Background Activation of inflammatory pathways plays an important contributory role in coronary plaque instability and subsequent rupture, which can lead to the development of acute coronary syndrome (ACS). Elevated levels of serum inflammatory markers such as C-reactive protein (CRP) represent independent risk factors for further cardiovascular events. Recent evidence indicates that in addition to lowering cholesterol levels, statins also decrease levels of inflammatory markers. Previous controlled clinical trials reporting the positive effects of statins in participants with ACS were designed for very early secondary prevention. To our knowledge, no controlled trials have evaluated the potential benefits of statin therapy, beginning immediately at the time of hospital admission. A previous pilot study performed by our group focused on early initiation of cerivastatin therapy. We demonstrated a highly significant reduction in levels of inflammatory markers (CRP and interleukin-6). Based on these preliminary findings, we are conducting a clinical trial to evaluate the efficacy of another statin, fluvastatin, as an early intervention in patients with ACS.
Background
Activation of inflammatory pathways plays an important contributory role in coronary plaque instability and subsequent rupture, which can lead to the development of acute coronary syndrome (ACS). Elevated levels of serum inflammatory markers such as C-reactive protein (CRP) represent independent risk factors for further cardiovascular events. Recent evidence indicates that in addition to lowering cholesterol levels, statins also decrease levels of inflammatory markers. Previous controlled clinical trials reporting the positive effects of statins in participants with ACS were designed for very early secondary prevention. To our knowledge, no controlled trials have evaluated the potential benefits of statin therapy, beginning immediately at the time of hospital admission. A previous pilot study performed by our group focused on early initiation of cerivastatin therapy. We demonstrated a highly significant reduction in levels of inflammatory markers (CRP and interleukin-6). Based on these preliminary findings, we are conducting a clinical trial to evaluate the efficacy of another statin, fluvastatin, as an early intervention in patients with ACS.Methods The FACS-trial (Fluvastatin in the therapy of Acute Coronary Syndrome) is a multicenter, randomized, double-blind, placebo-controlled study evaluating the effects of fluvastatin therapy initiated at the time of hospital admission. The study will enroll 1,000 participants admitted to hospital for ACS (both with and without ST elevation). The primary endpoint for the study is the influence of fluvastatin therapy on levels of inflammatory markers (CRP and interleukin-6) and on pregnancy associated plasma protein A (PAPP-A). A combined secondary endpoint is 30-day and one-year occurrence of death, nonfatal myocardial infarction, recurrent symptomatic ischemia, urgent revascularization, and cardiac arrest.
Methods
The FACS-trial (Fluvastatin in the therapy of Acute Coronary Syndrome) is a multicenter, randomized, double-blind, placebo-controlled study evaluating the effects of fluvastatin therapy initiated at the time of hospital admission. The study will enroll 1,000 participants admitted to hospital for ACS (both with and without ST elevation). The primary endpoint for the study is the influence of fluvastatin therapy on levels of inflammatory markers (CRP and interleukin-6) and on pregnancy associated plasma protein A (PAPP-A). A combined secondary endpoint is 30-day and one-year occurrence of death, nonfatal myocardial infarction, recurrent symptomatic ischemia, urgent revascularization, and cardiac arrest.Conclusion The primary objective of the FACS trial is to demonstrate that statin therapy, when started immediately after hospital admission for ACS, results in reduction of inflammation and improvement of prognosis. This study may contribute to new knowledge regarding therapeutic strategies for patients suffering from ACS and may offer additional clinical indications for the use of statins.
Conclusion
The primary objective of the FACS trial is to demonstrate that statin therapy, when started immediately after hospital admission for ACS, results in reduction of inflammation and improvement of prognosis. This study may contribute to new knowledge regarding therapeutic strategies for patients suffering from ACS and may offer additional clinical indications for the use of statins.

Background During the past decade, inflammation has often been cited as a major factor in the pathogenesis of atherosclerosis and its clinical sequelae, including ischemic heart disease. It was found that traditional risk factors such as hypertension, hypercholesterolemia, diabetes, and smoking could not fully account for the development of coronary stenosis in all patients suffering from ischemic heart disease. Intensive study of the pathogenesis of coronary plaque development and rupture led to the hypothesis that inflammatory factors contribute to this process. For example, T-lymphocytes and monocytes/macrophages have been repeatedly identified in plaque lesions; elevated levels of acute phase proteins (C-reactive protein, serum amyloid A, fibrinogen), cytokines (interleukin 1, interleukin 6, interleukin 8, tumor necrosis factor), and adhesive molecules (ICAM-1) correlate with the worse prognoses in patients with ischemic heart disease [ 1 - 5 ]. Furthermore, the increased level of C-reactive protein (CRP) is now widely recognized as being an independent risk factor for a higher incidence of non-fatal and fatal coronary events in patients with chronic ischemic heart disease and acute coronary syndromes [ 6 - 9 ]. Activation of the immune reaction in acute ischemic heart disease likely derives from: (i) pathological events occurring in the arterial wall where the lesion develops, leading to plaque rupture and the subsequent clinical consequences and (ii) myocardial necrosis, which triggers processes involved in removal of the necrotic mass and replacement with scar tissue. Whereas activation of plaque inflammation (as noted above) serves as a marker for plaque instability, elevation of inflammatory factors from the second source correlates with the extent of myocardial necrosis. Statins, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, lower cholesterol levels by decreasing the production of low-density lipoproteins (LDL) and up-regulating the expression of the LDL-receptor. These drugs are widely used in patients with hypercholesterolemia for primary and secondary prevention of coronary artery disease because of their efficacy in reducing cardiovascular morbidity and mortality [ 10 - 12 ]. Surprisingly, statin therapy also improves prognosis in patients with normal or low cholesterol levels[ 13 ]. Evaluation of the non-lipid effects of statins reveals a possible beneficial effect mediated by the reduction of inflammatory markers, namely, CRP. This effect seems to be independent of cholesterol level [ 1 , 14 ]. The mechanisms by which statins inhibit inflammation are not fully understood. It has been reported that they suppress production of monocyte chemotactic protein-1 (MCP-1) [ 15 ], as well as matrix metalloproteinases (MMPs) [ 5 , 16 , 17 ]. Statins also decrease macrophage expression of soluble ICAM-1 and secretion of IL-1, IL-6, TNF-alpha [ 5 , 18 - 23 ]. A number of large clinical trials have been designed to investigate the effect of statins in treating acute coronary syndromes (ACS) [ 24 - 30 ] Because patients in these studies were randomized later after being admitted to hospital, often after they had been clinically stabilized, these randomized, double blind trials focused more on early secondary prevention, as opposed to evaluating earlier therapy to target plaque instability in ACS. Furthermore, broad exclusion criteria in some of these trials, including coronary intervention during the index hospitalization visit, deter from the generalizability of their results to the majority of patients treated according to current clinical practice [ 24 ]. Nevertheless, these studies have shown promising results, despite the fact that the statins were administered after activation of the immune mechanisms was completed and after the inflammatory reaction was already fully developed. Little information is available on the efficacy of statins in treating ACS at an earlier phase, i.e., at the time of hospital admission. Recently published data from experimental projects [ 31 - 35 ] and from small clinical trials [ 36 - 38 ]) have shown a positive effect of statins when they are administered in the acute phase of ACS. Our preliminary results with cerivastatin treatment in patients with non-ST segment elevation ACS starting at the time of hospital admission have shown the safety of such a strategy as well as a decrease in inflammatory markers (CRP, IL-6) by 24-hour follow-up, as compared to the non-treated group [ 39 ]. Based on these pilot data, we are conducting a clinical trial to evaluate fluvastatin therapy administered to patients with ACS immediately at the time of admission (Figure  1 ). Figure 1 Design of the FACS trial in comparison to other trials evaluating statins in ACS patients. In the Pravastatin in Acute Coronary Treatment (PACT) trial statin, therapy was initiated within 24 hours of onset of ACS. In the Myocardial Ischemia Reduction With Aggressive Cholesterol Lowering (MIRACL) trial, patients were randomized 24 to 96 hours after ACS. In the Z-phase of the A-to-Z trial, simvastatin therapy was initiated within 5 days of the onset of ACS, after clinical stabilization. In the Pravastatin or Atorvastatin Evaluation and Infection Therapy (PROVE IT) trial, patients were randomized up to 10 days after ACS. In the Lipid-Coronary Artery Disease (L-CAD) trial, statin therapy was initiated up to ten days following the onset of ACS. In the Fluvastatin on Risk Diminishing After Acute Myocardial Infarction (FLORIDA) trial, patients were randomized up to two weeks after ACS.
