No association of the –2518 MCP-1 A/G promoter polymorphism with incidence and clinical course of IgA nephropathy

Oliver M. Steinmetz1, Ulf Panzer1, Sigrid Harendza1, Peter R. Mertens2, Tammo Ostendorf2, Jürgen Floege2, Udo Helmchen3 and Rolf A. K. Stahl1

1Medizinische Klinik IV, Universitätsklinikum Hamburg Eppendorf, 2Abteilung für Nephrologie und Klinische Immunologie, Universitätsklinikum Aachen and 3Institut für Pathologie, Universitätsklinikum Hamburg Eppendorf, Germany

Correspondence and offprint requests to: Oliver M. Steinmetz, MD, Zentrum für Innere Medizin, Medizinische Klinik IV, University of Hamburg, Martinistraße 52, D-20246 Hamburg, Germany. Email: o.steinmetz{at}uke.uni-hamburg.de



   Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. The clinical course of IgA nephropathy is highly variable, ranging from complete remission to progression with end-stage renal disease. Although the mechanisms involved in disease progression are not characterized in detail, loss of renal function is positively correlated with mononuclear cell infiltration. In general, chemokines play an important role in the directional recruitment of inflammatory cells. Recently, a polymorphism in the distal 5' regulatory region of the chemokine monocyte chemoattractant protein-1 (MCP-1), which affects gene expression, has been described (A/G at position –2518). The aim of our study was to evaluate a possible association of this polymorphism with disease progression in patients with IgA nephropathy, as well as susceptibility to this form of glomerulonephritis.

Methods. Blood samples from 207 patients with biopsy proven IgA nephropathy and 140 ethnically, age and sex-matched healthy controls were collected and genomic DNA was extracted. MCP-1 –2518 genotype was assessed by PCR, followed by restriction fragment length polymorphism analysis. Genotype distribution between the two groups was compared by {chi}2 test. Cumulative renal survival was assessed by Kaplan–Meier plot and log-rank analysis.

Results. 111 (53.6%) patients had the MCP-1 –2518 wild-type A/A, 83 (40.1%) were heterozygous for the G allele and 13 (6.3%) patients showed homozygosity. The allelic distribution was not significantly different in the control group of 140 healthy blood donors (P = 0.71). Renal survival analysis of patients did not reveal statistically significant differences in cumulative survival (P = 0.32), median survival time and 5 year survival rate between the wild-type group and carriers of the G allele. Furthermore, the number of infiltrating CD68-positive monocytes/macrophages into the kidneys of patients with IgA nephropathy was not statistically different between the groups.

Conclusion. Our data indicate that no association exists between the –2518 A/G polymorphism and susceptibility to IgA nephropathy or its clinical course.

Keywords: CCL2; chemokine; IgA nephropathy; inflammation; MCP-1



   Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
IgA nephropathy is the most frequent form of glomerulonephritis in the world. Its clinical course is characterized by a stable renal function in most patients. However, in one third of the patients a progression to end-stage renal disease (ESRD) can be observed [13]. The mechanisms responsible for disease progression are largely unknown. The observation of a different incidence of IgA nephropathy in ethnically distinct populations and the varying patterns of disease progression are strongly indicative of the involvement of genetic factors in susceptibility to this disease and its natural course.

Interestingly, a correlation between the presence of mononuclear cells (monocytes and lymphocytes) in the renal tissue and disease progression of IgA nephropathy was recently described [46]. In general, the directional recruitment of leucocytes is regulated by chemokines and their counteracting chemokine receptors [7]. In human and experimental models of glomerulonephritis it was shown that monocyte chemoattractant protein-1 (MCP-1), a ligand of the C-C chemokine receptor 2 (CCR2) plays a pivotal role in the attraction of monocytes into the renal tissue [811]. Therefore, genetic polymorphisms in the regulatory region of the MCP-1 gene that exert an impact on its expression could play a role in the susceptibility and progression of IgA nephropathy.

