Evidence for infection of the human embryo with adeno-associated virus in pregnancy

Tatiana Burguete1, Michèle Rabreau2, Marianne Fontanges-Darriet3, Edith Roset4, Hans-Dieter Hager5, Alexandra Köppel5, Paul Bischof4 and Jörg R. Schlehofer1,6

1 Deutsches Krebsforschungszentrum (DKFZ), Angewandte Tumorvirologie, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany, 2 Institut d'Histo Cyto Pathologie, 114 Av Léon Blum,F-33194 Le Bouscat, France, 3 Cabinet de Chirurgie Gynécologique et d'Obstétrique, Echographie et Diagnostic Ante-Natal, Polyclinique Jean Villar, Av Maryse-Bastié, F-33520 Bruges/Bordeaux, France, 4 Département de Gynecologie et Obstétrique, Hôpital Contonal Universitaire, 32bis, Bd. de la Cluse, CH-1211 Genève 14, Switzerland and 5 Institut für Humangenetik, Abteilung Zytogenetik, Universität Heidelberg, Im Neuenheimer Feld 328,D-69120 Heidelberg, Germany


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previous reports have demonstrated the presence of DNA of the human helper virus-dependent adeno-associated parvovirus (AAV) in uterine tissue and curettage material from early miscarriage. To examine infection of embryonic tissue during pregnancy, amnion fluids were analysed for the presence of AAV. Using polymerase chain reaction, AAV DNA was detected in 64 out of 238 DNA samples extracted from amnion cells. DNA of helper viruses were found in 12% (papillomavirus) and 18% (cytomegalovirus) of the samples (double infections with AAV in eight and nine cases, respectively). Furthermore, infectious AAV virions were found in 13 out of 43 AAV DNA-containing samples. In mothers with AAV DNA-positive amnion fluids, premature amniorrhexis and premature labour occurred significantly more frequently (P < 0.001). Using an immunofluorescence assay, 24% of newborn sera (unrelated to the amnion fluid samples) were found to contain IgM antibodies to AAV, in most cases paralleled by IgM antibodies in the mother's sera. The data demonstrate that AAV infection can occur in utero at early and at late stages of pregnancy. The observed complications at delivery should encourage studies to clarify possible pathological consequences of AAV infection in pregnancy and a possible latent infection of the fetus.

Key words: adeno-associated virus/amniocentesis/in-utero infection/trophoblast cells/virus infection in pregnancy


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The small, non-enveloped, single-stranded DNA viruses adeno-associated viruses (AAV) are members of the parvoviridae and usually require co-infecting helper adeno- or herpes or vaccinia viruses for replication (Cukor et al., 1984Go; Schlehofer et al., 1986Go). Recent data have added human papillomaviruses to the list of helpers for AAV (Walz et al., 1997Go). AAV types 2, 3 and 5 are human isolates (Blacklow et al., 1967Go; Parks et al., 1967Go, 1970Go; Bantel-Schaal and zur Hausen, 1984Go), and AAV infection seems to occur early in childhood (Blacklow et al., 1968aGo,bGo, 1971Go; Parks et al., 1970Go; Georg-Fries et al., 1984Go; Schlehofer et al., 1996Go). The lack of apparent pathogenicity, the observation of tumour-suppressive properties (Schlehofer, 1994Go), and the ability to integrate its DNA into the cellular genome in the absence of helper viruses at least in cell culture (Berns et al., 1975Go; Handa et al., 1977Go; Cheung et al., 1980Go; Laughlin et al., 1986Go; Kotin et al., 1990Go; Walz and Schlehofer, 1992Go) made these viruses candidate vectors for gene therapy (for review see Flotte and Carter, 1995Go).

In humans, DNA of AAV-2 has been detected at various frequencies in biopsies from the uterine cervix and from spontaneous abortion material (Tobiasch et al., 1994Go; Rabreau and Schlehofer, 1995Go; Friedman-Einat et al., 1997Go; Walz et al., 1997Go), in material from cervical brushings (Han et al., 1996Go) and at a very low percentage, also in blood samples (Grossman et al., 1992Go). Recently, infectious virions have been isolated from cervical biopsies (Walz et al., 1998Go).