Background
During the past decade, inflammation has often been cited as a major factor in the pathogenesis of atherosclerosis and its clinical sequelae, including ischemic heart disease. It was found that traditional risk factors such as hypertension, hypercholesterolemia, diabetes, and smoking could not fully account for the development of coronary stenosis in all patients suffering from ischemic heart disease. Intensive study of the pathogenesis of coronary plaque development and rupture led to the hypothesis that inflammatory factors contribute to this process. For example, T-lymphocytes and monocytes/macrophages have been repeatedly identified in plaque lesions; elevated levels of acute phase proteins (C-reactive protein, serum amyloid A, fibrinogen), cytokines (interleukin 1, interleukin 6, interleukin 8, tumor necrosis factor), and adhesive molecules (ICAM-1) correlate with the worse prognoses in patients with ischemic heart disease [ 1 - 5 ]. Furthermore, the increased level of C-reactive protein (CRP) is now widely recognized as being an independent risk factor for a higher incidence of non-fatal and fatal coronary events in patients with chronic ischemic heart disease and acute coronary syndromes [ 6 - 9 ].Activation of the immune reaction in acute ischemic heart disease likely derives from: (i) pathological events occurring in the arterial wall where the lesion develops, leading to plaque rupture and the subsequent clinical consequences and (ii) myocardial necrosis, which triggers processes involved in removal of the necrotic mass and replacement with scar tissue. Whereas activation of plaque inflammation (as noted above) serves as a marker for plaque instability, elevation of inflammatory factors from the second source correlates with the extent of myocardial necrosis.Statins, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, lower cholesterol levels by decreasing the production of low-density lipoproteins (LDL) and up-regulating the expression of the LDL-receptor. These drugs are widely used in patients with hypercholesterolemia for primary and secondary prevention of coronary artery disease because of their efficacy in reducing cardiovascular morbidity and mortality [ 10 - 12 ]. Surprisingly, statin therapy also improves prognosis in patients with normal or low cholesterol levels[ 13 ]. Evaluation of the non-lipid effects of statins reveals a possible beneficial effect mediated by the reduction of inflammatory markers, namely, CRP. This effect seems to be independent of cholesterol level [ 1 , 14 ]. The mechanisms by which statins inhibit inflammation are not fully understood. It has been reported that they suppress production of monocyte chemotactic protein-1 (MCP-1) [ 15 ], as well as matrix metalloproteinases (MMPs) [ 5 , 16 , 17 ]. Statins also decrease macrophage expression of soluble ICAM-1 and secretion of IL-1, IL-6, TNF-alpha [ 5 , 18 - 23 ].A number of large clinical trials have been designed to investigate the effect of statins in treating acute coronary syndromes (ACS) [ 24 - 30 ] Because patients in these studies were randomized later after being admitted to hospital, often after they had been clinically stabilized, these randomized, double blind trials focused more on early secondary prevention, as opposed to evaluating earlier therapy to target plaque instability in ACS. Furthermore, broad exclusion criteria in some of these trials, including coronary intervention during the index hospitalization visit, deter from the generalizability of their results to the majority of patients treated according to current clinical practice [ 24 ]. Nevertheless, these studies have shown promising results, despite the fact that the statins were administered after activation of the immune mechanisms was completed and after the inflammatory reaction was already fully developed.Little information is available on the efficacy of statins in treating ACS at an earlier phase, i.e., at the time of hospital admission. Recently published data from experimental projects [ 31 - 35 ] and from small clinical trials [ 36 - 38 ]) have shown a positive effect of statins when they are administered in the acute phase of ACS. Our preliminary results with cerivastatin treatment in patients with non-ST segment elevation ACS starting at the time of hospital admission have shown the safety of such a strategy as well as a decrease in inflammatory markers (CRP, IL-6) by 24-hour follow-up, as compared to the non-treated group [ 39 ]. Based on these pilot data, we are conducting a clinical trial to evaluate fluvastatin therapy administered to patients with ACS immediately at the time of admission (Figure  1 ).Methods Objectives The objectives of the FACS trial are to determine: (i) Whether initiation of fluvastatin therapy in patients with ACS immediately after hospital admission decreases levels of CRP, IL-6, and pregnancy-associated plasma protein A/ proform eosinophilic major basic protein (PAPP-A/proMBP), which represent indirect markers of plaque instability and indicators of poor prognosis; and (ii) Whether initiation of fluvastatin therapy decreases the occurrence of ischemic events (death, nonfatal myocardial infarction, recurrent symptomatic ischemia, urgent revascularization, cardiac arrest) in patients with ACS. Overview This is a prospective, 30-day, multicenter, randomized, double-blind, placebo-controlled study in 1,000 patients with ACS. Patients are enrolled from 10 sites in the Czech Republic and Slovakia. At each institution, the protocol and the informed consent form are reviewed and approved by the institutional ethics committee before study initiation. Eligible patients are randomized to one of two treatment groups immediately after hospital admission (within one hour). One group is assigned 80 mg/day fluvastatin (Lescol XL), with the other group receiving placebo. Participants are followed on an intention-to-treat basis. The primary endpoint relates to levels of CRP, IL-6, and PAPP-A/proMBP. The secondary endpoint is the occurrence of an ischemic event, defined as death, nonfatal myocardial infarction (MI), recurrent symptomatic myocardial ischemia, cardiac arrest with resuscitation, and urgent revascularization. Study population This study will enroll high-risk patients admitted to the hospital for ACS. Eligible patients with ST elevation ACS must have resting chest pain less than 12 hours before admission and either ≥ 1 mm ST-segment elevation in 2 or more continuous leads or new left bundle branch block on ECG. Those with non-ST elevation ACS must have resting chest pain during the previous 48 hours and either ≥ 1 mm ST segment depression or negative T waves in 2 or more continuous leads. Exclusion criteria Subjects are excluded from study participation if they are <18 years of age or if they have concomitant active liver disease or persistent elevation of transaminases (> 3 times the upper limit of normal), a history of lipid-lowering therapy less than 30 days before the index event or a known allergy to fluvastatin or to any additives present in the drug. Other exclusions include inability to ingest oral medication, unwillingness to be followed for the duration of the study, muscle disease (e.g., myositis), and creatine kinase ≥ 5 times the upper limit of normal due to conditions other than myocardial infarction. Women of childbearing potential who are pregnant, nursing or who are not using effective contraception will also be excluded. Follow-up After obtaining informed consent, blood samples are taken from patients for examination of inflammatory markers (CRP, IL-6, and PAPP-A/proMBP). Patients are then randomized to 80 mg fluvastatin (Lescol XL) or to placebo immediately p.o. Medical history and physical examination, standard 12-lead ECG, blood lipid profile, and liver function tests are performed as part of participants' routine admission care. Fluvastatin 80 mg or placebo are then taken once daily for 30 days. Follow-up measurement of inflammatory markers (CRP, IL-6, and PAPP-A/proMBP) is performed on day 2 and day 30. Follow-up visits are scheduled at pre-discharge, day 30, 90, 180 and 360. Blood liver function and creatine kinase tests are done at pre-discharge and at the 30-day visits. At day 30, the lipid profile is also examined, and study medication is withdrawn. All visits include assessment of ischemic events and recent medical history since the last follow-up visit, including use of concomitant medications (Figure  2 ). Figure 2 Study design of the FACS Trial. Patients admitted with acute coronary syndrome (ACS) are randomized to either fluvastatin 80 mg or placebo for 30 days. Patients are then followed for one year. Assessments of CRP, IL-6, and PAPP-A/proMBP (LAB) are performed at admission, on day 2, and day 30. During follow-up, no specific recommendations are made with respect to diagnostic and therapeutic strategy, except that other lipid-lowering drugs should not be given after randomization until day 30. All management decisions are left to the discretion of each patient's treating physician. Safety The principal safety concerns are hepatic dysfunction and myopathy. If a patient's serum transaminase levels are persistently elevated to > 3 times the upper limit of normal, the study medication is discontinued. Similarly, study medication is stopped if the patient develops muscle pain, weakness, or tenderness in association with a serum creatine kinase level > 10 times the upper limit of normal. Sample size The trial will enroll 1,000 patients, to ensure adequate power to detect significant treatment benefit of 80 mg fluvastatin (Lescol XL) with respect to the primary endpoint (30-day decrease of CRP and IL-6) and the combined secondary endpoint (death, nonfatal myocardial infarction, recurrent symptomatic ischemia, urgent revascularization, cardiac arrest). With 500 patients randomized to 80 mg fluvastatin (Lescol XL) and 500 patients randomized to placebo, the trial will have more than 80 % power to detect a decrease in CRP level by 1.36 μg/L and a decrease in IL-6 level by 1.09 ng/L. Calculations are based on a two-sample  t -test. The estimated combined secondary endpoint rate at 30-days is 20 %. Based on comparison of proportions with  p  = 0.05 test significance, the trial will have more than 80 % power to detect a 33% decrease in the combined secondary endpoint.
Methods
Objectives The objectives of the FACS trial are to determine: (i) Whether initiation of fluvastatin therapy in patients with ACS immediately after hospital admission decreases levels of CRP, IL-6, and pregnancy-associated plasma protein A/ proform eosinophilic major basic protein (PAPP-A/proMBP), which represent indirect markers of plaque instability and indicators of poor prognosis; and (ii) Whether initiation of fluvastatin therapy decreases the occurrence of ischemic events (death, nonfatal myocardial infarction, recurrent symptomatic ischemia, urgent revascularization, cardiac arrest) in patients with ACS.
