without gallstones (n=38)P-valueSex (female/male)17/527/11NS (χ2)Median age at the time of this
study (range), years33 (22–44)24 (18–40).0002Median age at the onset of the
disease (range), years23 (10–35)19 (8–36).015Median age at diagnosis (range),
years25 (18–35)21 (17–37)NSMedian disease duration (range),
years11.8 (8–15)1.8 (0–8.0).000001Median (IQR) SLEDAI, score7 (6–10)12 (10–20).000145Current steroid therapyMedian (IQR) current steroid
dosage, mg16.0 (10.0–18.5)2.1 (0–5.0).0001Median (IQR) steroid therapy
duration, years12 (8.0–15.0)0 (0–5.0).0001Median (IQR) monthly steroid dose,
mg/month16.1 (15.0–16.5)0 (0–8.0).0001Cumulative steroid dose (range), g848.0
(502.0–972.5)0 (0–65.7).0001Laboratory resultsTCh (range), mg/dL220.0
(172.0–251.0)177.0
(142.0–190.0).0004HDL-Ch (range), mg/dL46.0 (40.0–56.0)38.0 (34.0–42.0).008Ratio of TCh and HDL-Ch4.9 (4.1–5.5)4.3 (3.6–4.8).05LDL-Ch (range), mg/dL135.0
(103.0–173.0)111.2 (84.6–136.0).006ApoA1-HDL (range), mg/dL130.0
(115.0–151.0)112.0
(103.0–141.0).05ApoB-LDL (range), mg/dL108.0 (89.0–136.0)97.0 (77.0–119.0).005Fibrinogen (range), g/L3.8 (3.1–4.2)3.4 (2.6–4.2)NSTCh: Total cholesterol; HDL-Ch: HDL
cholesterol; LDL-Ch: LDL cholesterol; IQR: Interquartile range; NS: Nonsignificant.
All comparisons are Mann-WhitneyU-test unless where a chi-squared test is
indicated.


## 2.2. Laboratory Studies

### 2.2.1. Routine Clinical Samples

All samples
were collected after a written informed consent had been given under
institutional review board-approved protocols in accordance with the Helsinki
Declaration. Hemoglobin, leukocyte and platelet counts, fasting plasma glucose,
serum creatinine, serum liver functional tests (bilirubin, cholesterol,
aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase,
total protein, albumin, and globulin levels), urine microscopy, DNA antibodies,
complement profile (C3, C4, C50), and
antinuclear antibodies were measured as a part of the routine laboratory assessment.


### 2.2.2. Ultrasound Study

Ultrasonography was performed with a Combison 530 with 3.5 MHz convex (Kretz, Austria).
Gel was applied to the skin to provide an acoustic interface.


### 2.2.3. Lipids

Blood was sampled after a 14-hour of fasting. On the day of sampling, plasma and
serum were immediately separated, centrifuged, and stored at −20°C before
testing the lipid profile. TCh,
TG, and HDL-Ch were measured, by employing commercially available assays (Biocon,
Germany) on an Airone-200 analyzer (Crony, Italy). HDL-Ch
was measured after precipitation of LDL and very low-density lipoprotein
(VLDL), by using sodium 12-tungstophosphate (1.1 mmol/L) and magnesium chloride
(45 mmol/L)  (a final concentration)  [8]. The level of LDL-Ch was
calculated using the Friedewald formula [9]. Apolipoprotein AI
(Apo-AI) and apolipoprotein B (Apo-B) were measured by radial immunodiffusion.
All assays were repeated.