Rovin et al. [12] described a biallelic A/G polymorphism at position –2518 of the MCP-1 gene. The polymorphism proved functionally important, as peripheral blood mononuclear cells of individuals with G/G and A/G genotype produced significantly more MCP-1 after stimulation with IL-1ß than those with Caucasian wild-type A/A [12,13]. An association of the presence of G at position –2518 with the presence of cutaneous vasculitis could be shown in patients with systemic lupus erythematodes [14] as well as an association with development of coronary artery aneurysms after acute Kawasaki disease [13]. Furthermore, the G/G genotype was identified as a genetic risk factor for severe coronary artery disease [15] and a correlation between the incidence and severity of asthma and the G allele at position –2518 has been shown [16]. These findings suggest an important role for MCP-1 and the A/G polymorphism in its regulatory region in inflammatory processes.

The aim of our study was to evaluate whether the A/G polymorphism at position –2518 in the regulatory region of the MCP-1 gene is associated with the incidence and progression of IgA nephropathy in Caucasian patients.



   Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study cohort
Overall 207 patients with a primary form of IgA nephropathy and 140 healthy volunteers were analysed retrospectively in this study. More than 98% of the studied subjects were Caucasians. Patients were collected in the Medizinische Klinik of the Universitätskrankenhaus Hamburg Eppendorf, in various dialysis centres in Hamburg and in the University Hospital Aachen between February 2000 and April 2002. The inclusion criteria of the study were a histologically proven diagnosis, genomic DNA for adequate MCP-1 regulator polymorphism genotyping, and clinical follow-up data of at least 1 month starting from the time of renal biopsy.

Approval for the study was given by the local ethics committee on February 1, 2000 and has been conducted according to the Declaration of Helsinki principles.

MCP-1 promoter genotyping
Genomic DNA was isolated from peripheral blood leucocytes using a commercial kit (QIAamp DNA Blood Mini Kit). The identification of the polymorphism was carried out using PCR, followed by a restriction fragment length polymorphism (RFLP) assay, using a PvuII site, which is introduced by the presence of the G nucleotide. The regulatory region of the MCP-1 gene (–1817 to –2746) was amplified by PCR, resulting in a 930 bp fragment. Primers used were 5'-CCGAGATGTTCCCAGCACAG-3' (forward) and 5'-CTGCTTTGCTTGTGCCTCTT-3' (reverse). Five microlitres of genomic DNA (~50 ng) were added to 20 µl of amplification buffer containing 12.7 µl of H2O, 2.5 µl of 10x PCR buffer, 1.5 µl of MgCl2 (50 mM), 1 µl of dNTPs (10 mM each), 0.3 µl of Taq DNA polymerase (5 U/µl), 1.5 µl of forward and 1.5 µl of reverse primer (10 pmol/µl each). PCR was run for 40 cycles using the following temperature profile: denaturation at 94°C for 60 s, annealing at 55°C for 60 s, extension at 72°C for 1 min 30 s, followed by a single final extension step at 72°C for 10 min. Eight microlitres of the PCR products were digested with 10 U of PvuII in 10x buffer and H2O up to a final volume of 20 µl at 37°C for 2 h 30 min.

The resulting products were separated by gel-electrophoresis in 1.5% agarose gels, containing ethidium bromide in a final concentration of 0.5 µg/ml. Samples showing only a 930 bp band were assigned as A/A, samples showing two bands of 708 and 222 bp were considered G/G and samples showing three bands at 930, 708 and 222 bp were typed A/G [12]. Of 42 randomly selected samples from the IgA collective (20%), 21 were amplified and digested twice and 21 were analysed by automatic DNA sequencing after PCR amplification as a control for possible mismatches introduced by the Taq polymerase. All 42 samples were in agreement (data not shown).

Statistical methods and design
Allelic and genotypic frequencies were obtained by direct counting. Statistical comparison of genotypic frequencies between patients with IgA nephropathy and healthy controls was carried out by {chi}2 test. For survival studies, due to the small number of individuals with the G/G genotype, these patients were included in the same group as patients with the A/G genotype and compared with the group with the Caucasian wild-type genotype A/A. Differences of nominal variables (proteinuria, hypertension and sex) between the two groups were compared by {chi}2 test. Differences of continuous variables (creatinine, age and time of follow-up) were calculated by Mann–Whitney U-test. The study was designed to have a power of 80% to detect a difference in hazard ratio between the two groups of 2.5 or more with a two-sided significance level of P = 0.05 referring to the log-rank test. Cumulative renal survival was analysed by Kaplan–Meier plot and log-rank test. Analysis of covariables was carried out according to the Cox proportional hazard model. The number of infiltrating CD68-positive cells into renal biopsies of patients with IgA nephropathy and A/A, A/G or G/G genotype was compared by Kruskal–Wallis test. SPSS 10.1.3 was used for statistical analyses.