Objectives
The objectives of the FACS trial are to determine:(i) Whether initiation of fluvastatin therapy in patients with ACS immediately after hospital admission decreases levels of CRP, IL-6, and pregnancy-associated plasma protein A/ proform eosinophilic major basic protein (PAPP-A/proMBP), which represent indirect markers of plaque instability and indicators of poor prognosis; and(ii) Whether initiation of fluvastatin therapy decreases the occurrence of ischemic events (death, nonfatal myocardial infarction, recurrent symptomatic ischemia, urgent revascularization, cardiac arrest) in patients with ACS.Overview This is a prospective, 30-day, multicenter, randomized, double-blind, placebo-controlled study in 1,000 patients with ACS. Patients are enrolled from 10 sites in the Czech Republic and Slovakia. At each institution, the protocol and the informed consent form are reviewed and approved by the institutional ethics committee before study initiation. Eligible patients are randomized to one of two treatment groups immediately after hospital admission (within one hour). One group is assigned 80 mg/day fluvastatin (Lescol XL), with the other group receiving placebo. Participants are followed on an intention-to-treat basis. The primary endpoint relates to levels of CRP, IL-6, and PAPP-A/proMBP. The secondary endpoint is the occurrence of an ischemic event, defined as death, nonfatal myocardial infarction (MI), recurrent symptomatic myocardial ischemia, cardiac arrest with resuscitation, and urgent revascularization.
Overview
This is a prospective, 30-day, multicenter, randomized, double-blind, placebo-controlled study in 1,000 patients with ACS. Patients are enrolled from 10 sites in the Czech Republic and Slovakia. At each institution, the protocol and the informed consent form are reviewed and approved by the institutional ethics committee before study initiation. Eligible patients are randomized to one of two treatment groups immediately after hospital admission (within one hour). One group is assigned 80 mg/day fluvastatin (Lescol XL), with the other group receiving placebo. Participants are followed on an intention-to-treat basis. The primary endpoint relates to levels of CRP, IL-6, and PAPP-A/proMBP. The secondary endpoint is the occurrence of an ischemic event, defined as death, nonfatal myocardial infarction (MI), recurrent symptomatic myocardial ischemia, cardiac arrest with resuscitation, and urgent revascularization.Study population This study will enroll high-risk patients admitted to the hospital for ACS. Eligible patients with ST elevation ACS must have resting chest pain less than 12 hours before admission and either ≥ 1 mm ST-segment elevation in 2 or more continuous leads or new left bundle branch block on ECG. Those with non-ST elevation ACS must have resting chest pain during the previous 48 hours and either ≥ 1 mm ST segment depression or negative T waves in 2 or more continuous leads. Exclusion criteria Subjects are excluded from study participation if they are <18 years of age or if they have concomitant active liver disease or persistent elevation of transaminases (> 3 times the upper limit of normal), a history of lipid-lowering therapy less than 30 days before the index event or a known allergy to fluvastatin or to any additives present in the drug. Other exclusions include inability to ingest oral medication, unwillingness to be followed for the duration of the study, muscle disease (e.g., myositis), and creatine kinase ≥ 5 times the upper limit of normal due to conditions other than myocardial infarction. Women of childbearing potential who are pregnant, nursing or who are not using effective contraception will also be excluded.
Study population
This study will enroll high-risk patients admitted to the hospital for ACS. Eligible patients with ST elevation ACS must have resting chest pain less than 12 hours before admission and either ≥ 1 mm ST-segment elevation in 2 or more continuous leads or new left bundle branch block on ECG. Those with non-ST elevation ACS must have resting chest pain during the previous 48 hours and either ≥ 1 mm ST segment depression or negative T waves in 2 or more continuous leads.Exclusion criteria Subjects are excluded from study participation if they are <18 years of age or if they have concomitant active liver disease or persistent elevation of transaminases (> 3 times the upper limit of normal), a history of lipid-lowering therapy less than 30 days before the index event or a known allergy to fluvastatin or to any additives present in the drug. Other exclusions include inability to ingest oral medication, unwillingness to be followed for the duration of the study, muscle disease (e.g., myositis), and creatine kinase ≥ 5 times the upper limit of normal due to conditions other than myocardial infarction. Women of childbearing potential who are pregnant, nursing or who are not using effective contraception will also be excluded.
Exclusion criteria
Subjects are excluded from study participation if they are <18 years of age or if they have concomitant active liver disease or persistent elevation of transaminases (> 3 times the upper limit of normal), a history of lipid-lowering therapy less than 30 days before the index event or a known allergy to fluvastatin or to any additives present in the drug. Other exclusions include inability to ingest oral medication, unwillingness to be followed for the duration of the study, muscle disease (e.g., myositis), and creatine kinase ≥ 5 times the upper limit of normal due to conditions other than myocardial infarction. Women of childbearing potential who are pregnant, nursing or who are not using effective contraception will also be excluded.Follow-up After obtaining informed consent, blood samples are taken from patients for examination of inflammatory markers (CRP, IL-6, and PAPP-A/proMBP). Patients are then randomized to 80 mg fluvastatin (Lescol XL) or to placebo immediately p.o. Medical history and physical examination, standard 12-lead ECG, blood lipid profile, and liver function tests are performed as part of participants' routine admission care. Fluvastatin 80 mg or placebo are then taken once daily for 30 days. Follow-up measurement of inflammatory markers (CRP, IL-6, and PAPP-A/proMBP) is performed on day 2 and day 30. Follow-up visits are scheduled at pre-discharge, day 30, 90, 180 and 360. Blood liver function and creatine kinase tests are done at pre-discharge and at the 30-day visits. At day 30, the lipid profile is also examined, and study medication is withdrawn. All visits include assessment of ischemic events and recent medical history since the last follow-up visit, including use of concomitant medications (Figure  2 ). Figure 2 Study design of the FACS Trial. Patients admitted with acute coronary syndrome (ACS) are randomized to either fluvastatin 80 mg or placebo for 30 days. Patients are then followed for one year. Assessments of CRP, IL-6, and PAPP-A/proMBP (LAB) are performed at admission, on day 2, and day 30. During follow-up, no specific recommendations are made with respect to diagnostic and therapeutic strategy, except that other lipid-lowering drugs should not be given after randomization until day 30. All management decisions are left to the discretion of each patient's treating physician.
Follow-up
After obtaining informed consent, blood samples are taken from patients for examination of inflammatory markers (CRP, IL-6, and PAPP-A/proMBP). Patients are then randomized to 80 mg fluvastatin (Lescol XL) or to placebo immediately p.o. Medical history and physical examination, standard 12-lead ECG, blood lipid profile, and liver function tests are performed as part of participants' routine admission care. Fluvastatin 80 mg or placebo are then taken once daily for 30 days. Follow-up measurement of inflammatory markers (CRP, IL-6, and PAPP-A/proMBP) is performed on day 2 and day 30. Follow-up visits are scheduled at pre-discharge, day 30, 90, 180 and 360. Blood liver function and creatine kinase tests are done at pre-discharge and at the 30-day visits. At day 30, the lipid profile is also examined, and study medication is withdrawn. All visits include assessment of ischemic events and recent medical history since the last follow-up visit, including use of concomitant medications (Figure  2 ).During follow-up, no specific recommendations are made with respect to diagnostic and therapeutic strategy, except that other lipid-lowering drugs should not be given after randomization until day 30. All management decisions are left to the discretion of each patient's treating physician.Safety The principal safety concerns are hepatic dysfunction and myopathy. If a patient's serum transaminase levels are persistently elevated to > 3 times the upper limit of normal, the study medication is discontinued. Similarly, study medication is stopped if the patient develops muscle pain, weakness, or tenderness in association with a serum creatine kinase level > 10 times the upper limit of normal.
Safety
The principal safety concerns are hepatic dysfunction and myopathy. If a patient's serum transaminase levels are persistently elevated to > 3 times the upper limit of normal, the study medication is discontinued. Similarly, study medication is stopped if the patient develops muscle pain, weakness, or tenderness in association with a serum creatine kinase level > 10 times the upper limit of normal.Sample size The trial will enroll 1,000 patients, to ensure adequate power to detect significant treatment benefit of 80 mg fluvastatin (Lescol XL) with respect to the primary endpoint (30-day decrease of CRP and IL-6) and the combined secondary endpoint (death, nonfatal myocardial infarction, recurrent symptomatic ischemia, urgent revascularization, cardiac arrest). With 500 patients randomized to 80 mg fluvastatin (Lescol XL) and 500 patients randomized to placebo, the trial will have more than 80 % power to detect a decrease in CRP level by 1.36 μg/L and a decrease in IL-6 level by 1.09 ng/L. Calculations are based on a two-sample  t -test. The estimated combined secondary endpoint rate at 30-days is 20 %. Based on comparison of proportions with  p  = 0.05 test significance, the trial will have more than 80 % power to detect a 33% decrease in the combined secondary endpoint.