### 2.2.4. Composition and Structure of Gallstones

Color
cathodoluminescence scanning electron microscopy (CCL-SEM) was employed to
study the composition and structure of gallstones. A “Stereoscan MK-IIA” SEM
(Cambridge Instruments, UK) was equipped with a CCL-attachment (developed by
GVS, SKO, and PVI at the Department of
Physics, Moscow State University, Russia) and operated in the “real-color mode”
at an electron energy of under 20 keV and a beam current of 10 nA. As a result,
color images of the samples under examination were electron-induced luminescence
of major chemical gallstone components located onto the section surface. “Adobe
Photoshop” software was used to separate colors and to have intensity
histograms. The method and its possible fields of application were
characterized in detail in our earlier publications [10–12]. Ten
gallstones obtained from 4 SLE patients on 10- to 20-year prednisolone
treatment at cholecystectomy were selected for CCL-SEM evaluation of their
composition, microarchitectonics, and
microstructure. All samples of the gallstones were cut in half through
their centers.The standard
commercial unconjugated bilirubin (C33H36N4O6,
Merck, Germany), unesterified cholesterol (C22H46O Serva,
Germany), high molecular-weight protein, the prototype of a bile mucine
component (Serva, Germany) were used to obtain control CCL-SEM images of major
bile constituents. The color images of the control samples were as follows: the
unconjugated bilirubin luminiscented under electronic rays was colored
reddish-orange (Figure 1(a)), the crystals of unesterified cholesterol were
blue (Figure 1(b)), and high molecular weight protein was yellowish-green (Figure
1(c)). The color micrographs of gallstone samples were compared with those of
the control ones. The application of the computer program (software) and color
contrast by the CCL-SEM technique permitted the determination of cholesterol,
bilirubin, protein, and their relationships within the stone [12].CCL-SEM micrographs of bilirubin (a), cholesterol (b), high molecular weight protein (c) and their corresponding histograms obtained after color separation.
(a)(b)(c)


### 2.2.5. Statistical Analysis

The results
for continuous variables were expressed as mean ± SD. Nonparametric tests were carried
out due to the skewed distribution of the Gaussian curve and the small number
of samples. Differences between groups were examined by the Mann-WhitneyU-test. Proportions were determined using the standard chi-square test (χ2) or the Fisher exact test.
The values of P<.05 were considered significant. Univariate analysis using the individual factors was
carried out for the association with gallstones. It was used to compare all
clinical and lipid profile variables between 2 groups (the gallstone group and
the nongallstone group). The variables found significant by univariate analysis
were considered to be predictors of gallstone formation in SLE patients. All
baseline variables that have found on univariate analysis significant at P<.05
were entered into a backward elimination variable selection procedure by the
multivariate logistic regression analysis. A multivariate logistic regression
analysis was used to assess the relationship between the predictor variables
and the presence of gallstones (present or not). The results were expressed as
odds ratio (95% confidence interval) (Table 3). The predictors with a P<.05 were considered as independent predictors of gallstone formation in SLE
patients. Discrimination analysis was used to detect the signs predicting the
development of gallstone disease in SLE patients.Table 3
Univariable association of predisposing factors to gallstone formations in the
study SLE patients.
VariableSLE patients withSLE patients withoutORLn ORSE ln OR95%  CIP-value (χ2)gallstone (n=22)gallstone (n=38)present,nnot,npresent,nnot,nDemographic characteristicsAge at the onset of the disease (≥25.1 years)Age at the time of this study (≥30.3 years)Prednisolone therapyPrednisolone therapy duration (≥7.0 months)Current steroid dosage (≥8.66 mg.)Minimum maintenance dosage of
prednisolone (≥10.2 mg)Maximum maintenance dosage of prednisolone (≥28.5 mg)Monthly steroid dose (≥10.4 mg)Cumulative steroid dose (≥498.5 mg)Pregnancy historyPregnancy in past historySLE-related featuresEnanthema of mucosal soft palateThrombocytopeniaHemolytic anemiaCentral nervous system lesionLupus nephritisIgG-anticardiolipin antibody (≤11.6)Antiphospholipid syndromeLaboratory resultsTCh (≥169.0 mg/dL)HDL-Ch (≤45.5 mg/dL)ApoB-LDL(≤74.0 mg/dL)LDL-Ch (≤136.0 mg/dL)ApoA1-HDL (≥116.5 mg/dL)Ratio of TCh and HDL-Ch (>4.5)Ratio of HDL-Ch and HDL-PhL (≥0.504)TCh: Total cholesterol; HDL-Ch: HDL
cholesterol; LDL-Ch: LDL cholesterol; HDL-PhL: HDL phospholipids.


## 2.2.1. Routine Clinical Samples

All samples
were collected after a written informed consent had been given under
institutional review board-approved protocols in accordance with the Helsinki
Declaration. Hemoglobin, leukocyte and platelet counts, fasting plasma glucose,
serum creatinine, serum liver functional tests (bilirubin, cholesterol,
aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase,
total protein, albumin, and globulin levels), urine microscopy, DNA antibodies,
complement profile (C3, C4, C50), and
antinuclear antibodies were measured as a part of the routine laboratory assessment.