In samples from early miscarriage, AAV DNA and proteins were localized predominantly in the syncytiotrophoblast cell layer of the placenta (Tobiasch et al., 1994Go). Furthermore, DNA of AAV-2 was recently detected in cell lines derived from human trophoblast or amnion (Dutheil et al., 1997Go). In order to assess infection of embryonic cells with AAV we tested amniotic fluids taken from pregnant women for diagnostic reasons for the presence of AAV DNA, using polymerase chain reaction (PCR) and cell culture assays for infectious virus.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Amnion fluids
Non-selected amnion fluid samples collected at three hospitals, in France, Switzerland and Germany, were obtained by transabdominal amniocentesis. Amniocentesis was performed for various reasons including high maternal and fetal abnormalities. Samples were kept frozen (–20°C) until analysis. In France (Bruges, Bordeaux), the age range of the pregnant women was 22–44 years and amniocentesis was performed between the 15th and 25th week of gestation. The cases from Germany (Heidelberg) were 18–46 years old, and amniocentesis took place between the 14th and 21st week of gestation. In Switzerland (Geneva), amniocentesis was between weeks 15 and 19, in 27–42 year old patients. Weeks of pregnancy were determined by the last menstrual period and by ultrasonic scan.

Trophoblast material
Trophoblast cells and syncytiotrophoblasts [separated by gradient centrifugation (Bischof et al., 1991Go)] from induced abortions were from the Laboratoire d'Hormonologie of the University Hospital of Geneva. Trophoblast cells were maintained as a short-term culture in Dulbecco's modified Eagle's medium (DMEM) (Gibco BRL, Eggenstein, Germany), supplemented with 10% fetal calf serum (FCS).

Trophoblast cultures
Specimen of amniotic fluid cells (cultivated for prenatal diagnostic purposes) obtained from the Institut für Humangenetik (University of Heidelberg) were grown in plastic flasks in AmnioMax-C100 Basal Medium with AmnioMax-C100 supplement (Gibco BRL).

DNA extraction
DNA was extracted from pelleted amnion fluid samples using the QIAamp Kit (QIAgen, Hilden, Germany).

Polymerase chain reaction (PCR)
DNA extractions and PCR were performed in a laboratory in which experiments involving AAV infection or plasmid preparations are not carried out. To amplify AAV DNA in DNA extracted from the biopsy specimen two sets of primers were chosen to detect AAV types 2, 3 and 5 (Table IGo) (Tobiasch et al., 1994Go, 1998Go; Han et al., 1996Go). PCR primers for adenovirus (Ad) and herpes simplex virus (HSV, types 1 and 2) were chosen as described (Malhomme et al., 1997Go). For the detection of human cytomegalovirus (HCMV), a nested PCR was performed according to Arai et al. (Arai et al., 1995Go). Primers amplifying HPV DNA sequences were as described (van den Brule et al., 1989Go, 1990Go; Armbruster-Moraes et al., 1994Go). Primer sequences to distinguish between the different AAV types were chosen from the published AAV-3 sequence (Muramatsu et al., 1996Go), and from a partial AAV-5 sequence (Tobiasch et al., 1998Go). The reaction mixture for PCR contained ~600 ng DNA, 22 mmol/l Tris–HCl (pH 8.4), 55 mmol/l KCl, 1.65 mmol/l MgCl2, 220 mmol/l deoxynucleoside triphosphates, 100 ng of each primer and 1 U Taq DNA polymerase (Gibco BRL): PCR was done in a PTC-100 thermal cycler (MJ Research Inc., USA). Genomic DNA of HeLa or SiHa cells and of AAV-2 DNA containing HA16 cells (Walz and Schlehofer, 1992Go) or choriocarcinoma cell lines (Dutheil et al., 1997Go) were used as AAV DNA-negative and -positive controls, respectively. Controls for HPV DNA sequences were DNA from SiHa or HeLa cells (containing HPV16 or HPV18 DNA, respectively). DNA from MRC-5 cells infected with HCMV, from HeLa cells infected with Adenovirus type 2, or with HSV-1 or HSV-2 were used as positive controls for the respective viruses. In order to test whether the samples contained amplifiable DNA, AAV DNA negative samples were analysed with primers detecting ß-globin DNA (Tobiasch et al., 1994Go). In all PCR, amplified bands were confirmed by Southern blots hybridized with 5'-labelled oligonucleotides specific for the respective viruses (see Table IGo, for AAV), or using nested PCR primers (for HCMV, see above). DNA extractions and PCR analysis for AAV DNA were done twice (using different primer pairs, see above), starting from original material.