Sample size
The trial will enroll 1,000 patients, to ensure adequate power to detect significant treatment benefit of 80 mg fluvastatin (Lescol XL) with respect to the primary endpoint (30-day decrease of CRP and IL-6) and the combined secondary endpoint (death, nonfatal myocardial infarction, recurrent symptomatic ischemia, urgent revascularization, cardiac arrest). With 500 patients randomized to 80 mg fluvastatin (Lescol XL) and 500 patients randomized to placebo, the trial will have more than 80 % power to detect a decrease in CRP level by 1.36 μg/L and a decrease in IL-6 level by 1.09 ng/L. Calculations are based on a two-sample  t -test. The estimated combined secondary endpoint rate at 30-days is 20 %. Based on comparison of proportions with  p  = 0.05 test significance, the trial will have more than 80 % power to detect a 33% decrease in the combined secondary endpoint.tpConclusion The FACS trial is the first multicenter, randomized, double-blind, placebo-controlled trial investigating the effects of fluvastatin therapy started immediately after hospital admission in patients with ACS.
Conclusion
The FACS trial is the first multicenter, randomized, double-blind, placebo-controlled trial investigating the effects of fluvastatin therapy started immediately after hospital admission in patients with ACS.Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contributions PO has made substantial contributions to the concept and design of the trial and has drafted the manuscript. DA, PH, JVej, JK, MMat, and JVes have made substantial contributions to the concept and design of the study and have been involved in revising the manuscript. PB, MW, OA, JS, EN, FH, MR, and JC have been involved in the acquisition and analysis of data. MMac and MK have given final approval of the version to be published.
Authors' contributions
PO has made substantial contributions to the concept and design of the trial and has drafted the manuscript. DA, PH, JVej, JK, MMat, and JVes have made substantial contributions to the concept and design of the study and have been involved in revising the manuscript. PB, MW, OA, JS, EN, FH, MR, and JC have been involved in the acquisition and analysis of data. MMac and MK have given final approval of the version to be published.


Background Possible methods for distinguishing receptor binding models and analysing their parameters are considered.
Background
Possible methods for distinguishing receptor binding models and analysing their parameters are considered.Results and Discussion The conjugate gradients method is shown to be optimal for solving problems of the kind considered. Convergence with experimental data is rapidly achieved with the appropriate model but not with alternative models.
Results and Discussion
The conjugate gradients method is shown to be optimal for solving problems of the kind considered. Convergence with experimental data is rapidly achieved with the appropriate model but not with alternative models.Conclusion Lack of convergence using the conjugate gradients method can be taken to indicate inconsistency between the receptor binding model and the experimental data. Thus, the conjugate gradients method can be used to distinguish among receptor binding models.
Conclusion
Lack of convergence using the conjugate gradients method can be taken to indicate inconsistency between the receptor binding model and the experimental data. Thus, the conjugate gradients method can be used to distinguish among receptor binding models.

Background Most medicinal preparations and biologically active substances do not penetrate into cells and must therefore exert their influence on intracellular processes by interaction with specific protein molecules at the cell surface [ 1 - 3 ], for which the name "receptors" is in common use. Hormones and drugs that interact with receptors are known as "ligands". Data from research in molecular biology, and also results from indirect studies, have established the following schemes of ligand-receptor interaction [see [ 4 - 6 ] represented by the general models: Non-cooperative interaction between ligand and receptor : where  R  is the receptor molecule,  L  is the ligand molecule,  RL  is the ligand-receptor complex, and  k +1  and  k -1  are respectively the kinetic constants of formation and dissociation of the complex. Cooperative interaction between ligand and receptor Interaction of one ligand with N types of binding sites Let us note that the ligand-receptor interaction can also involve a combination of all three of these schemes. The most frequently used method for studying ligand-receptor interactions is the radioreceptor method [ 7 ], based on measuring the amount of radioactively labelled ligand bound in some defined manner to the appropriate receptor. Thus, experimentally, direct measurements of ligand-receptor complex concentration, [ RL ] are determined. The investigator has to solve two basic interrelated problems [ 6 ]: 1. discrimination among the ligand-receptor binding models (1–3 or modifications thereof); 2. determination of parameters that adequately relate the model to the experimental data. From a pharmacological point of view, the most important parameters are the following: [ R 0 ] (initial receptor concentration), and K d  =  k -1 / k +1  (dissociation constant) [ 7 ] The concentration of receptors and the dissociation constant can be changed. Modification of these parameter values can occur in many physiological and pathophysiological situations. For instance, the receptor concentration can reflect functional receptor modifications, and the dissociation constant can reflect genetic alterations of the receptor [ 6 ]. To solve the two interrelated problems a series of graphic methods can be deployed, of which the most frequently used is the Scatchard method [ 7 , 8 ]. However, the application of graphic methods in many cases is limited because of experimental errors and/or receptor binding complexity [ 9 , 10 ]. In particular, graphic methods are inapplicable for definition of the cooperative binding parameters and for analysis of non-equilibrium binding. Regression methods can be found for the measurement of ligand-receptor interaction constants [ 11 ]. As a matter of fact, these procedures computerize the graphic methods. Therefore, both regression methods and graphic methods are of limited applicability. The present paper argues that it is very difficult or impossible to discriminate reliably among receptor binding models or to analyse the parameters by traditional analytical methods.
Background
Most medicinal preparations and biologically active substances do not penetrate into cells and must therefore exert their influence on intracellular processes by interaction with specific protein molecules at the cell surface [ 1 - 3 ], for which the name "receptors" is in common use. Hormones and drugs that interact with receptors are known as "ligands". Data from research in molecular biology, and also results from indirect studies, have established the following schemes of ligand-receptor interaction [see [ 4 - 6 ] represented by the general models:Non-cooperative interaction between ligand and receptor :Non-cooperative interaction between ligand and receptorwhere  R  is the receptor molecule,  L  is the ligand molecule,  RL  is the ligand-receptor complex, and  k +1  and  k -1  are respectively the kinetic constants of formation and dissociation of the complex.RLRLkkCooperative interaction between ligand and receptorInteraction of one ligand with N types of binding sitesLet us note that the ligand-receptor interaction can also involve a combination of all three of these schemes. The most frequently used method for studying ligand-receptor interactions is the radioreceptor method [ 7 ], based on measuring the amount of radioactively labelled ligand bound in some defined manner to the appropriate receptor. Thus, experimentally, direct measurements of ligand-receptor complex concentration, [ RL ] are determined. The investigator has to solve two basic interrelated problems [ 6 ]:RL1. discrimination among the ligand-receptor binding models (1–3 or modifications thereof);2. determination of parameters that adequately relate the model to the experimental data.From a pharmacological point of view, the most important parameters are the following:[ R 0 ] (initial receptor concentration), andRK d  =  k -1 / k +1  (dissociation constant) [ 7 ]KdkkThe concentration of receptors and the dissociation constant can be changed. Modification of these parameter values can occur in many physiological and pathophysiological situations. For instance, the receptor concentration can reflect functional receptor modifications, and the dissociation constant can reflect genetic alterations of the receptor [ 6 ].To solve the two interrelated problems a series of graphic methods can be deployed, of which the most frequently used is the Scatchard method [ 7 , 8 ]. However, the application of graphic methods in many cases is limited because of experimental errors and/or receptor binding complexity [ 9 , 10 ]. In particular, graphic methods are inapplicable for definition of the cooperative binding parameters and for analysis of non-equilibrium binding.Regression methods can be found for the measurement of ligand-receptor interaction constants [ 11 ]. As a matter of fact, these procedures computerize the graphic methods. Therefore, both regression methods and graphic methods are of limited applicability. The present paper argues that it is very difficult or impossible to discriminate reliably among receptor binding models or to analyse the parameters by traditional analytical methods.Materials and methods Let us write the law of mass action for each ligand-receptor interaction scheme as: For the scheme  (1) But [ R ] = [ R 0 ] - [ RL ], [ L ] = [ L 0 ] - [ RL ]. So equation (4) can be rewritten: This differential equation relates to the class of  Rikkatty  equations. It can be solved analytically with the help of a special substitution [ 12 ], but in all other cases the substitutions [ R ] = [ R 0 ] - [ RL ], [ L ] = [ L 0 ] - [ RL ] do not generate analytically soluble equations. Therefore, all equations of this form were solved numerically using the Runge-Kutta method [ 13 , 14 ]. The differential equations are as follows: For scheme  (2): For scheme  (3): Numerical solution of equations (5–7) was carried out to determine [ RL ] u . Random error assuming the normal distribution law was superimposed on the magnitude of [ RL ] u , and was calculated at 5, 10, 20 or 100 points. The magnitude [ RT ] m  was calculated using parameters other than [ RL ] u  from models (1–3). These parameters were applied to the determination of [ RL ] u  by the following functional minimization: Φ = ([ RL ] u  - [ RL ] m ) 2 .     (8) For functional minimization as per equation (8), Newton's method and its variants (the conjugate gradients method and coordinate descent method in various modifications) were used [ 15 - 17 ]. The iteration procedure stopped, when Φ/[ RL ] u  was constant on the next iteration step. It is clear from the literature [ 6 ] that [ R 0 ] and  K d  cannot be <10 -15  M or >10 -5  M. Hence the iteration procedure could be improved by re-scaling these parameters logarithmically, making 10 -15  M equivalent to -1 on the new scale and 10 -5  M equivalent to 1.