## 2.2.2. Ultrasound Study

Ultrasonography was performed with a Combison 530 with 3.5 MHz convex (Kretz, Austria).
Gel was applied to the skin to provide an acoustic interface.


## 2.2.3. Lipids

Blood was sampled after a 14-hour of fasting. On the day of sampling, plasma and
serum were immediately separated, centrifuged, and stored at −20°C before
testing the lipid profile. TCh,
TG, and HDL-Ch were measured, by employing commercially available assays (Biocon,
Germany) on an Airone-200 analyzer (Crony, Italy). HDL-Ch
was measured after precipitation of LDL and very low-density lipoprotein
(VLDL), by using sodium 12-tungstophosphate (1.1 mmol/L) and magnesium chloride
(45 mmol/L)  (a final concentration)  [8]. The level of LDL-Ch was
calculated using the Friedewald formula [9]. Apolipoprotein AI
(Apo-AI) and apolipoprotein B (Apo-B) were measured by radial immunodiffusion.
All assays were repeated.


## 2.2.4. Composition and Structure of Gallstones

Color
cathodoluminescence scanning electron microscopy (CCL-SEM) was employed to
study the composition and structure of gallstones. A “Stereoscan MK-IIA” SEM
(Cambridge Instruments, UK) was equipped with a CCL-attachment (developed by
GVS, SKO, and PVI at the Department of
Physics, Moscow State University, Russia) and operated in the “real-color mode”
at an electron energy of under 20 keV and a beam current of 10 nA. As a result,
color images of the samples under examination were electron-induced luminescence
of major chemical gallstone components located onto the section surface. “Adobe
Photoshop” software was used to separate colors and to have intensity
histograms. The method and its possible fields of application were
characterized in detail in our earlier publications [10–12]. Ten
gallstones obtained from 4 SLE patients on 10- to 20-year prednisolone
treatment at cholecystectomy were selected for CCL-SEM evaluation of their
composition, microarchitectonics, and
microstructure. All samples of the gallstones were cut in half through
their centers.The standard
commercial unconjugated bilirubin (C33H36N4O6,
Merck, Germany), unesterified cholesterol (C22H46O Serva,
Germany), high molecular-weight protein, the prototype of a bile mucine
component (Serva, Germany) were used to obtain control CCL-SEM images of major
bile constituents. The color images of the control samples were as follows: the
unconjugated bilirubin luminiscented under electronic rays was colored
reddish-orange (Figure 1(a)), the crystals of unesterified cholesterol were
blue (Figure 1(b)), and high molecular weight protein was yellowish-green (Figure
1(c)). The color micrographs of gallstone samples were compared with those of
the control ones. The application of the computer program (software) and color
contrast by the CCL-SEM technique permitted the determination of cholesterol,
bilirubin, protein, and their relationships within the stone [12].CCL-SEM micrographs of bilirubin (a), cholesterol (b), high molecular weight protein (c) and their corresponding histograms obtained after color separation.
(a)(b)(c)