Immunohistochemical staining of CD68 in biopsies from patients with IgA nephropathy
Paraffin tissue sections (3–4 µm thick) were protease treated for antigen retrieval and subsequently incubated with anti-CD68 (KP1 Zymed, 1:2) for 30 min. Sections were then incubated with rabbit anti-mouse-IgG (Z 259 DAKO, 1:135) for 15 min, followed by application of APAAP complex (Progen, 1:300) for 15 min. The last two steps were carried out twice, followed by staining with neufuchsin for 30 min.

The infiltration of CD68-positive cells into the tubulointerstitium of kidneys from patients with IgA nephropathy was quantified by counting all positive cells in 30 high-power fields (x1000) for each biopsy section. The number of CD68-positive cells in glomeruli was quantified by counting all positive infiltrating cells in all glomerular cross sections of each biopsy section and subsequent calculation of the mean value per glomerular cross section.



   Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Demographic and clinical data of the study cohort
The following demographic and clinical data were assessed for the patients with IgA nephropathy at the time of biopsy: age, gender, presence of hypertension (systolic blood pressure > 140 mmHg or antihypertensive treatment), proteinuria (>1 g/24 h) and serum creatinine (mg/dl) (Table 1). Healthy controls were ethnically, age and sex matched.


View this table:
[in this window]
[in a new window]
 
Table 1. Demographic and clinical data of the study cohort for (A) patients with IgA nephropathy and (B) healthy controls

 
The study endpoint was ESRD with start of renal replacement therapy. Renal survival was defined as absence of renal replacement therapy in the follow-up period. Follow-up was defined as the time from renal biopsy establishing the diagnosis of IgA nephropathy until the last follow-up of the patients with remaining kidney function or until the start of renal replacement therapy in those patients who reached the endpoint of the study. The high rate of ESRD among the study population can be explained by recruitment of many patients in dialysis centres.

Genotyping of MCP-1 promoter polymorphism
The MCP-1 –2518 genotype was identified by PCR, followed by RFLP analysis (Figure 1). The genotypic frequency of the 207 IgA nephropathy patients was as follows: 111 (53.6%) patients had the A/A genotype, which can be regarded as wild-type for a Caucasian population; 83 (40.1%) patients had the A/G genotype; 13 (6.3%) patients had the G/G genotype; the frequency of the G allele was 26.3% (109/414). The distribution in the group of healthy controls was not statistically different: 81 (57.9%) patients showed the A/A, 50 (35.7%) patients the A/G and nine (6.4%) patients the G/G genotype ({chi}2 test: P = 0.71). The frequency of the G allele was 24.29% (68/280). Thus, the distribution of the genotypes in the two collectives was according to the Hardy–Weinberg equilibrium and no association of the A/G polymorphism and susceptibility to IgA nephropathy was detectable.



View larger version (39K):
[in this window]
[in a new window]
 
Fig. 1. Example of genotyping for MCP-1 –2518 A/G promoter polymorphism. PvuII digestion after PCR amplification of the distal regulatory region yields different fragments according to genotype: A/A, 930 bp; A/G, 930, 708 and 222 bp; G/G, 708 and 222 bp.