Materials and methods
Let us write the law of mass action for each ligand-receptor interaction scheme as:For the scheme  (1)For the schemeBut [ R ] = [ R 0 ] - [ RL ], [ L ] = [ L 0 ] - [ RL ].RRRLLLRLSo equation (4) can be rewritten:This differential equation relates to the class of  Rikkatty  equations. It can be solved analytically with the help of a special substitution [ 12 ], but in all other cases the substitutions [ R ] = [ R 0 ] - [ RL ], [ L ] = [ L 0 ] - [ RL ] do not generate analytically soluble equations. Therefore, all equations of this form were solved numerically using the Runge-Kutta method [ 13 , 14 ].RikkattyRRRLLLRLThe differential equations are as follows:For scheme  (2):For schemeFor scheme  (3):For schemeNumerical solution of equations (5–7) was carried out to determine [ RL ] u . Random error assuming the normal distribution law was superimposed on the magnitude of [ RL ] u , and was calculated at 5, 10, 20 or 100 points.RLuRLuThe magnitude [ RT ] m  was calculated using parameters other than [ RL ] u  from models (1–3). These parameters were applied to the determination of [ RL ] u  by the following functional minimization:RTmRLuRLuΦ = ([ RL ] u  - [ RL ] m ) 2 .     (8)RLuRLmFor functional minimization as per equation (8), Newton's method and its variants (the conjugate gradients method and coordinate descent method in various modifications) were used [ 15 - 17 ]. The iteration procedure stopped, when Φ/[ RL ] u  was constant on the next iteration step.RLuIt is clear from the literature [ 6 ] that [ R 0 ] and  K d  cannot be <10 -15  M or >10 -5  M. Hence the iteration procedure could be improved by re-scaling these parameters logarithmically, making 10 -15  M equivalent to -1 on the new scale and 10 -5  M equivalent to 1.RKdResults and discussion The functional (8) contour plots are shown in fig.  1 . From this figure, the degree of correlation between the parameters [ R 0 ]  K d  can be seen. Therefore the magnification of the random error in evaluating the magnitude of [ RL ] u  displaces the functional (8) global maximum from its true values. In a sufficiently large neighbourhood of the global maximum, the functional magnitude (8) is practically invariant. However, this modification becomes more essential for evaluating the ratio of the functional (8) to basis vector of values [ RL ] u . Therefore this ratio was used with the inhibiting criterion choice. Figure 1 The functional (8) contour plot. The various methods of functional minimization are illustrated: a. The second derivative Newton method b. The conjugate gradients method c. The coordinate descent method The Newton method converges only in the close neighbourhood of the global maximum. However, modifications of the Newton method using second derivatives allow convergence to the global maximum after 1–2 iterations (fig.  1 , line 1). The conjugate gradients method converged after 2–3 iterations (fig.  1 , line 2). When magnification of the random error in the evaluation of [ RL ] u  was taken into account, the convergence of the conjugate gradients method varied less than that of the Newton method. The coordinate descent method required an indeterminately large number of iterations before satisfactory convergence was reached. Use of the exhausting coordinate descent method accelerated the convergence procedure, but the number of iterative steps remained large (fig.  1 , line 3). It can be shown that 5 points suffice to identify the parameters of model (1) using the conjugate gradients method, whereas this method required >10 points for identifying the parameters in a more complicated model. The Newton methods required >7 and 12 points respectively, and the coordinate descent method required >10 and 18 points. Functional (8) behaviour was analysed with respect to the evaluation of [ RL ] m  using an incorrect binding model. In particular (see fig.  2 ), the functional (8) contour plot for model (1) with the attempt to approximate the given model by scheme (2). It follows from the figure that a discordant receptor binding model results in functional (8) contour plot modification. Figure 2 The functional (8) contour plot with an inadequate choice of receptor-binding model. Thus, the modification of the functional (8) contour plot from the type in fig.  1  to the type in fig.  2  can be used as the criterion for choosing a receptor binding model. With the right choice, the contour plot is similar to that represented in fig.  1 . With the incorrect choice, the contour plot is similar to that shown in fig.  2 . It appears that when an incorrect choice of the receptor binding model has been made, the conjugate gradients method does not lead to convergence, whereas in some cases the Newton method converges to one of the local minima. Therefore, lack of convergence using the conjugate gradients method suggests an incorrect choice of receptor binding model.
Results and discussion
The functional (8) contour plots are shown in fig.  1 . From this figure, the degree of correlation between the parameters [ R 0 ]  K d  can be seen. Therefore the magnification of the random error in evaluating the magnitude of [ RL ] u  displaces the functional (8) global maximum from its true values. In a sufficiently large neighbourhood of the global maximum, the functional magnitude (8) is practically invariant. However, this modification becomes more essential for evaluating the ratio of the functional (8) to basis vector of values [ RL ] u . Therefore this ratio was used with the inhibiting criterion choice.RKdRLuRLuThe Newton method converges only in the close neighbourhood of the global maximum. However, modifications of the Newton method using second derivatives allow convergence to the global maximum after 1–2 iterations (fig.  1 , line 1).The conjugate gradients method converged after 2–3 iterations (fig.  1 , line 2). When magnification of the random error in the evaluation of [ RL ] u  was taken into account, the convergence of the conjugate gradients method varied less than that of the Newton method.RLuThe coordinate descent method required an indeterminately large number of iterations before satisfactory convergence was reached. Use of the exhausting coordinate descent method accelerated the convergence procedure, but the number of iterative steps remained large (fig.  1 , line 3).It can be shown that 5 points suffice to identify the parameters of model (1) using the conjugate gradients method, whereas this method required >10 points for identifying the parameters in a more complicated model. The Newton methods required >7 and 12 points respectively, and the coordinate descent method required >10 and 18 points.Functional (8) behaviour was analysed with respect to the evaluation of [ RL ] m  using an incorrect binding model. In particular (see fig.  2 ), the functional (8) contour plot for model (1) with the attempt to approximate the given model by scheme (2). It follows from the figure that a discordant receptor binding model results in functional (8) contour plot modification.RLmThus, the modification of the functional (8) contour plot from the type in fig.  1  to the type in fig.  2  can be used as the criterion for choosing a receptor binding model. With the right choice, the contour plot is similar to that represented in fig.  1 . With the incorrect choice, the contour plot is similar to that shown in fig.  2 .It appears that when an incorrect choice of the receptor binding model has been made, the conjugate gradients method does not lead to convergence, whereas in some cases the Newton method converges to one of the local minima. Therefore, lack of convergence using the conjugate gradients method suggests an incorrect choice of receptor binding model.Conclusion Possible methods have been explored for discriminating among models for receptor binding model and for defining the relevant parameters. The procedure devised allows one to determine the receptor binding model and its parameters,  even when the application of graphical methods is difficult or impossible . As seen here, lack of convergence in the conjugate gradients method indicates that an incorrect choice of model has been made. It is also shown that for the defining the parameters of the correct model, 5–10 data points are sufficient.
Conclusion
Possible methods have been explored for discriminating among models for receptor binding model and for defining the relevant parameters. The procedure devised allows one to determine the receptor binding model and its parameters,  even when the application of graphical methods is difficult or impossible . As seen here, lack of convergence in the conjugate gradients method indicates that an incorrect choice of model has been made. It is also shown that for the defining the parameters of the correct model, 5–10 data points are sufficient.even when the application of graphical methods is difficult or impossible


Background Scleromyxoedema is a rare chronic skin disease of obscure origin, which may often be associated with severe internal co-morbidity. Even though different casuistic treatment modalities have been described, to date, curing still seems to be impossible.
Background
Scleromyxoedema is a rare chronic skin disease of obscure origin, which may often be associated with severe internal co-morbidity. Even though different casuistic treatment modalities have been described, to date, curing still seems to be impossible.Case presentation We report a 44-year-old Caucasian female presenting with remarkable circumscribed, erythematous to skin-coloured, indurated skin eruptions at the forehead, arms, shoulders, legs and the gluteal region. Routine histology and Alcian blue labelling confirmed a massive deposition of acid mucopolysaccharides. Immunohistochemical investigations revealed proliferating fibroblasts and a discrete lymphocytic infiltration as well as increased dermal expression of MIB-1 +  and anti-mastcell-tryptase +  cells. Bone marrow biopsies confirmed a monoclonal gammopathy of undetermined significance without morphological characteristics of plasmocytoma; immunofixation unveiled the presence of IgG-kappa paraproteins.
Case presentation
We report a 44-year-old Caucasian female presenting with remarkable circumscribed, erythematous to skin-coloured, indurated skin eruptions at the forehead, arms, shoulders, legs and the gluteal region. Routine histology and Alcian blue labelling confirmed a massive deposition of acid mucopolysaccharides. Immunohistochemical investigations revealed proliferating fibroblasts and a discrete lymphocytic infiltration as well as increased dermal expression of MIB-1 +  and anti-mastcell-tryptase +  cells. Bone marrow biopsies confirmed a monoclonal gammopathy of undetermined significance without morphological characteristics of plasmocytoma; immunofixation unveiled the presence of IgG-kappa paraproteins.Conclusions Taking all data into account, our patient exhibited a complex form of lichen mxyoedematosus, which could most likely be linked a variant of scleromyxoedema. Experimental treatment with methotrexate resulted in a stabilisation of clinical symptoms but no improvement after five months of therapy. A subsequent therapeutic attempt by the use of medium-dose ultraviolet A1 cold-light photomonotherapy led to a further stabilisation of clinical symptoms, but could not induce a sustained amelioration of skin condition.