## 2.2.5. Statistical Analysis

The results
for continuous variables were expressed as mean ± SD. Nonparametric tests were carried
out due to the skewed distribution of the Gaussian curve and the small number
of samples. Differences between groups were examined by the Mann-WhitneyU-test. Proportions were determined using the standard chi-square test (χ2) or the Fisher exact test.
The values of P<.05 were considered significant. Univariate analysis using the individual factors was
carried out for the association with gallstones. It was used to compare all
clinical and lipid profile variables between 2 groups (the gallstone group and
the nongallstone group). The variables found significant by univariate analysis
were considered to be predictors of gallstone formation in SLE patients. All
baseline variables that have found on univariate analysis significant at P<.05
were entered into a backward elimination variable selection procedure by the
multivariate logistic regression analysis. A multivariate logistic regression
analysis was used to assess the relationship between the predictor variables
and the presence of gallstones (present or not). The results were expressed as
odds ratio (95% confidence interval) (Table 3). The predictors with a P<.05 were considered as independent predictors of gallstone formation in SLE
patients. Discrimination analysis was used to detect the signs predicting the
development of gallstone disease in SLE patients.Table 3
Univariable association of predisposing factors to gallstone formations in the
study SLE patients.
VariableSLE patients withSLE patients withoutORLn ORSE ln OR95%  CIP-value (χ2)gallstone (n=22)gallstone (n=38)present,nnot,npresent,nnot,nDemographic characteristicsAge at the onset of the disease (≥25.1 years)Age at the time of this study (≥30.3 years)Prednisolone therapyPrednisolone therapy duration (≥7.0 months)Current steroid dosage (≥8.66 mg.)Minimum maintenance dosage of
prednisolone (≥10.2 mg)Maximum maintenance dosage of prednisolone (≥28.5 mg)Monthly steroid dose (≥10.4 mg)Cumulative steroid dose (≥498.5 mg)Pregnancy historyPregnancy in past historySLE-related featuresEnanthema of mucosal soft palateThrombocytopeniaHemolytic anemiaCentral nervous system lesionLupus nephritisIgG-anticardiolipin antibody (≤11.6)Antiphospholipid syndromeLaboratory resultsTCh (≥169.0 mg/dL)HDL-Ch (≤45.5 mg/dL)ApoB-LDL(≤74.0 mg/dL)LDL-Ch (≤136.0 mg/dL)ApoA1-HDL (≥116.5 mg/dL)Ratio of TCh and HDL-Ch (>4.5)Ratio of HDL-Ch and HDL-PhL (≥0.504)TCh: Total cholesterol; HDL-Ch: HDL
cholesterol; LDL-Ch: LDL cholesterol; HDL-PhL: HDL phospholipids.


## 3. Results

### 3.1. Gallstone Disease in SLE Patients

Table1 gives the data characterizing the SLE
patients being examined, gallstone disease was found in  22 (36.7%) patients of the 60 examinees, by
ultrasonography. The size of gallstones varied from 2 to 8 mm in
diameter. Gallstone disease was found only in patients on prednisolone therapy.
Twenty two (68.8%) out of the 32 SLE patients on prednisolone therapy had cholelithiasis. The later
was absent in 28 prednisolone-untreated patients (SLE had been first diagnosed
at the examination)  (χ2=20.6; P<.001).
Further analysis of 32 corticosteroid-treated SLE patients demonstrated the
association of the gallstone formation with the dosage of prednisolone and its
treatment duration. Significantly higher mean monthly doses of prednisolone (P<.001),
its higher cumulative dose (P<.001), and its longer treatment use (P<.001)
were observed in patients with gallstone disease. Almost every patient who had
taken prednisolone for more than 5 years had gallstones. Parameters, such as age, disease duration, and SLEDAI score, did
not differ significantly between the corticosteroid-treated patients with and
without gallstones.To evaluate the impact of various factors on
the development of cholelithiasis in SLE patients, all clinical signs and
laboratory data were compared in the gallstone and nongallstone groups. Table2 shows the results
of comparing the individual variables in these groups. Both groups did
not differ significantly in gender and median age at diagnosis. The SLE
patients with gallstones had statistically a longer history of the disease (P<.000001)
and statistically lower SLEDAI scores (P<.000145). There were the
most significant differences between the groups in both the duration of therapy
with prednisolone (P<.0001), its mean daily (P<.0001), mean
monthly (P<.0001),
and cumulative doses (P<.0001).The signs showing the significance of group
differences of less than 0.05 were regarded as risk factors for gallstone
disease in SLE patients. For univariate analysis, the most informative signs were
chosen from all the variables
included into the study and found to differ significantly. The frequency of
this variable (present, absent) in a group, odds ratio (OR), natural logarithm
of the odds ratio (ln OR), standard error in the odds ratio (SE ln OR), 95%
confidence interval (CI) for the odds ratio, and significance P by theχ2 test (Table 3) were derived
for each variable.The data given in Table3 were used to
calculate two logistic regressions that allow us to define with an accuracy of
100% the signs by which the patients are divided into two groups (SLE patients with gallstones and SLE patients without gallstones). Such variables were age at the
onset of the disease; cumulative steroid dose; monthly steroid dose; current
steroid dosage; prednisolone therapy duration.The values of
the study variables atP<.05 in SLE patient with and without
gallstones underwent discrimination analysis. This yielded for the examined
patient cohort following discrimination equation: G(X)=0.298 (age at the
onset of the disease)  + 0.746 (current steroid dosage)  + 0.908 (prednisolone
therapy duration). The equation predicts the development of cholelithiasis in SLE patients if the
condition G(X)≥19.0 is met. If G(X) is <19.0, the
prediction of development of gallstone disease is negative. The 100% validity
of the classification of all the patients by the gallstone group and nongallstone one was confirmed.Table4 gives
the data obtained using the Fisher
exact test for the examined SLE patients, which showing that age, mean daily
dose of prednisolone and duration of its therapy are the most likely predictors
of cholelithiasis in SLE patients.Table 4
The predisposing factors to gallstone formation in the study SLE patients.
VariableThe Fisher exact testP-valueAge at the onset of the disease93.8<.000001Current steroid dosage109.2<.0000001Steroid therapy duration152.0<.0000001