 
Effects of the MCP-1 –2518 A/G promoter polymorphism on renal survival in patients with IgA nephropathy
Comparison of renal survival by means of the log-rank test showed no difference in development of ESRD between the group of IgA nephropathy patients with the wild-type genotype A/A and the group including the A/G and G/G genotypes (Figure 2, median of renal survival time in months: A/A, 46, A/G and G/G, 42; 5-year survival rate: A/A, 43.4%, A/G and G/G, 44.2%; log-rank test: P = 0.32). Differences of clinical data between groups were not statistically significant ({chi}2 for nominal variables, Mann–Whitney U-test for continuous variables). Subgroup analysis of slow progressing patients with remaining kidney function after 5, 8 and 10 years revealed no significant differences in genotypic frequency (A/A, A/G or G/G) of these three groups compared with the overall genotype distribution in IgA nephropathy patients ({chi}2 test). Univariate analysis of six different factors (MCP-1 –2518 genotype, gender, age, hypertension, proteinuria and creatinine at biopsy) using the Cox proportional hazard model revealed creatinine at biopsy (P<0.005) as a risk factor for progression to ESRD. The hazard ratio (95% confidence interval) was 1.49 (1.36–1.63) for each mg/dl of creatinine. Multivariate analysis with stepwise inclusion of covariables, however, identified creatinine (P <0.001) and proteinuria (P = 0.04) as influencing factors with hazard ratios (95% confidence intervals) of 1.54 (1.39–1.7) for each mg/dl of creatinine and 1.76 (1.03–3.03) for patients with proteinuria compared with patients without proteinuria.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2. Kaplan–Meier plot of renal survival for up to 12 years with subsequent log-rank analysis revealed no statistical difference between groups with the Caucasian wild-type genotype A/A compared with carriers of the G allele, A/G plus G/G genotype (P = 0.32). The number of patients at risk is indicated below the plot.

 
These data indicate that the presence of G at position –2518 in the MCP-1 promoter region does not influence the clinical course of IgA nephropathy.

CD68 immunohistochemistry
To analyse whether the MCP-1 –2518 genotype affects the recruitment of monocytes/macrophages into the renal tissue, immunohistochemical examination of renal biopsies from 23 patients with IgA nephropathy was performed. The results revealed no significant differences in interstitial and glomerular infiltration of CD68-positive monocytes/macrophages between the three groups [cells/glomerular cross section: A/A (n = 9) 9.2±5.1; A/G (n = 9) 10.7±4.9; G/G (n = 5) 8.4±3.7; P = 0.71; cells/30 high power fields: A/A 380±127; A/G 447±184; G/G 403±104; P = 0.71] (Figures 3 and 4). Patients in the three groups did not differ significantly in clinical basis data (age at biopsy, creatinine at biopsy, proteinuria, hypertension and gender) (analysis by Kruskal–Wallis resp. {chi}2 test, data not shown).



View larger version (89K):
[in this window]
[in a new window]
 
Fig. 3. CD68 staining of renal biopsies of patients with IgA nephropathy and A/A (x200), A/G (x200) or G/G (x200) MCP-1 –2518 genotype (inlets are x1000). Positive cells show red staining.

 


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 4. The number of CD68-positive monocytes/macrophages infiltrating per glomerular cross section (A) and the tubulointerstitium (B) of kidneys from patients with IgA nephropathy was not statistically different between the three genotypes.

 


   Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
As IgA nephropathy is the most common form of glomerulonephritis in the world, it is a rewarding subject for detailed studies. In the majority of patients the clinical course of the disease is benign. In approximately one third, however, progression to ESRD can be observed. The early identification of patients who belong to the latter group and therefore who would potentially benefit from therapy is of great interest. Several risk factors for disease progression have been identified to date, like the serum creatinine, proteinuria >1 g/24 h and the presence of hypertension at the time of biopsy [17]. Further predictors of a clinically progressive course are needed nevertheless to optimize early identification of patients for whom a therapy would be beneficial.

The present study is, to the best of our knowledge, the first to investigate a potential influence of the MCP-1 A/G promoter polymorphism at position –2518 on the incidence and clinical course of IgA nephropathy. The presence of G at position –2518 has previously been shown to be associated with a higher production of MCP-1 by peripheral blood mononuclear cells after stimulation with IL-1ß [12,13]. The G allele was found in 22% of African Americans, 29% of Caucasians and 47% of Mexicans and Asians, suggesting ethnic variation. Knowing that the progression of IgA nephropathy correlates with the presence of mononuclear cells in the kidney [46], an enhanced production of the chemokine MCP-1, which is known to be a major chemoattractant for monocytes and T lymphocytes [811,18], might therefore exert an effect on the clinical course. Mice lacking MCP-1 presented with a strongly impaired recruitment of monocytes and an ameliorated disease severity in experimental models of glomerulonephritis [19,20], stressing the unique importance of MCP-1, despite the existence of other chemokines that bind to CCR2 and/or attract monocytes in vitro [18]. Furthermore, other studies have reported a significant impact of this polymorphism on other inflammatory processes. An association of the presence of G at position –2518 with cutaneous vasculitis among patients with systemic lupus erythematodes [14], severity of asthma in children [16] and coronary artery aneurysm after acute Kawasaki disease [13] has been shown.

We studied the MCP-1 –2518 A/G polymorphism in 207 patients with biopsy proven primary IgA nephritis and a group of 140 matched controls. The distribution of genotypic frequencies was neither statistically different between these two groups nor between three subgroups of patients with a slower progression rate of IgA nephropathy and the total collective of patients with IgA nephropathy. These findings strongly argue against a role of the MCP-1 –2518 A/G polymorphism for susceptibility to IgA nephropathy and against an influence on the progression rate of this disease.

In addition, immunohistochemical staining of biopsies for CD68 from the different genotypes showed no significant differences in monocyte/macrophage infiltration into glomeruli and tubulointerstitium. The results provide evidence against a major pathophysiological role of the MCP-1 –2518 A/G polymorphism in renal monocyte/macrophage recruitment.

Due to collection in dialysis centres, our study group contains a high number of patients who reached ESRD. In this collective, no association between the renal survival and the MCP-1 –2518 genotype could be detected.

Certain aspects, however, remain unclear and require further studies. The group of patients homozygous for the G allele included in this study was too small for separate statistical analysis and was therefore included in the group of patients with the A/G genotype. This approach leaves unanswered whether a possible effect of homozygosity on renal survival exists. This question could be addressed by investigation of a population with IgA nephropathy from an ethnic group with a high incidence of the G allele at position –2518, e.g. Asians. Several previous studies, however, have revealed a very similar 10 year renal survival of adult patients with IgA nephropathy in many populations with a low frequency of the G allele, e.g. Germany and on the other hand Japan, where the G allele and thus, homozygotes, are much more frequent [3].

As it has previously been shown that urinary MCP-1 levels correlate with disease severity of IgA nephropathy [21], the effect of the –2518 promotor polymorphism on intrarenal expression of MCP-1 would be another interesting subject to study.

In summary, our data suggest that the MCP-1 –2518 A/G gene polymorphism is not associated with the incidence and progression of IgA nephropathy in a Caucasian population.



   Acknowledgments
 
We thank M. Rezska, A. Peters and U. Kneissler for their excellent technical help as well as V. Schoder for help with statistical analyses. This work was supported by a grant of the Deutsche Forschungsgemeinschaft (RAKS+UP STA 193/7-1).

Conflict of interest statement. None declared.



   Notes
 
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.



   References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Donadio JV, Grande JP. Medical progress: IgA nephropathy. New Engl J Med 2002; 347: 738–748[Free Full Text]
  2. Floege J, Feehally J. IgA nephropathy: recent developments. J Am Soc Nephrol 2000; 11: 2395–2403[Free Full Text]
  3. D’Amico G. Natural history of idiopathic IgA nephropathy: role of clinical and histological prognostic factors. Am J Kidney Dis 2000; 36: 227–237[ISI][Medline]
  4. Falk MC, Ng G, Zhang GY et al. Infiltration of the kidney by alpha beta and gamma T cells: effects on progression in IgA nephropathy. Kidney Int 1995; 47: 177–185[ISI][Medline]
  5. Alexopoulos E, Seron D, Hartley RB, Nolasco F, Cameron JS. The role of the interstitial infiltrates in IgA nephropathy: a study with monoclonal antibodies. Nephrol Dial Transplant 1989; 4: 187–195[Abstract]
  6. Li HL, Hancock WW, Hooke DH, Dowling JP, Atkins RC. Mononuclear cell activation and decreased renal function in IgA nephropathy with crescents. Kidney Int 1990; 37: 1552–1556[ISI][Medline]
  7. Baggiolini, M. Chemokines and leukocyte traffic. Nature 1998; 392: 565–568[CrossRef][ISI][Medline]
  8. Wenzel U, Schneider A, Valente AJ et al. Monocyte chemoattractant protein-1 mediates monocyte/macrophage influx in antithymocyte antibody-induced glomerulonephritis. Kidney Int 1997; 51: 770–776[ISI][Medline]
  9. Panzer U, Thaiss F, Zahner G et al. Monocyte chemoattractant protein-1 and osteopontin differentially regulate monocyte recruitment in experimental glomerulonephritis. Kidney Int 2001; 59: 1762–1769[CrossRef][ISI][Medline]
  10. Noris M, Bernasconi S, Casiraghi F et al. Monocyte chemoattractant protein-1 is excreted in excessive amounts in the urine of patients with lupus nephritis. Lab Invest 1995; 73: 804–809[ISI][Medline]
  11. Rovin BH, Rumancik M, Tan L, Dickerson J. Glomerular expression of monocyte chemoattractant protein-1 in experimental and human glomerulonephritis. Lab Invest 1994; 71: 536–542[ISI][Medline]
  12. Rovin BH, Lu L, Saxena R. A novel polymorphism in the MCP-1 gene regulatory region that influences MCP-1 expression. Biochem Biophys Res Commun 1999; 259: 344–348[CrossRef][ISI][Medline]
  13. Toshiaki J, Masaru T, Masayuki S et al. Monocyte chemoattractant protein-1 gene regulatory region polymorphism and serum levels of monocyte chemoattractant protein-1 in Japanese patients with Kawasaki disease. Arthritis Rheum 2001; 44: 2211–2212[CrossRef][ISI][Medline]
  14. Aguilar F, González-Escribano MF, Sánchez-Román J, Nunez-Roldán A. MCP-1 promotor polymorphism in Spanish patients with systemic lupus erythematosus. Tissue Antigens 2001; 58: 335–338[CrossRef][ISI][Medline]
  15. Szalai C, Duba J, Prohászka Z et al. Involvement of polymorphisms in the chemokine system in the susceptibility for coronary artery disease (CAD). Coincidence of elevated Lp (a) and MCP-1 –518 G/G genotype in CAD patients. Atherosclerosis 2001; 158: 233–239[CrossRef][ISI][Medline]
  16. Szalai C, Kozma GT, Nagy A et al. Polymorphism in the gene regulatory region of MCP-1 is associated with asthma susceptibility and severity. J Allergy Clin Immunol 2001; 108: 75–81
  17. Bartosik LP, Lajoie G, Sugar L, Cattran DC. Predicting progression in IgA nephropathy. Am J Kidney Dis 2001; 38: 728–735[ISI][Medline]
  18. Lu BB, Rutledge BJ, Gu L et al. Abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein-1 deficient mice. J Exp Med 1998; 187: 601–608[Abstract/Free Full Text]
  19. Tesch GH, Maifert S, Schwarting A, Rollins BJ, Kelley VR. Monocyte chemoattractant protein-1 dependent leukocytic infiltrates are responsible for autoimmune disease in MRL-Fas (lpr) mice. J Exp Med 1999; 190: 1813–1824[Abstract/Free Full Text]
  20. Tesch GH, Schwarting A, Kinoshita K, Lan HY, Rollins BJ, Kelley VR. Monocyte chemoattractant protein-1 promotes macrophage-mediated tubular injury, but not glomerular injury, in nephrotoxic serum nephritis. J Clin Invest 1999; 103: 73–80[Abstract/Free Full Text]
  21. Saitoh A, Suzuki Y, Takeda M, Kubota K, Itoh K, Tomino Y. Urinary levels of monocyte chemoattractant protein-1 (MCP-1) and disease activity in patients with IgA nephropathy. J Clin Lab Anal 1998; 12: 1–5[CrossRef][ISI][Medline]
Received for publication: 12. 5.03
Accepted in revised form: 1.10.03





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Disclaimer
Request Permissions
Google Scholar
Articles by Steinmetz, O. M.
Articles by Stahl, R. A. K.
PubMed
PubMed Citation
Articles by Steinmetz, O. M.
Articles by Stahl, R. A. K.