Conclusions
Taking all data into account, our patient exhibited a complex form of lichen mxyoedematosus, which could most likely be linked a variant of scleromyxoedema. Experimental treatment with methotrexate resulted in a stabilisation of clinical symptoms but no improvement after five months of therapy. A subsequent therapeutic attempt by the use of medium-dose ultraviolet A1 cold-light photomonotherapy led to a further stabilisation of clinical symptoms, but could not induce a sustained amelioration of skin condition.

Background Lichen myxoedematosus (LM) represents a rare chronic skin disorder of unknown aetiology, which may often be accompanied by severe internal co-morbidity such as haematological involvement including paraproteinaemia, neurologic syndromes, gastrointestinal complications or cardiac abnormalities [ 1 - 4 ]. Clinically and histologically, LM is characterised by papular eruptions caused by an extensive dermal deposition of glycosaminoglycans [ 5 ]. Scleromyxoedema (SCL) is a variant of LM exhibiting erythematous, sclerotic and stiffed lesions beside lichenoid papules with only little tendency of spontaneous remission [ 6 , 7 ]. Even though various experimental treatment modalities have been described, to date, curing of SCL is still not possible. The oncoming case presentation focuses on a progressive variant of SCL as referred to clinical, immunohistochemical and laboratory investigations, followed by low-dose methotrexate (MTX) and subsequent medium-dose ultraviolet A1 (UVA1) cold-light treatment.
Background
Lichen myxoedematosus (LM) represents a rare chronic skin disorder of unknown aetiology, which may often be accompanied by severe internal co-morbidity such as haematological involvement including paraproteinaemia, neurologic syndromes, gastrointestinal complications or cardiac abnormalities [ 1 - 4 ]. Clinically and histologically, LM is characterised by papular eruptions caused by an extensive dermal deposition of glycosaminoglycans [ 5 ]. Scleromyxoedema (SCL) is a variant of LM exhibiting erythematous, sclerotic and stiffed lesions beside lichenoid papules with only little tendency of spontaneous remission [ 6 , 7 ]. Even though various experimental treatment modalities have been described, to date, curing of SCL is still not possible.The oncoming case presentation focuses on a progressive variant of SCL as referred to clinical, immunohistochemical and laboratory investigations, followed by low-dose methotrexate (MTX) and subsequent medium-dose ultraviolet A1 (UVA1) cold-light treatment.Case presentation We report a 44-year-old Caucasian woman who initially presented in 2003 with a multitude of progressive lichenoid 2–4 mm papules starting two years ago, particularly marked on the forehead right above both eyebrows, on the dorsal aspects of the forearms, shoulders, legs as well as on the gluteal region, accompanied by severe pruritus (Fig.  1 ). Clinically, the papules were judged as discrete, circumscribed, erythematous to skin-coloured, firm skin eruptions associated with an induration and stiffening of the affected lesions. Furthermore, the patient complained about a progressive thickening of the glabella. Even though we cannot ensure continuous clinical deterioration, at the time of the initiation of therapy, there was no hint for a beginning stabilisation or even improvement of symptoms. Otherwise, she felt healthy and well. The general examination was without pathological findings. Neither a topical nor a systemic therapy was yet applied. Figure 1 Lichenoid papules beside thickened skin on the dorsal aspect of the left shoulder. Skin biopsies were taken from the left forearm/wrist, both legs and the left shoulder. Routine histological examination including haematoxylin-eosin, PAS labelling and Alcian blue staining revealed marked mucinous deposition within the upper and mid dermis beside an increased appearance of fibroblasts, collagen bundles and a discrete inflammatory infiltration. Additionally, immunohistochemical investigations were performed in order to enumerate CD4 +  (T helper cells), CD68 +  (macrophages), anti-mastcell-tryptase +  (mastcells), decorin +  (collagen fibril stability protein), MIB-1 +  (Ki-67 +  proliferating cells), CD20 +  (B lymphocytes), and FGF-R +  (fibroblast growth factor receptor bearing cells) cells taking consecutive sections (Table  1 ). A punch skin biopsy measuring 3 mm in diameter was taken from affected skin of the right forearm. 5 μm paraffin-embedded sections were cut, mounted on slides and routinely preserved. Prior to the single immunolabelling, different pretreatments were performed for antigen retrieval (Table  1 ). The alkaline phosphatase anti-alkaline phosphatase (APAAP) technique using the labelled streptavidin-biotin (LSAB) method was used to enumerate immunopositive cells at an individual dilution (Table  1 ) taking consecutive sections. The alkaline phosphatase fast red detection kit utilised a biotinylated secondary antibody that binds to the primary antibody. This step was followed by the addition of an streptavidin enzyme conjugate binding to the biotin present on the secondary antibody. Afterwards the specific antibody-secondary-antibody-streptavidin-enzyme-complex was detected using a precipitating enzyme reaction product. Each step was incubated for a precise time and temperature. The alkaline phosphatase was used as enzyme; the chromogene fast red could be visualised. Cells were evaluated semiquantitatively (absent (-), rare (o), moderate (+), frequent (++)) directly below the dermoepidermal junction. Immunopositive cells were evaluated 'blind' separately in two view fields in a row (0.25 mm × 0.25 mm each) directly below the dermoepidermal junction resulting in a length of 0.25 mm and 0.50 mm in depth. In order to avoid a sampling error, a number of sections were randomly reevaluated by a second observer. In case of a significant difference, the sections had to be recounted by both observers. In brief, immunolabelling revealed occasional perivascular CD4 +  and CD20 +  lymphocytes located in the papillary dermis and a high number of anti-mastcell-tryptase +  cells within the subepithelial perivascular infiltrate revealing a continuing decrease with increasing depth (Fig.  2 ). Simultaneously, an increased dermal expression of MIB-1 +  cells (Fig.  3 ), morphologically predominantly fibroblasts, within the upper and mid dermis and sporadic FGF-R +  cells in an unspecific distribution could be detected. CD68 immunohistochemistry and intradermal decorin levels did not alter remarkably as compared to healthy controls (data not shown). Exact results of all immunohistochemical stainings are detailed in Table  2 . Table 1 Overview about the performed immunohistochemistry (alkaline phosphatase anti-alkaline phosphatase (APAAP) technique using the labelled streptavidin-biotin (LSAB) method) Antibody Source Pretreatment* Dilution Incubation time CD4 Novocastra Loxo, Dossenheim, Germany H 1:60 30 min CD68 Dako, Hamburg, Germany P 1:25 30 min Tryptase Dako, Hamburg, Germany P 1:400 28 min MIB-1 Dako, Hamburg, Germany H 1:10 32 min FGF-R Oncogene Research, San Diego, USA N 1:10 30 min Decorin Oncogene Research, San Diego, USA N 1:10 30 min CD20 Novocastra Loxo, Dossenheim, Germany H 1:50 30 min *N = none, P = protease digestion, H = heat (microwave-3-step-technique) Figure 2 Immunhistochemistry unveiling sporadic lymphocytes beside a high number of anti-mastcell-tryptase +  cells within the subepithelial perivascular infiltrate. Figure 3 Mucinous deposition of the upper and mid-dermis accompanied by an elevated occurrence of MIB-1 +  dermal fibroblasts. Table 2 Semiquantitative data* of the immunohistochemical studies on a patient with scleromyxoedema Antibody CD4 CD68 Tryptase MIB-1 FGF-R Decorin CD20 Upper dermis + o ++ + o - o Mid-dermis - - + + - - - Lower dermis o - o o - - - *- = absent, o = rare, + = moderate, ++ = frequent Complete laboratory measurements unveiled the following pathological results: leucocytes 10870 μL -1 , lymphocytes 13.2%, IgG 2000 mg/dl. There was no increase in B cell count. Immunoelectrophoresis disclosed albumin 51.4%, alpha-2 globuline 10.5%, gamma globuline 22.9%. Cranial x-ray, x-ray of the thorax, ultrasound of the abdominal organs, electrocardiography and urinary investigations were unremarkable. Blood smear cytological evaluation revealed beginning qualitative but still no quantitative changes as defined by leukocytic aberrations pointing towards a leftward shift. Serum immunofixation demonstrated an IgG-kappa paraproteinaemia. No elevation of the IgG-lambda paraprotein was assessed. Bone marrow biopsies displaying reactive lymphoid infiltration including minimal extension of plasma cells with monoclonal immunoglobuline production provided evidence for monoclonal gammopathy of undetermined significance (MGUS) without distinct morphological characteristics of a plasmocytic plasmocytoma or plasmoblasts. Initially, 20 MHz ultrasound scanning producing cross section images of the skin was established in order to measure both structure and thickness of the skin at the dorsal aspects of the left wrist and the right forearm. The total thickness of the skin was measured from the entrance echo to the border between the dermis and the subcutaneous tissue. A cutaneous diameter of 2291 μm at the left wrist (lesional skin) and of 1106 μm at the right forearm (non-lesional skin) could be assessed. In our unit, an experimental treatment modality using oral MTX 12.5 mg once per week followed by a subsequent folic acid application on the following day for a 6-months-period was subsequently initiated. MTX was well tolerated by our patient. After the first three months, the continuous progress of skin lesions during the last two years could be stopped and our patient experienced subjectively an improvement and objectively a stable clinical outcome without new lesions. Subjective impression of amelioration could not be confirmed by means of ultrasound measurement. Within the following two months, no further improvement could be evaluated, whereas no further progression such as formation of new lesions or increase of stiffness could be observed. Due to the unsatisfying clinical results and declining acceptance by our patient, MTX treatment was stopped and a subsequent therapeutic attempt with medium-dose UVA1 cold-light phototherapy was initiated. Irradiation consisting of 50 J/cm 2  single-dose UVA1 (CL 300000 liquid, Photomed, Hamburg, Germany) was performed four times a week for three weeks followed by two times a week for further two weeks resulting in a cumulative dose of 800 J/cm 2  after five weeks. Meanwhile, the skin status again remained stable, whereas no improvement could be observed. Therefore, our patient broke up phototherapy. To date, skin condition has slightly worsened without any current treatment modality.