### 3.2. Lipid-Protein Blood Spectrum and Gallstone Disease in SLE Patients

As we
expected, the levels of plasma lipids, ApoB-LDL (P=.005) and ApoAI-HDL (P=.05)
differed significantly in SLE patients with and without gallstones. SLE
patients with gallstones had significantly higher levels of TCh (P=.0004),
LDL-Ch (P=.006), HDL-Ch (P=.008) than those without gallstones.When the
group of SLE patients treated with corticosteroids were additionally compared,
significantly higher levels of TCh (P<.02) and LDL-Ch (P<.03)
were observed in patients with gallstones than in those without gallstones.
Significantly reduced levels of HDL-Ch (P<.02) and ApoAI-HDL (P<.04)
were found in SLE patients treated with corticosteroids without gallstone
disease.Interestingly,
the levels TG and ApoB-LDL, as well as TCh/HDL-Ch and LDL-Ch/HDL-Ch ratios, did
not differ significantly in corticosteroid-treated SLE patients as compared
with patients who had never been treated with corticosteroids.


### 3.3. The Composition and Structure of Gallstones in SlLE Patients

Series of
CCL-SEM color images of 10 gallstones were taken for analysis. Each image was
compared with control images to distinguish the deposition of bilirubin,
cholesterol, and protein components. The major components of the gallstones
under examination were cholesterol (Figure2(b)) and protein (Figure 2(c)).
They were all detected over the entire surface of the scanned gallstone while
rare bilirubin insertions (Figure 2(a)) were seen only at the periphery of the
gallstone. All 10 of examined gallstones from 4 SLE patients with
cholelithiasis due to long-term prednisolone therapy were structurally
identical. The findings have provided evidence for the protein-cholesterol
composition of the gallstones in corticosteroid-treated patients with SLE.CCL-SEM micrographs demonstrating the structural organization and chemical composition of a gallstone obtained from SLE patient: (A) general gallstone structure before color separation; (a) bilirubin inclusions; (b) cholesterol component; (c) protein.