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Table I. Primers used to detect adeno-associated virus (AAV) DNA
 
Analysis for the presence of infectious AAV particles
Supernatants of amnion fluid samples (in some cases concentrated by centrifugation [50 000 rpm for 2.5 h in a Beckman TL-100 swing-out rotor]) or supernatants of trophoblast cells (cultured for 2 weeks) were inoculated onto semiconfluent HeLa cells followed by superinfection with adenovirus type 2 (Ad2) as a helper. Controls were HeLa cells inoculated with AAV DNA-negative amnion fluid samples and superinfected with Ad2. After onset of adenovirus-induced cytopathic effect (CPE) detached cells as well as culture supernatants were collected (fraction 1), and medium was replenished to allow cell division and further progress of CPE. After complete CPE, medium and trypsinized cells were combined (fraction 2). Fractions 1 and 2 were analysed for the presence of replicated AAV DNA by the dispersed cell assay, i.e. adsorption to GeneScreen membranes (Dupont, Boston, MA, USA) by suction followed by hybridization with pTAV2 DNA (Heilbronn et al., 1990Go), labelled with ([32P]dCTP (Amersham, Braunschweig, Germany) or digoxigenin-dUTP (Boehringer, Mannheim, Germany) as described (Schlehofer et al., 1983aGo,bGo).

Detection of IgM-type serum antibodies against AAV
Samples of sera from newborns (<1 week of age) taken for other clinical reasons at the University Hospital of Mainz, were analysed for IgG and IgM antibodies against AAV. For IgM tests, sera were treated with FrekaFluor [Fresenius, Bad Homburg, Germany; removes IgG and is commonly used in IgM serology (removal of IgG was checked for {alpha}-AAV IgG positive sera)] to exclude IgG-mediated signals. Sera were diluted (1:10) and applied to slides with methanol–acetone (1:2)-fixed 293T cells previously transfected with a plasmid expressing AAV type 2 capsid proteins (Wistuba et al., 1997Go). After washing, the cells were incubated with goat anti-human IgG or IgM antibodies, respectively (Dianova, Hamburg, Germany) labelled with fluorescein isothiocyanate and analysed using a fluorescence microscope.

Statistics
The main purpose of the statistical analysis was to analyse a possible correlation between the presence of AAV DNA and complications during delivery (birthweight, gestation age, premature labour, premature amniorrhexis). For this analysis, the Fisher's exact test ({chi}2) was used as proposed by the statisticians of the Epidemiology Division of the DKFZ.


    Results
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 Materials and methods
 Results
 Discussion
 References
 
DNA extracted from amnion fluids, taken for diagnostic reasons, were tested for the presence of AAV DNA. The women were aged between 18 and 46 years; amniocentesis was between the 14th and 25th weeks of gestation. Samples were checked for the presence of DNA by PCR using primers amplifying ß-globin DNA sequences. For detection of AAV DNA, PCR was performed using primers detecting DNA of the AAV types 2, 3 and 5 [pan 1, pan 3 (Tobiasch et al., 1994Go) and, for confirmation, R78.1, R78.2 (Han et al., 1996Go; Tobiasch et al., 1998Go)], and amplified products were confirmed to be AAV-specific using radiolabelled probes hybridizing within the respective amplified fragments (Table IGo). In some cases, AAV DNA-positive samples were analysed with primers discriminating between the different AAV types (Tobiasch et al., 1998Go).