Case presentation
We report a 44-year-old Caucasian woman who initially presented in 2003 with a multitude of progressive lichenoid 2–4 mm papules starting two years ago, particularly marked on the forehead right above both eyebrows, on the dorsal aspects of the forearms, shoulders, legs as well as on the gluteal region, accompanied by severe pruritus (Fig.  1 ). Clinically, the papules were judged as discrete, circumscribed, erythematous to skin-coloured, firm skin eruptions associated with an induration and stiffening of the affected lesions. Furthermore, the patient complained about a progressive thickening of the glabella. Even though we cannot ensure continuous clinical deterioration, at the time of the initiation of therapy, there was no hint for a beginning stabilisation or even improvement of symptoms. Otherwise, she felt healthy and well. The general examination was without pathological findings. Neither a topical nor a systemic therapy was yet applied.Skin biopsies were taken from the left forearm/wrist, both legs and the left shoulder. Routine histological examination including haematoxylin-eosin, PAS labelling and Alcian blue staining revealed marked mucinous deposition within the upper and mid dermis beside an increased appearance of fibroblasts, collagen bundles and a discrete inflammatory infiltration.Additionally, immunohistochemical investigations were performed in order to enumerate CD4 +  (T helper cells), CD68 +  (macrophages), anti-mastcell-tryptase +  (mastcells), decorin +  (collagen fibril stability protein), MIB-1 +  (Ki-67 +  proliferating cells), CD20 +  (B lymphocytes), and FGF-R +  (fibroblast growth factor receptor bearing cells) cells taking consecutive sections (Table  1 ). A punch skin biopsy measuring 3 mm in diameter was taken from affected skin of the right forearm. 5 μm paraffin-embedded sections were cut, mounted on slides and routinely preserved. Prior to the single immunolabelling, different pretreatments were performed for antigen retrieval (Table  1 ). The alkaline phosphatase anti-alkaline phosphatase (APAAP) technique using the labelled streptavidin-biotin (LSAB) method was used to enumerate immunopositive cells at an individual dilution (Table  1 ) taking consecutive sections. The alkaline phosphatase fast red detection kit utilised a biotinylated secondary antibody that binds to the primary antibody. This step was followed by the addition of an streptavidin enzyme conjugate binding to the biotin present on the secondary antibody. Afterwards the specific antibody-secondary-antibody-streptavidin-enzyme-complex was detected using a precipitating enzyme reaction product. Each step was incubated for a precise time and temperature. The alkaline phosphatase was used as enzyme; the chromogene fast red could be visualised. Cells were evaluated semiquantitatively (absent (-), rare (o), moderate (+), frequent (++)) directly below the dermoepidermal junction. Immunopositive cells were evaluated 'blind' separately in two view fields in a row (0.25 mm × 0.25 mm each) directly below the dermoepidermal junction resulting in a length of 0.25 mm and 0.50 mm in depth. In order to avoid a sampling error, a number of sections were randomly reevaluated by a second observer. In case of a significant difference, the sections had to be recounted by both observers. In brief, immunolabelling revealed occasional perivascular CD4 +  and CD20 +  lymphocytes located in the papillary dermis and a high number of anti-mastcell-tryptase +  cells within the subepithelial perivascular infiltrate revealing a continuing decrease with increasing depth (Fig.  2 ). Simultaneously, an increased dermal expression of MIB-1 +  cells (Fig.  3 ), morphologically predominantly fibroblasts, within the upper and mid dermis and sporadic FGF-R +  cells in an unspecific distribution could be detected. CD68 immunohistochemistry and intradermal decorin levels did not alter remarkably as compared to healthy controls (data not shown). Exact results of all immunohistochemical stainings are detailed in Table  2 .Overview about the performed immunohistochemistry (alkaline phosphatase anti-alkaline phosphatase (APAAP) technique using the labelled streptavidin-biotin (LSAB) method)*N = none, P = protease digestion, H = heat (microwave-3-step-technique)Semiquantitative data* of the immunohistochemical studies on a patient with scleromyxoedema*- = absent, o = rare, + = moderate, ++ = frequentComplete laboratory measurements unveiled the following pathological results: leucocytes 10870 μL -1 , lymphocytes 13.2%, IgG 2000 mg/dl. There was no increase in B cell count. Immunoelectrophoresis disclosed albumin 51.4%, alpha-2 globuline 10.5%, gamma globuline 22.9%. Cranial x-ray, x-ray of the thorax, ultrasound of the abdominal organs, electrocardiography and urinary investigations were unremarkable. Blood smear cytological evaluation revealed beginning qualitative but still no quantitative changes as defined by leukocytic aberrations pointing towards a leftward shift. Serum immunofixation demonstrated an IgG-kappa paraproteinaemia. No elevation of the IgG-lambda paraprotein was assessed. Bone marrow biopsies displaying reactive lymphoid infiltration including minimal extension of plasma cells with monoclonal immunoglobuline production provided evidence for monoclonal gammopathy of undetermined significance (MGUS) without distinct morphological characteristics of a plasmocytic plasmocytoma or plasmoblasts.Initially, 20 MHz ultrasound scanning producing cross section images of the skin was established in order to measure both structure and thickness of the skin at the dorsal aspects of the left wrist and the right forearm. The total thickness of the skin was measured from the entrance echo to the border between the dermis and the subcutaneous tissue. A cutaneous diameter of 2291 μm at the left wrist (lesional skin) and of 1106 μm at the right forearm (non-lesional skin) could be assessed.In our unit, an experimental treatment modality using oral MTX 12.5 mg once per week followed by a subsequent folic acid application on the following day for a 6-months-period was subsequently initiated. MTX was well tolerated by our patient. After the first three months, the continuous progress of skin lesions during the last two years could be stopped and our patient experienced subjectively an improvement and objectively a stable clinical outcome without new lesions. Subjective impression of amelioration could not be confirmed by means of ultrasound measurement. Within the following two months, no further improvement could be evaluated, whereas no further progression such as formation of new lesions or increase of stiffness could be observed. Due to the unsatisfying clinical results and declining acceptance by our patient, MTX treatment was stopped and a subsequent therapeutic attempt with medium-dose UVA1 cold-light phototherapy was initiated. Irradiation consisting of 50 J/cm 2  single-dose UVA1 (CL 300000 liquid, Photomed, Hamburg, Germany) was performed four times a week for three weeks followed by two times a week for further two weeks resulting in a cumulative dose of 800 J/cm 2  after five weeks. Meanwhile, the skin status again remained stable, whereas no improvement could be observed. Therefore, our patient broke up phototherapy. To date, skin condition has slightly worsened without any current treatment modality.Conclusions The population prevalence of SCL is known to be extremely low. Skin lesions are characterised by an increased deposition of acid mucopolysaccharides within the papillary and upper reticular dermis [ 8 ]. Even today, aetiology and pathogenesis remain hypothetic. Aberrant dermal deposition of monoclonal paraproteins predominantly of the IgG subtype combined with elevated IgG serum levels indirectly stimulating fibroblast activity are frequently found in LM patients [ 9 - 11 ]. Nevertheless, fibromucinous lesions of LM without the presence of paraprotein accumulations have also been described [ 12 ]. Beside typical skin eruptions, LM might also be associated with severe internal and neurological abnormalities such as cardiac irregularities, paralysis, hemiparesis or even progress to coma [ 3 , 4 , 13 ]. Despite sporadic case reports introducing new therapeutic strategies in LM and SCL, common treatment modalities are still disappointing and unsatisfactory. Topical treatment including hyaluronidase and triamcinolone as well as systemic efforts by the use of corticosteroids, cyclophosphamide, electron-beam therapy, hydroxychloroquine, PUVA, extracorporeal photopheresis, plasmapheresis or high-dose intravenous immunoglobulin partly displayed only limited success in individual patients [ 12 , 14 - 20 ]. In our patient, Alcian blue staining disclosed a remarkable deposition of mucinous material within the upper dermal layers combined with an increased appearance of proliferating MIB-1 +  and occasional FGF-R +  fibroblasts in immunohistochemistry. Ki-67 (MIB-1) and fibroblast growth factors are involved in a variety of mitogen and proliferative activities [ 21 ]. Thus, the enhanced appearance of positive cells might represent an increased overall activation probably resulting in an aberrant release of mucopolysaccharides. Decorin contributes to the collagen fibril stability and high levels of decorin seem to be closely linked to dermal fibrotic stages as known from systemic sclerosis [ 22 ]. Here, an increased intradermal