## 3.1. Gallstone Disease in SLE Patients

Table1 gives the data characterizing the SLE
patients being examined, gallstone disease was found in  22 (36.7%) patients of the 60 examinees, by
ultrasonography. The size of gallstones varied from 2 to 8 mm in
diameter. Gallstone disease was found only in patients on prednisolone therapy.
Twenty two (68.8%) out of the 32 SLE patients on prednisolone therapy had cholelithiasis. The later
was absent in 28 prednisolone-untreated patients (SLE had been first diagnosed
at the examination)  (χ2=20.6; P<.001).
Further analysis of 32 corticosteroid-treated SLE patients demonstrated the
association of the gallstone formation with the dosage of prednisolone and its
treatment duration. Significantly higher mean monthly doses of prednisolone (P<.001),
its higher cumulative dose (P<.001), and its longer treatment use (P<.001)
were observed in patients with gallstone disease. Almost every patient who had
taken prednisolone for more than 5 years had gallstones. Parameters, such as age, disease duration, and SLEDAI score, did
not differ significantly between the corticosteroid-treated patients with and
without gallstones.To evaluate the impact of various factors on
the development of cholelithiasis in SLE patients, all clinical signs and
laboratory data were compared in the gallstone and nongallstone groups. Table2 shows the results
of comparing the individual variables in these groups. Both groups did
not differ significantly in gender and median age at diagnosis. The SLE
patients with gallstones had statistically a longer history of the disease (P<.000001)
and statistically lower SLEDAI scores (P<.000145). There were the
most significant differences between the groups in both the duration of therapy
with prednisolone (P<.0001), its mean daily (P<.0001), mean
monthly (P<.0001),
and cumulative doses (P<.0001).The signs showing the significance of group
differences of less than 0.05 were regarded as risk factors for gallstone
disease in SLE patients. For univariate analysis, the most informative signs were
chosen from all the variables
included into the study and found to differ significantly. The frequency of
this variable (present, absent) in a group, odds ratio (OR), natural logarithm
of the odds ratio (ln OR), standard error in the odds ratio (SE ln OR), 95%
confidence interval (CI) for the odds ratio, and significance P by theχ2 test (Table 3) were derived
for each variable.The data given in Table3 were used to
calculate two logistic regressions that allow us to define with an accuracy of
100% the signs by which the patients are divided into two groups (SLE patients with gallstones and SLE patients without gallstones). Such variables were age at the
onset of the disease; cumulative steroid dose; monthly steroid dose; current
steroid dosage; prednisolone therapy duration.The values of
the study variables atP<.05 in SLE patient with and without
gallstones underwent discrimination analysis. This yielded for the examined
patient cohort following discrimination equation: G(X)=0.298 (age at the
onset of the disease)  + 0.746 (current steroid dosage)  + 0.908 (prednisolone
therapy duration). The equation predicts the development of cholelithiasis in SLE patients if the
condition G(X)≥19.0 is met. If G(X) is <19.0, the
prediction of development of gallstone disease is negative. The 100% validity
of the classification of all the patients by the gallstone group and nongallstone one was confirmed.Table4 gives
the data obtained using the Fisher
exact test for the examined SLE patients, which showing that age, mean daily
dose of prednisolone and duration of its therapy are the most likely predictors
of cholelithiasis in SLE patients.Table 4
The predisposing factors to gallstone formation in the study SLE patients.
VariableThe Fisher exact testP-valueAge at the onset of the disease93.8<.000001Current steroid dosage109.2<.0000001Steroid therapy duration152.0<.0000001


## 3.2. Lipid-Protein Blood Spectrum and Gallstone Disease in SLE Patients

As we
expected, the levels of plasma lipids, ApoB-LDL (P=.005) and ApoAI-HDL (P=.05)
differed significantly in SLE patients with and without gallstones. SLE
patients with gallstones had significantly higher levels of TCh (P=.0004),
LDL-Ch (P=.006), HDL-Ch (P=.008) than those without gallstones.When the
group of SLE patients treated with corticosteroids were additionally compared,
significantly higher levels of TCh (P<.02) and LDL-Ch (P<.03)
were observed in patients with gallstones than in those without gallstones.
Significantly reduced levels of HDL-Ch (P<.02) and ApoAI-HDL (P<.04)
were found in SLE patients treated with corticosteroids without gallstone
disease.Interestingly,
the levels TG and ApoB-LDL, as well as TCh/HDL-Ch and LDL-Ch/HDL-Ch ratios, did
not differ significantly in corticosteroid-treated SLE patients as compared
with patients who had never been treated with corticosteroids.


## 3.3. The Composition and Structure of Gallstones in SlLE Patients

Series of
CCL-SEM color images of 10 gallstones were taken for analysis. Each image was
compared with control images to distinguish the deposition of bilirubin,
cholesterol, and protein components. The major components of the gallstones
under examination were cholesterol (Figure2(b)) and protein (Figure 2(c)).
They were all detected over the entire surface of the scanned gallstone while
rare bilirubin insertions (Figure 2(a)) were seen only at the periphery of the
gallstone. All 10 of examined gallstones from 4 SLE patients with
cholelithiasis due to long-term prednisolone therapy were structurally
identical. The findings have provided evidence for the protein-cholesterol
composition of the gallstones in corticosteroid-treated patients with SLE.CCL-SEM micrographs demonstrating the structural organization and chemical composition of a gallstone obtained from SLE patient: (A) general gallstone structure before color separation; (a) bilirubin inclusions; (b) cholesterol component; (c) protein.


## 4. Discussion

Gallstone
disease is one the most common disorders of the digestive system. In several
past decades, there has been a tendency toward the increased incidence of
gallstone disease. It can be explained by better diagnosis and by the new
noninvasive ultrasound techniques being introduced into clinical practice.Any
publications on the incidence and mechanisms of cholelithiasis in SLE have not
been found in the literature. This study has examined the relationship between
gallstone disease and corticosteroid treatment in SLE. As far as we know, this
is the first study to show the high frequency of gallstone disease in SLE
patients treated with prednisolone. Regression and discrimination analyses
reveal a strong association of gallstone formation in SLE patients with age at
the onset of the disease, current steroid dosage, and duration of prednisolone
therapy. Cholesterol metabolic disturbance is one of the possible causes of
gallstone formation in SLE patients on prednisolone therapy.Since there
is a strong correlation between cholelithiasis and blood lipid-protein spectral
changes in patients on corticosteroid therapy, we hypothesize that cholesterol
metabolism occurring onP450 cytochrome of the agranular endoplasmic
reticulum is altered and may be involved in gallstone formation.Our findings suggest
that higher total cholesterol levels in SLE patients with gallstones are most
likely to be caused by cholesterol catabolic disorders. Up to 80% of the total
cholesterol pool is oxidized to bile acids [13]. Bile acids are
synthesized from cholesterol, by involving P450 cytochrome of the
hepatic agranular endoplasmic reticulum [13]. They are the final
products of cholesterol metabolism and one of the most important ways of its
excretion from the body [13].The effects
of corticosteroids on the lipid-protein blood spectrum have been studied
before. The plasma lipid abnormalities reported in SLE corticosteroid treatment
patients are similar to those in other dyslipoproteinemias [14–16].Our SLE
patients with gallstones had significantly elevated LDL-Ch and HDL-Ch levels,
as compared to those without gallstones (Table2). The increased level of HDL
responsible for cholesterol transport from tissues and organs to the liver may
be accounted for by diminished cholesterol biotransformation to bile acids,
which should stimulate cholesterol delivery to hepatocytes through the feedback
system. Since HDL-cholesterol, both from the intestine and peripheral sources,
is the preferred type of cholesterol for biliary secretion, increased HDL
transport to the liver can also cause cholesterol hypersecretion in bile [17].
Enhanced cholesterol outflow from the peripheral tissues and cells by HDL
causes an increase in LDL levels via the feedback system to maintain the normal
levels of cholesterol in the tissues and cells in SLE patients long taking
prednisolone. Interestingly, the ApoB-LDL levels and the LDL-C/HDL-C ratio are
in the normal range, and do not differ significantly in the patients untreated
or treated with prednisolone. ApoB-LDL plays a major regulatory role in the
response of biliary cholesterol secretion to high-dietary cholesterol [18].
These findings suggest that cholesterol transport by means of lipoproteins is
unchanged in SLE patients receiving corticosteroids long.Recent
advances in bile molecular biology and biochemistry have demonstrated that
gallstone disease is a complex disorder: cholesterol supersaturation, decreased
bile acid/cholesterol ratio, hydrophobic bile salts, pronucleating proteins,
inflammation changes, and impaired gallbladder motility [19]. The SLE
patients are prone to biliary sludge and stone formation because of biliary
dyskinesia caused by ischemia of the gallbladder. Increased incidence of
cholelithiasis in SLE patients may also be related to vasculitis and
thrombosis. Gallbladder bile from gallstone patients contains factors that
stimulate formation of macroscopic cholesterol crystals and cholesterol stones [17, 20]. Supersaturation of bile with cholesterol is generally considered to
be a major trigger of cholesterol precipitation [20, 21].Steroid hormone catabolism also takes
place in the hepatocytes through theP450 cytochrome-dependent
hydroxylation. It is one of the principal pathways of their hepatic
inactivation in mammals [13, 22].We assume
that prednisolone competitively inhibits hepatocytic alpha-hydroxylases and
thus decelerates cholesterol transformation into bile acids. Subsequently, it
raises the serum levels of total cholesterol, LDL-Ch, and HDL-Ch, and also
increases cholesterol excretion into bile. Simultaneously, enhanced cholesterol
excretion is accompanied with the reduced hepatocytic secretion of bile acids
into the bile capillaries, resulting in a decrease in the bile acid/cholesterol
ratio. In SLE patients receiving long steroid hormones, gallbladder
inflammatory changes and immunological changes in the presence of abnormal
cholesterol and protein metabolisms may initiate gallstone formation (Figure3).Figure 3
Diagram of prednisolone (steroid hormones)-induced inhibition of cholesterol catabolism (see the text for comments).Cholesterol
gallstones are more common in women than in men, and exposure to oral
contraceptive steroids and conjugated estrogens increases the risk for
gallstones [23]. Sex hormones are most likely to be responsible for
the increased risk. Estrogen increases biliary cholesterol secretion causing
cholesterol supersaturation of bile. The hepatic estrogen receptor alpha, but
not estrogen receptor beta, plays a critical role in  17β-estradiol-induced formation of  cholesterol gallstones [23]. Thus, hormone replacement therapy in
postmenopausal women and the use of oral contraceptives have also been
described to be associated with an increased risk for gallstone disease [24].
Steroid (sex hormones, glucocorticoids, and mineralocorticoids) hormones can
competitively inhibit alpha-hydroxylases of hepatocytes and thus decelerates
cholesterol transformation into bile acids (Figure 3). Concomitant inflammatory
diseases of the gallbladder and its motor dysfunction may lead to the
development of gallstone disease. If there is evidence for this assumption, it
tells about drug-induced gallstone disease.There are
also reports on that high biliary protein concentration in lithogenic bile was associated with
cholesterol gallstone formation [25–27]. Mucin
is supposed to accelerate the crystallization of cholesterol in model bile [28].
Wilhelmi et al. demonstrated a dose-dependent effect of human but not of bovine
gallbladder mucin on the formation of cholesterol monohydrate crystals in
gallbladder bile of patients with cholesterol stones [28]. An
inflammation-dependent epidermal growth factor receptor cascade causes
overproduction of the gel-forming mucin MUC5AC, which accumulates in
cholesterol gallstone disease [29].Despite
the importance of high protein and cholesterol concentrations on the sequence
of events in gallstone formation, very little is known about the relationship
between cholesterol and protein to the
microarchitectonics of gallstones [27]. The standard technique for visualization of cholesterol stones
is scanning electron microscopy (SEM)  [30, 31]. However, this
technique does not permit identification and localization of chemical organic
deposits within the gallstone.In present study, we used the novel electron microscopy
technique, color cathodoluminescence scanning electron microscopy (CCL-SEM), to
investigate the chemical composition and microstructure of gallstones obtained
from patients with SLE.The main advantage of CCL-SEM over SEM is its capability to
visualize different organic and inorganic components and their distribution
over the gallstone section surface [12]. This information
can potentially contribute to the elucidation of the mechanisms of gallstone
formation.The CCL-SEM technology allowed us to generate CCL-mapping of
color images of 3 constituents of studied gallstones, unesterified cholesterol,
unconjugated bilirubin, and protein (Figure2). Comparative analysis with
control images (see Figure 1) has identified cholesterol (Figure 2(b)) and
protein (Figure 2(c)) as major components with homogeneous distribution from
the center to the periphery of the gallstone. In contrast, rare bilirubin
insertions (Figure 2(a)) were observed only at the periphery. CCL-SEM of all
study gallstones from SLE patients with gallstone disease demonstrated their
similar structure and chemical composition. The protein-cholesterol nature of
gallstones in SLE patients may serve as circumstantial evidence for the above
assumption that gallstone disease develops due to impaired cholesterol and
protein metabolisms during cholepoiesis under the action of corticosteroids.In conclusion, gallstone disease was detected in 36.7% of the examinees; 68.8% of SLE
patients on corticosteroid therapy had cholelithiasis. Age at the onset of the
disease, current steroid dosage, and steroid therapy duration are factors that
predispose to gallstone formation. The CCL-SEM study identified predominantly
the protein-cholesterol structure of gallstones. Multicenter-randomized studies
are required to support the aforesaid assumption that long-term steroid
hormones therapy is a model of cholesterol lithogenesis. Moreover, if there is
evidence that cholelitiasis is induced by steroids, this will provide impetus
to the search for and design of new drugs and therapeutic approaches to treating
autoimmune diseases.



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*Source: 123481-2009-03-25.xml*