In 64 out of 238 amnion samples, DNA sequences of AAV were detected by PCR (Table IIA, Figure 1GoGo). In Figure 1Go the amnion fluid samples no. 28, 30, and 32 were found to contain AAV DNA (confirmed by Southern blot hybridization with an internal probe; Table IGo). Out of 15 AAV DNA containing specimens tested, two contained AAV-3 DNA whereas in the other cases type AAV-2 DNA was identified, using discriminating primers (data not shown, see Tobiasch et al., 1998Go). Since AAV is a helper-dependent parvovirus we checked in addition for the presence of possible helper viruses (Table IIA,BGoGo). In 32 out of 183 cases, HCMV DNA was amplified. Out of these, nine samples contained both AAV and HCMV DNA. DNA of herpes simplex virus (HSV, types 1 or 2) was not detected. In two cases, there were faint hints for the presence of DNA of adenovirus. Because of shortage of material this could not be confirmed definitively. Twenty-five out of 208 tested samples contained DNA of human papillomaviruses (12%). In eight samples (3.9%), DNA of HPV could be identified in addition to AAV-2 DNA. HPV DNA was also detected in 18 samples not containing AAV-2 DNA.


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Table IIA. Presence of viral DNA in amniotic fluid samples
 


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Figure 1. Representative agarose gel analysis of polymerase chain reaction products from amniotic fluid DNAs amplified with adeno-associated virus (AAV)-specific primers (pan1/pan3; 337 bp). Positive controls were DNA samples from the AAV DNA-containing cell lines, JEG, JAr and BeWo (Dutheil et al., 1997Go), and of HA16 cells (Walz and Schlehofer, 1992Go); DNA samples from SiHa and HeLa cells were AAV DNA-negative controls. pBR322/TaqI was used as molecular size marker.

 

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Table IIB. Identification of HPV types
 
For most samples, it was possible to determine the HPV type (Table IIBGo). HPV-18 was found in 13 cases and HPV-16 in nine cases.

For 43 samples, sufficient material was available to inoculate the centrifuged supernatant in adenovirus-infected HeLa cells. Under these conditions, replication of AAV DNA could be detected in 13 cases, indicating the presence of infectious AAV particles in the amnion fluid (Figure 2Go) at a frequency (30%) similar to the detection of AAV DNA by PCR (27%). With the exception of one case which contained HPV-18 DNA, the samples with infectious AAV were negative (by PCR) for the presence of helper virus DNA.



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Figure 2. Detection of infectious virions of adeno-associated virus (AAV) in amniotic fluids. Suspended pellets of amnion fluids were inoculated on HeLa cells cultured in 6-well plates. The cells were then super-infected with adenovirus type 2 (Ad-2) and incubated (30 min, 37°C). After 3 days the cells were harvested by trypsination, trapped by solution onto nylon filters and hybridized with digoxigenin–dUTP-labelled pTAV-2. Filter # 1: HeLa cells infected with AAV-2 and Ad-2 (positive control); filters # 2, 3, 4, 5: HeLa cells inoculated with AAV DNA containing (PCR data) amniotic fluids (pellets), and super-infected with Ad-2; filter # 6: HeLa cells inoculated with a pellet of an AAV DNA-negative (PCR) amniotic fluid. The signals on filters # 3 and # 5 indicate replication of DNA of AAV virions present in the amniotic fluids.

 
Information on the course and outcome of the pregnancies was available only for 24 cases from Bordeaux, and 32 cases from Geneva. For the cases from Heidelberg, it was not possible to obtain clinical data since testing was done on anonymous amnion fluid samples or trophoblast cultures.

In three of the Bordeaux cases, the pregnancy was interrupted because of fetal anomalies [For one of these (a fetus with meningo-myelocele), AAV DNA was present in the amniotic fluid.] For the remaining 21 pregnancies, limited information about the newborns was available. On average, the babies were born at week 37.6 (weeks after amenorrhoea). The average birthweight was 3175 g. However, when analysing the data of the six newborns for whom AAV DNA had been detected in amnion fluids, an average birthweight of 2990 g was found. For these six babies, the average week of delivery was 38.8. Hence, AAV detection was accompanied by a birthweight reduced by 185 g. When correcting for the gestational age, this difference is even higher at 385 g.

There was no indication of an influence of the presence of helper virus DNA (HPV sequences were found in two cases) or of the presence of infectious AAV (two cases, others than the HPV-positive ones).

In the cases from Geneva, the average birthweight of children from AAV-infected mothers was 2720 g as opposed to 3201 g for infants from mothers whose amnion fluid did not contain AAV DNA. However, the differences in the birthweights were statistically significant neither for the Bordeaux (P = 0.14) nor for the Geneva (P = 0.27) cases.

For the Geneva cases, more detailed clinical information was available. (However, the Geneva samples could not be tested for infectious virions.) Despite the small numbers it was interesting to note that in 71% of the AAV-infected mothers, premature amniorrhexis occurred, in contrast to 8% in non-infected women (statistically significant difference, P = 0.001; Table IIIGo). In the AAV DNA-positive cases, also a significantly higher frequency of premature labour was observed [57% versus 8%; P = 0.01 (Table IIIGo)]. Four of the five AAV DNA-positive cases with premature rupture of membranes were positive also for helper virus DNA; the two AAV DNA-negative cases had no signs of HCMV or HPV infection. However, among the AAV-negative samples (without delivery complications), four contained HCMV DNA and three were HPV-positive.


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Table III. Perinatal status (Geneva cases)
 
In order to substantiate the findings of in-utero infection with AAV, we analysed human newborn sera from the University Hospital of Mainz (Germany) which were unrelated to the collected amnion fluid samples. Using an immunofluorescence assay, IgM type antibodies to AAV capsid proteins were detected in nine out of 38 sera (Table IVGo). For 20 newborns, sera were also available from the mother. In four cases, IgM antibodies to AAV were present in both the mother's and the child's serum. In one case, only the child had IgM antibodies against AAV. In seven cases, IgM antibodies were found in maternal sera, with no anti-AAV IgM antibodies in the newborns. (As expected, IgG antibodies against AAV were found in all sera.)


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Table IV. Presence of IgM type serum antibodies against adeno-associated virus (AAV)
 

    Discussion
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 Materials and methods
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The recent detection of a persistent infection with adeno-associated virus (AAV) of the female genital tract (Tobiasch et al., 1994Go; Han et al., 1996Go; Friedman-Einat et al., 1997Go) and the detection of virus DNA and proteins in the placental villi in some cases of miscarriage (Tobiasch et al., 1994Go) prompted us to assess the possibility of an intrauterine infection with these helper virus-dependent parvoviruses. This possibility is further underlined by the recent finding that human choriocarcinoma-derived trophoblast cell lines and the amnion-derived cell line, FL, contain integrated AAV DNA sequences (Dutheil et al., 1997Go). In addition, AAV DNA (together with HPV DNA) has been reported to be present in decidual gland cells from miscarriages (Rabreau et al., 1995Go).

Since our previously reported PCR data on the presence of AAV DNA in curettage material from spontaneous abortion might be overestimated due to the—then unknown—presence of AAV DNA in the cervix uteri of the majority of women (Tobiasch et al., 1994Go; Han et al., 1996Go; Walz et al., 1997Go), we assessed the issue of an in-utero infection with AAV by analysing material from transabdominal amniocentesis.

AAV DNA sequences were detected in 27% of amnion fluids. Furthermore, infectious virus particles could be demonstrated in 30% of testable AAV DNA-positive cases. In addition, infectious AAV could be rescued from cultured trophoblasts. Though `contamination' with maternal tissue cannot be formally excluded for all amniocentesis samples, the detection of AAV in trophoblasts clearly demonstrates infection of embryo cells.

Since these data point to in-utero infection with AAV, we analysed serum from newborns for IgM type antibodies to AAV (not crossing the placenta, hence indicative of in-utero infection). In one third of such serum samples (which were unrelated to the amnion samples), we demonstrated IgM type antibodies to AAV, strongly indicating that AAV infection also takes place later in pregnancy (the embryo is not able to respond with Ig synthesis prior to the 20th week of gestation). The presence of IgM antibodies in the corresponding maternal blood suggests reactivation of persistent AAV infection of the uterus tissue (Tobiasch et al., 1994Go; Walz et al., 1997Go) or re-infection during pregnancy. In-utero infection may lead to an incomplete immune response or to a partial immuno-tolerance. This could explain our previous data on the detection of IgM antibodies in adults (in the presence or in the absence of IgG antibodies) (Tobiasch et al., 1994Go), suggesting a re-infection with AAV or a re-activation of persisting virus. It is interesting to note that in the cited study, IgM antibodies were more frequently detected in women with HPV-related cervical lesions or in women with miscarriage. It is tempting to speculate that specific (hormonal?) changes in the uterine tissue might influence re-infection with AAV or re-activation of locally persisting virus, and may favour infection of embryo or fetal cells during pregnancy.

We have recently shown that HPV can act as helper viruses for AAV replication (Walz et al., 1997Go) and that HPV DNA is frequently detectable in genital tissues containing AAV DNA in the absence of other helpers such as adenovirus or herpes simplex virus (Malhomme et al., 1997Go). The data presented herein indicate that co-infections of AAV with HCMV and also HPV occur in amniotic cells. Detection of HCMV and HPV in amnion fluids has also been observed by others (Armbruster-Moraes et al., 1993Go; Armbruster-Moraes et al., 1994Go; Ruellan-Eugene et al., 1996Go), and reactivation of these helper viruses for AAV is known to occur during pregnancy (Schneider et al., 1987Go; Rabreau and Schlehofer, 1995Go; Lipitz et al., 1997Go). However, it cannot be excluded that embryonic cells may provide a milieu allowing replication of AAV DNA or expression of AAV functions in the absence of helper viruses. It is interesting to note that recent reports indicate that differentiating trophoblasts are more susceptible to infection with AAV than with other viruses (Parry et al., 1997Go, 1998Go).

To date, we have no conclusive indication of pathological conditions possibly resulting from in-utero infection with AAV. It is interesting to note that in cases in which AAV DNA was found in the amnion fluid, premature rupture of membranes and/or premature labour were more frequently observed. However, there is no evidence of a causal relationship between AAV infection and those perinatal conditions. A pathogenic role of AAV or (co-)infection with the other viruses tested for these conditions is still obscure because of the small numbers of cases that could be assessed.

It has to be taken into account, in addition, that women undergoing amniocentesis are a selected population, often with gestational risk conditions related to pregnancy. Therefore there might be a bias concerning the role of infections in pregnancy problems. Hence, several confounding variables might play a role: older women may have risk factors other than AAV infection for the observed conditions. However, the data presented herein give direct evidence that AAV infection of human embryo cells does occur and this should encourage investigations addressing the question of pathological consequences in clinical follow-up studies on AAV infection in pregnancy. It remains to be determined if the presence of AAV together with one of the possible helpers (HCMV or HPV) has pathological outcomes other than the mere presence of AAV DNA. Perhaps organs latently infected with AAV respond differently to infections or pathological conditions occurring later in life. Elucidation of this question and determination of the target organs of natural infection with AAV (in utero or later in life) may also be important when contemplating the use of these viruses as vectors in gene therapy.


    Acknowledgments
 
We thank A.Queisser-Luft for newborn sera, J.Kleinschmidt for an AAV-2 capsid proteins expressing plasmid, used in transfected cells for the immunofluorescence antibody test, K.Erles for advice in AAV serology, M.Blettner and T.Scheuchenpflug for statistical analysis, and K.Yürekci for skilful technical assistance. Suggestions of P.Schnitzler for the HCMV PCR as well as advice from O.Malhomme for specific PCR questions are gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft.


    Notes
 
6 To whom correspondence should be addressed Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Submitted on April 1, 1999; accepted on June 10, 1999.