decorin expression could not be demonstrated. Simultaneously, in our patient the mucinous deposition was accompanied by a decreased presence of collagenous bundles. Interestingly, immunohistochemistry also revealed a number of CD4 +  and CD20 +  dermal inflammatory lymphocytes as well as anti-human mast cell tryptase +  cells, which may profoundly contribute to mucinosis formation [ 23 ]. Unfortunately, we were not able to provide a longitudinal analysis of the different stainings due to missing consent of the patient to perform additional experimental biopsies within the course of therapy. By considering clinical appearance, laboratory findings, immunofixation, bone marrow biopsy and histological evaluation, our patient presumably exhibited a complex variant of SCL. Fibroblast activity was supposed to be increased reflected by a corresponding high number of MIB-1 +  and FGF-R +  cells within the upper dermis beside a massive mucinous deposition. MTX therapy is approved in the treatment of malignant lymphoma. Additionally, low-dose MTX therapy has been established as a potent regimen in the treatment of T cell related skin diseases associated with a subsequent elevated fibroblast activation status (e.g. progressive systemic sclerosis) [ 24 ]. As in our patient paraproteinaemia of the IgG-kappa class, morphological signs of MGUS, proliferating dermal fibroblasts and a discrete T cell weighted lymphocytic dermal infiltration could be verified, we first decided to start a therapeutic attempt by the use of 12.5 mg MTX weekly in order to suppress local cellular activity following the promising reports about application of the anti-metabolite melphalan and the alkylating agent cyclophosphamide in previous studies [ 12 , 14 ]. Follow-up examinations were performed monthly. Three months after initiation of MTX therapy, an encouraging stable clinical outcome as well as an decline of pruritus without further progression of the disease was observed. However, the 5-months-follow-up revealed no apparent improvement of skin status leading to the joint decision of breaking up MTX treatment. UVA1 phototherapy has been shown to be effective in a number of inflammatory and fibrotic skin disorders by the induction of T cell apoptosis, collagenase activity and antiproliferative pathways [ 25 ]. Therefore, we decided to initiate a second attempt by the usage of a common medium-dose UVA1 irradiation protocol. Nevertheless, our patient broke up this regimen even after a 5-weeks-period due to the absence of immediate clinical improvement and an unfavourable time/benefit ratio, while follow-up examinations during this time revealed a stop of the progress anyway. However, in order to interrupt the clinical progression or even therapy-resistance as reflected by our case presentation and by considering the stable skin conditions following UVA1 phototherapy, we are currently discussing a new therapeutic attempt applying extracorporeal photochemotherapy, as proposed by Krasagakis et al. [ 16 ], in order to stabilise or even improve the present slight aggravation without any potent therapy.
Conclusions
The population prevalence of SCL is known to be extremely low. Skin lesions are characterised by an increased deposition of acid mucopolysaccharides within the papillary and upper reticular dermis [ 8 ]. Even today, aetiology and pathogenesis remain hypothetic. Aberrant dermal deposition of monoclonal paraproteins predominantly of the IgG subtype combined with elevated IgG serum levels indirectly stimulating fibroblast activity are frequently found in LM patients [ 9 - 11 ]. Nevertheless, fibromucinous lesions of LM without the presence of paraprotein accumulations have also been described [ 12 ].Beside typical skin eruptions, LM might also be associated with severe internal and neurological abnormalities such as cardiac irregularities, paralysis, hemiparesis or even progress to coma [ 3 , 4 , 13 ]. Despite sporadic case reports introducing new therapeutic strategies in LM and SCL, common treatment modalities are still disappointing and unsatisfactory. Topical treatment including hyaluronidase and triamcinolone as well as systemic efforts by the use of corticosteroids, cyclophosphamide, electron-beam therapy, hydroxychloroquine, PUVA, extracorporeal photopheresis, plasmapheresis or high-dose intravenous immunoglobulin partly displayed only limited success in individual patients [ 12 , 14 - 20 ].In our patient, Alcian blue staining disclosed a remarkable deposition of mucinous material within the upper dermal layers combined with an increased appearance of proliferating MIB-1 +  and occasional FGF-R +  fibroblasts in immunohistochemistry. Ki-67 (MIB-1) and fibroblast growth factors are involved in a variety of mitogen and proliferative activities [ 21 ]. Thus, the enhanced appearance of positive cells might represent an increased overall activation probably resulting in an aberrant release of mucopolysaccharides. Decorin contributes to the collagen fibril stability and high levels of decorin seem to be closely linked to dermal fibrotic stages as known from systemic sclerosis [ 22 ]. Here, an increased intradermal decorin expression could not be demonstrated. Simultaneously, in our patient the mucinous deposition was accompanied by a decreased presence of collagenous bundles. Interestingly, immunohistochemistry also revealed a number of CD4 +  and CD20 +  dermal inflammatory lymphocytes as well as anti-human mast cell tryptase +  cells, which may profoundly contribute to mucinosis formation [ 23 ]. Unfortunately, we were not able to provide a longitudinal analysis of the different stainings due to missing consent of the patient to perform additional experimental biopsies within the course of therapy.By considering clinical appearance, laboratory findings, immunofixation, bone marrow biopsy and histological evaluation, our patient presumably exhibited a complex variant of SCL. Fibroblast activity was supposed to be increased reflected by a corresponding high number of MIB-1 +  and FGF-R +  cells within the upper dermis beside a massive mucinous deposition.MTX therapy is approved in the treatment of malignant lymphoma. Additionally, low-dose MTX therapy has been established as a potent regimen in the treatment of T cell related skin diseases associated with a subsequent elevated fibroblast activation status (e.g. progressive systemic sclerosis) [ 24 ]. As in our patient paraproteinaemia of the IgG-kappa class, morphological signs of MGUS, proliferating dermal fibroblasts and a discrete T cell weighted lymphocytic dermal infiltration could be verified, we first decided to start a therapeutic attempt by the use of 12.5 mg MTX weekly in order to suppress local cellular activity following the promising reports about application of the anti-metabolite melphalan and the alkylating agent cyclophosphamide in previous studies [ 12 , 14 ]. Follow-up examinations were performed monthly. Three months after initiation of MTX therapy, an encouraging stable clinical outcome as well as an decline of pruritus without further progression of the disease was observed. However, the 5-months-follow-up revealed no apparent improvement of skin status leading to the joint decision of breaking up MTX treatment. UVA1 phototherapy has been shown to be effective in a number of inflammatory and fibrotic skin disorders by the induction of T cell apoptosis, collagenase activity and antiproliferative pathways [ 25 ]. Therefore, we decided to initiate a second attempt by the usage of a common medium-dose UVA1 irradiation protocol. Nevertheless, our patient broke up this regimen even after a 5-weeks-period due to the absence of immediate clinical improvement and an unfavourable time/benefit ratio, while follow-up examinations during this time revealed a stop of the progress anyway.However, in order to interrupt the clinical progression or even therapy-resistance as reflected by our case presentation and by considering the stable skin conditions following UVA1 phototherapy, we are currently discussing a new therapeutic attempt applying extracorporeal photochemotherapy, as proposed by Krasagakis et al. [ 16 ], in order to stabilise or even improve the present slight aggravation without any potent therapy.List of abbreviations LM: Lichen myxoedematosus; SCL: Scleromyxoedema; MTX: methotrexate; UVA1: ultraviolet A1; MGUS: monoclonal gammopathy of undetermined significance
List of abbreviations
LM: Lichen myxoedematosus; SCL: Scleromyxoedema; MTX: methotrexate; UVA1: ultraviolet A1; MGUS: monoclonal gammopathy of undetermined significanceCompeting interests None declared.
Competing interests
None declared.Authors' contributions F.B. participated in the design of the study, carried out the immunohistochemistry, performed the statistical analysis and drafted the manuscript. S.R. carried out 20 MHz ultrasound scanning. A.K. conceived of the study. M.F., M.S. and P.A. participated in histology,  design and coordination. All authors read and approved the final manuscript.
Authors' contributions
F.B. participated in the design of the study, carried out the immunohistochemistry, performed the statistical analysis and drafted the manuscript. S.R. carried out 20 MHz ultrasound scanning. A.K. conceived of the study. M.F., M.S. and P.A. participated in histology,  design and coordination.All authors read and approved the final manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here: