Helicobacter pylori-antigen-binding fragments expressed on the filamentous M13 phage prevent bacterial growth

Biochimica et Biophysica Acta 1474 (2000) 107^113

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Helicobacter pylori-antigen-binding fragments expressed on the ¢lamentous M13 phage prevent bacterial growth Jun Cao a , Yi-qian Sun a , Thomas Berglindh b , Bjo«rn Mellga®rd b , Zhao-qi Li a , Bibbi Ma®rdh a , Sven Ma®rdh a; * a

Department of Biomedicine and Surgery, Division of Cell Biology, Faculty of Health Sciences, Linko«ping University, Linko«ping, Sweden b Astra Ha«ssle AB, Mo«lndal, Sweden Received 29 September 1999; received in revised form 14 December 1999; accepted 11 January 2000

Abstract Colonization of the human stomach by Helicobacter pylori is associated with the development of gastritis, duodenal ulcer, mucosaassociated lymphoid tissue (MALT) lymphoma, and gastric cancer. H. pylori-antigen-binding single-chain variable fragments (ScFv) were derived from murine hybridomas producing monoclonal antibodies and expressed as a g3p-fusion protein on a filamentous M13 phage. The recombinant ScFv-phage reacted specifically with a 30-kDa monomeric protein of a H. pylori surface antigen preparation and by means of immunofluorescence microscopy the phage was shown to bind to both the spiral and coccoid forms of the bacterium. In vitro, the recombinant phage exhibited a bacteriocidal effect and inhibited specifically the growth of all the six strains of H. pylori tested. When H. pylori was pretreated with the phage 10 min before oral inoculation of mice, the colonization of the mouse stomachs by the bacterium was significantly reduced (P 6 0.01). The results suggest that genetic engineering may be used to generate therapy-effective phages. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Bactericidal ; Helicobacter pylori infection; Monoclonal antibody; Mouse model; Phage speci¢city; Recombinant bacteriophage ; Single-chain fragment variable (ScFv)

1. Introduction Helicobacter pylori infection is one of the most prevalent infections in humans. About 50% of the adults in the industrialized world and more than 90% of the population in developing countries are infected [1]. H. pylori is a Gram-negative bacterium that was ¢rst isolated and cultured in 1983 [2]. H. pylori colonizes the human stomach and is now accepted as a major cause of gastritis, duodenal ulcer, and mucosa-associated lymphoid tissue (MALT) lymphoma, and a risk factor for gastric cancer [3^8]. Eradication of H. pylori is possible by treatment with antibiotics and this is associated with healing or regression of the disease [9^11]. The general development of various bacteria that become resistant to antibiotics correlates with a generous use of antibiotics. This is a global problem of increasing medical concern. There is a need for alternative methods

* Corresponding author. Fax: +46-13-224254 ; E-mail : [email protected]

to eradicate bacteria in order to circumvent the problem of such resistance. From a practical perspective, the e¡ectiveness of antimicrobial agents has diminished the interest in vaccines. The development of vaccines against H. pylori is still at an early stage, but this ¢eld of research has attracted much attention and the feasibility of both prophylactic and therapeutic immunization is considered [12]. Hybridoma technology described in 1975 [13] resulted in the production of a variety of monoclonal antibodies (MAbs). MAbs have been modi¢ed by genetic engineering to include chimeric MAbs, humanized MAbs, a variety of antibody fragments, and phage antibodies. Such modi¢ed MAbs comprise novel reagents for in vivo diagnosis and therapy [14]. A bacteriophage is a virus which speci¢cally infects bacteria and which reprograms the bacteria for phage replication [15]. In some countries in Eastern Europe, phage therapy was earlier an alternative to treatment of bacterial infections with antibiotics [16], while in Western countries, phage therapy has been virtually unknown in the daily practice. Due to the increasing problem with resistance to antibiotics, phage therapy is becoming increasingly in-

0304-4165 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 0 ) 0 0 0 0 5 - 2

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teresting [17,18]. Recombinant phages are frequently used as vectors to produce large quantities of DNA, or peptides. Phage therapy, especially using genetically improved phages, might be an alternative method to control and combat pathogenic bacteria [17]. In the present investigation, we used a ¢lamentous phage display system [19]. MAbs against H. pylori surface antigens were developed and the variable fragments of these MAbs were displayed on a ¢lamentous M13 phage in order to study the initial binding and the e¡ects of the phage on H. pylori. In vitro the recombinant M13 phage speci¢cally inhibited the growth of all six strains of H. pylori tested. In a mouse model of H. pylori infection, the recombinant anti-H. pylori phage gave a signi¢cant protection. 2. Materials and methods 2.1. Materials QuickPrep mRNA puri¢cation kit, Recombinant phage antibody system kit (Mouse ScFv module, Expression module, Detection module), S¢I and NotI restriction enzymes, T4 DNA ligase, and horseradish peroxidase (HRP) anti-M13 conjugate were obtained from Pharmacia Biotech (Uppsala, Sweden). dNTPs mix, 10UPCR bu¡er and AmpliTaq DNA polymerase were purchased from Perkin Elmer (Sundbyberg, Sweden). Spermidine (N-[3aminopropyl]-1,4-butanediamine), thiamine hydrochloride, hexamine cobalt chloride, triethylamine, kanamycin, phenol:chloroform:isoamyl alcohol (25:24:1), anti-sheep IgG FITC conjugate, and anti-mouse IgG FITC conjugate were from Sigma (St Louis, MO, USA). ECL detection kit was obtained from Amersham (Buckinghamshire, UK). SlowFade antifade reagent was obtained from Molecular Probes (Eugene, OR, USA). Horseradish peroxidase antimouse IgG conjugate was obtained from Jackson ImmunoResearch Laboratories, (West Grove, PA, USA). Nitrocellulose paper was from Schleicher and Schuell (Dassel, Germany). Anaerocult C mini was obtained from Merck (Darmstadt, Germany) and BBL CampyPak Plus Microaerophilic System Envelopes with Palladium Catalyst was obtained from Becton Dickinson (Cockeysville, MD, USA). Bacto yeast extract, Bacto tryptone, and Bacto agar were purchased from Difco Laboratories (Detroit, MI, USA). Columbia agar plates and brucella broth were obtained from the Division of Microbiology (Linko«ping University, Sweden). Other chemicals were of analytical grade and commercially available. 2.2. Animals The study was approved by the local Ethics Committees of Animal Welfare (Astra Ha«ssle AB and Linko«ping). The mice were purchased from Bomholt Breeding Center

(Denmark). They were kept in makrolon cages with free supply of water and food. The animals were 4^6 weeks old at arrival. 2.3. Antigen preparation H. pylori strains 17874, 25, 66, 253, and 1139 (obtained from Astra Ha«ssle, Sweden) were cultured on columbia agar supplemented with 8.5% horse blood, 10% horse serum, 1% isovitalex under microaerophilic condition with Anaerocult at 37³C. Procedures described previously were followed to prepare surface proteins of H. pylori [20]. Approximately 4 g (wet weight) of the ¢ve strains of H. pylori was incubated for 15 min at room temperature in 100 ml of 0.2 M glycine-HCl (pH 2.2). The solution was then neutralized with NaOH and the antigen preparation was centrifuged at 10 000Ug for 10 min at 4³C. The pellet was discarded and the supernatant was dialyzed overnight against distilled water at 4³C and stored at 320³C until further used. 2.4. Enzyme-linked immunosorbent assay (ELISA) Nunc-Immuno plates were coated with 50 Wl of 0.05 M Na2 CO3 /NaHCO3 bu¡er, pH 9.8, containing indicated antigen (10 Wg/ml) and incubated overnight at 4³C. Free binding sites were blocked with PBS containing 0.05% Tween-20 (PBS-T) at 37³C for 1 h. Goat anti-mouse IgG peroxidase conjugate was used as a secondary antibody. Washing with PBS-T was performed three times between each incubation. Bound peroxidase was detected with 0.04% (w/v) oPD and 14 mM hydrogen peroxide in 0.1 M citric acid/0.2 M NaHPO4 , pH 5. The reaction was stopped by the addition of 50 Wl 2 M H2 SO4 and the plates were read at 490 nm. In phage ELISA, Nunc-Immuno plates were coated with 50 Wl of 0.05 M Na2 CO3 /NaHCO3 bu¡er, pH 9.8, containing H. pylori surface antigen (10 Wg/ml) and incubated overnight at 4³C. Phage suspension (2U1010 transforming units/ml (tfu/ml)) was added to each well and incubated at 37³C for 1 h. Bound phage was then linked to a secondary HRP anti-M13 conjugate. The reaction was developed in diammonium 2P2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) plus H2 O2 and was read at 405 nm. 2.5. Production of monoclonal antibodies Immunization procedure [21,22] and the production of H. pylori-speci¢c MAbs [22] were carried out as described. By means of immuno£uorescence microscopy, eight of the MAbs were shown to stain speci¢cally H. pylori taken from an agar plate culture. MAbs from hybridoma clones designated 2H6, 5D8, 5F8 and 5C4 gave a stronger reaction against H. pylori than others and these clones were chosen for construction of a phage antibody.

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2.6. Construction of phage antibody The Recombinant phage antibody system (Pharmacia Biotech) was used to produce recombinant phage antibody. Total mRNA was extracted from the hybridomas and puri¢ed. The puri¢ed mRNA was reverse-transcribed into cDNA using murine reverse transcriptase and random hexa-deoxyribo-nucleotides. The cDNAs corresponding to the heavy and light chain genes were ampli¢ed by the polymerase chain reaction (PCR). The genes of the heavy and light chains were then assembled as a single-chain fragment variable (ScFv) gene with a linker (corresponding peptide (Gly4 Ser)3 ). The ScFv gene was then digested by restriction enzymes NotI and S¢I, and subsequently inserted in the phagemid vector pCANTAB5 that had been predigested with the same enzymes. The ligation product was introduced into Escherichia coli (TG1) by heating and chilling and transformed cells were grown at 30³C in a medium containing glucose and ampicillin. In the next rescue step, phagemid-containing bacterial colonies were infected with M13KO7 helper phage to yield recombinant phage that display antibody ScFv-fragments. Phage-displayed antibodies capable of binding H. pylori antigen were selected by panning against the antigen as follows: a 25-cm2 culture £ask was coated at 4³C with 5 ml of H. pylori surface protein (15 Wg/ml in 0.05 M sodium carbonate bu¡er, pH 9.6) overnight. After three washes with PBS, the £ask containing 10 ml of 1% BSA (w/v) in PBS was incubated at 37³C for 1 h. Following three washes with PBS, the £ask was incubated at 37³C for 2 h with the ScFv-phage. The £ask was then washed 20 times with PBS containing 0.1% (w/v) Tween-20 and 20 times with PBS. Bound phage particles were released by the addition of 1 ml of 0.1 M triethylamine and gentle shaking for 10 min, and then immediately neutralized with 0.5 ml of 1 M Tris-HCl, pH 7.4. The eluted phage was used to reinfect log-phase E. coli TG1 cells on SOBAG agar containing 2% (w/v) Bacto tryptone, 0.5% (w/v) Bacto yeast extract, 0.05% (w/v) NaCl, 0.001 M MgCl2 , 0.01% (w/v) glucose and 0.01% (w/v) ampicillin. Colonies were picked, transferred, grown and rescued again with M13KO7. These steps of rescue, panning and reinfection were repeated to improve the binding a¤nity of the ScFv-phage. The ScFv-phage was concentrated by precipitation with polyethylene glycol as described in the instruction manual (Expression module/Recombinant phage antibody system) and stored for further use. Approximate concentrations of phage are expressed as tfu/ml. The value is obtained by infecting TG1 cells with serial dilutions of the recombinant phage and subsequent culture on SOBAG agar containing antibiotics. 2.7. Immuno£uorescence microscopy The suspension of recombinant phage (2U1012 tfu/ml)

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was added to a smear of H. pylori (CCUG 17874) which had been cultured in brucella broth for 1 day, or for 8 days. After incubation for 1 h at room temperature, bound phage was linked to a sheep anti-M13 IgG, and an anti-sheep IgG/FITC conjugate, which was visualized by means of £uorescence microscopy using a Nikon Labophot 2 microscope with a £uorescence attachment unit. A mercury lamp was used for excitation with ¢lters 465^495 nm (FITC). Emitted £uorescence was detected with the band pass ¢lters 515^555 nm. Images were digitalized using a CCD video camera (DEI-470B, PAL) and an image analysis equipment from Bergstro«m Instrument AB (Stockholm, Sweden). 2.8. Western blotting Surface antigens of H. pylori (15 Wg/well) was subjected to SDS-polyacrylamide gel electrophoresis [23]. The separated proteins were transferred to a nitrocellulose ¢lter by electroblotting. The ScFv-phage (2U1012 tfu/ml) was then applied to the ¢lter, and incubated for 1 h. HRP/anti-M13 IgG (1:4000 dilution) was added as a secondary antibody. Alternatively, a mix of parental MAbs was added to the ¢lter and HRP/anti-mouse IgG conjugate was added as a secondary antibody. Detection of binding was carried out by using the ECL detection kit. All incubation steps were performed at room temperature. Bovine serum albumin (10% w/v) in PBS-0.05% (v/v) Tween-20 was used as diluting and blocking bu¡er. 2.9. E¡ect of the ScFv-phage on growth of H. pylori in vitro The e¡ect of the ScFv-phage on H. pylori 17874 cultured in brucella broth for 1 and 2 days was investigated. The ScFv-phage was added (106 tfu/ml) at either day 0, or at day 0 and day 1. The control condition was brucella broth in the absence of phage. In other experiments, H. pylori laboratory strains 17874, 1139, 253, 244, 66 and 25, Staphylococcus aureus (ATTC 29213), Streptococcus (Raf M87), and E. coli (TG1) were cultured for 24 h in brucella broth in the absence and presence of the ScFv-phage (¢nal concentration 108 tfu/ ml). 2.10. E¡ect of the ScFv-phage on growth of H. pylori in a mouse model In initial experiments, the H. pylori strain 244 was proven to be a good colonizer of the mouse stomach. The bacteria were adopted to female SPF BALB/c mice by passage at least 3 times through the mice. Bacteria from a stock kept at 370³C were grown overnight in brucella broth at 37³C in a microaerophilic atmosphere (10% CO2 , 5% O2 ). In order to enhance the colonization of bacteria and to protect the phage from exposure to gastric acid, the animals were given an oral dose of omeprazole (400 Wmol/

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kg) 3^5 h prior to the oral inoculation of H. pylori (approximately 107 tfu/animal). The mice were inoculated with either freshly grown H. pylori in the absence of phage, or H. pylori pre-mixed with the recombinant ScFv-phage. Mixtures were incubated 10 min at room temperature before inoculation of the animals. There were six animals in each group. After 10 days, the mice were killed by CO2 and cervical dislocation and analyzed for the presence of H. pylori in the gastric mucosa. The abdomen and chest cavity was opened, and the stomach was removed. The stomach was cut along the greater curvature, rinsed in saline and cut into two pieces. An area of 25 mm2 of the mucosa from both the antrum and corpus was scraped separately with a surgical blade. The mucosa scraping was suspended in brucella broth, diluted and plated onto blood Skirrow agar plates. The plates were incubated under microaerophilic conditions for 3^5 days and the number of colonies was counted. The identity of H. pylori was ascertained by urease and catalase tests, and by direct microscopy with Gram staining.

Fig. 1. Agarose gel electrophoresis of PCR products. (A) The genes of the variable heavy (VH ) and light (VL ) chains from hybridomas secreting MAbs were ampli¢ed by PCR. Lane 1, base pair markers; lane 2, heavy chain fragments (340 bp); lane 3, light chain fragments (325 bp). (B) The VH and VL genes were assembled with a linker as the ScFvgene. Lane 1, base pair markers ; lane 2, ScFv-gene (750 bp); lane 3, ScFv-marker (1000 and 750 bp).

bp) genes were visualized by agarose gel electrophoresis (Fig. 1A). The ScFv-gene (750 bp) was prepared by assembling the heavy and light chains with the linker (Fig. 1B). Many recombinant phagemid clones were rescued and packaged into the M13 phage. More than 200 clones were tested for binding to H. pylori in ELISA. Four ScFvphage reacted with the H. pylori antigen. After two steps of rescue^panning^reinfection, the percentage of positive clones analyzed by ELISA increased 5-fold.

2.11. Presentation of data and statistical calculation All experiments were reproduced at least 2^3 times and representative results are shown. The data from the mouse model were evaluated using Wilcoxon^Mann^Whitney sign rank test. The data are presented as means þ S.E. (six mice/group). 3. Results

3.2. Binding of the ScFv-phage

3.1. Construction and selection of ScFv-phage

Judged by immuno£uorescence, the ScFv-phage bound to the spiral form of H. pylori taken from 1-day broth culture (Fig. 2A), and to the coccoid form of the bacterium from 8-day broth culture (Fig. 2B). A control phage, M13KO7, did not stain H. pylori (not shown). In Western blots under non-reducing conditions, the

A total of 11 Wg of mRNA was obtained from the hybridoma (2H6, 5D8, 5F8 and 5C4) that were secreting H. pylori-speci¢c MAbs. The cDNA transcribed from the mRNA was ampli¢ed by means of PCR. The variable regions of the heavy chain (340 bp) and light chain (325

Table 1 H. pylori laboratory strains 17874, 1139, 253, 244, 66 and 25, Staphylococcus aureus (ATTC 29213), Streptococcus (Raf M87), and E. coli (TG1) cultured for 24 h in brucella broth in the absence or presence of the ScFv-phage (108 tfu/ml), or with control phage (M13KO7) Medium H. pylori 17874 1139 253 244 66 25 Staphylococcus Streptococcus E. coli

+ScFv-phage

+M13KO7

0h

24 h

0h

24 h

0h

24 h

2.1U104 4.7U104 1.1U104 104 1.4U104 1.4U104 7.8U107 2.3U107 2.6U106

5.8U106 1.5U107 1.7U107 3.1U105 2.1U107 9U107 1010 5.6U107 4.4U108

2.8U104 4.3U104 1.3U104 104 1.4U104 7U103 2.1U108 1.9U107 2.6U106

4U102 102 4U105 103 4U104 3U102 8U109 5.8U107 4.2U108

3.9U104 ^ ^ ^ ^ ^ ^ ^ -

4U106 ^ ^ ^ ^ ^ ^ ^ -

Values are cfu/ml.

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Fig. 3. SDS-PAGE and immunoblotting of glycine-acid extract of H. pylori. Lane A, Coomassie brilliant blue staining of the major protein bands. Lane B, immunostaining with the ScFv-phage. Lane C, immunostaining with a pool of the parental MAbs.

Fig. 2. Immuno£uorescence micrographs of H. pylori (CCUG 17874). (A) ScFv-phage immunostaining of H. pylori taken from 1-day brucella broth culture. (B) ScFv-phage immunostaining of H. pylori taken from 8-day brucella broth culture. Scale bar: 5 Wm.

ScFv-phage and the parental MAbs stained a 30-kDa H. pylori protein, but also a 60-kDa protein after long exposure time (Fig. 3). When the antigen was reduced by dithioteitrol and alkylated by iodoacetamide before the electrophoresis (not shown), only the 30-kDa protein was obtained which suggested the monomeric structure of the antigen. A control phage, M13KO7, did not react with the antigens (not shown).

29213), Streptococcus (Raf M87), and E. coli (TG1) were cultured in the absence and presence of phage (107 tfu/ml) for 24 h. The concentration of bacteria (cfu/ml) was analyzed at time 0 and at 24 h (Table 1). The recombinant ScFv-phage decreased the cfu of all the strains of H. pylori, but did not inhibit the growth of Staphylococcus, Streptococcus, or E. coli. H. pylori 17874 was not a¡ected by the phage M13KO7 used as a control. 3.4. E¡ect of the ScFv-phage on the growth of H. pylori in a mouse model In antrum, the number of H. pylori 244 from control animals was 7.8U103 þ 1.8U103 (cfu/25 mm2 ; (mean þ S.E., n = 6) and 1.6U103 þ 4.9U102 in the H. pylori plus the ScFv-phage animal group (P 6 0.01) (Fig. 5). In corpus, the number of H. pylori was 1.0U104 þ 2.8U103 in the control mice (cfu/25 mm2 ; mean þ S.E., n = 6) and

3.3. E¡ect of the ScFv-phage on the growth of H. pylori in vitro The concentration (cfu/ml) of H. pylori 17874 increased by four orders of magnitude when cultured for 2 days (Fig. 4). When the ScFv-phage (106 tfu/ml) was added at day 0, the phage reduced the concentration of bacteria by two orders of magnitude in the following day, but growth recovered slightly at day 2. When the phage, however, was added at both day 0 and day 1, the cfu value of H. pylori was zero at day 2. H. pylori laboratory strains 17874, 1139, 253, 244, 66, and 25 together with Staphylococcus aureus (ATCC

Fig. 4. E¡ect of the ScFv-phage on the growth of H. pylori in vitro. H. pylori (CCUG 17874) was cultured in brucella broth. Arrows indicate the addition of 106 tfu/ml of the ScFv-phage.

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Fig. 5. E¡ect of the ScFv-phage on the growth of H. pylori in a mouse model 10 days after oral inoculation. Open bars, inoculation with H. pylori only; hatched bars, inoculation with H. pylori pre-mixed with ScFv-phage. *P 6 0.01, mean þ S.E., n = 6.

2.1U103 þ 5.3U102 in the H. pylori plus ScFv-phage group (P 6 0.01). 4. Discussion We describe a ¢lamentous M13 phage with an ScFvpeptide expressed on its surface. The pCANTAB5 phagemid vector allowed the expression of the ScFv gene cloned between the leader sequence and the gene 3. Many recombinant phagemid clones were produced and packaged into the M13 phage. Four ScFv-phage clones of more than 200 tested by ELISA bound to the H. pylori surface antigens. Immuno£uorescence microscopy, ELISA and Western blots, showed that the binding speci¢city of the parental MAbs was conserved in the ScFv-phage which reacted speci¢cally with 30- and 60-kDa proteins in the H. pylori antigen preparation. It bound speci¢cally to both the spiral and coccoid forms of the bacterium. In vitro, the recombinant ScFv-phage inhibited the growth of all strains of H. pylori tested and when added two to three orders of magnitude in excess in relation to the bacterium, the ScFv-phage exhibited an evident bactericidal e¡ect by yet unknown mechanisms. Also, the colonization of H. pylori in the mouse stomach was lowered signi¢cantly by the ScFv-phage. The ScFv-phage is not a `lytic' phage and its bactericidal activity was not expected. Yamaguchi et al. [24] described a monoclonal antibody to heat-shock protein 60. This MAb exhibited a bactericidal activity against H. pylori. Although antigens of 30 and 60 kDa are described as antigens in the present investigations, electrophoresis with reduction and alkylation of the antigens indicates that the major antigen of the parental MAbs is a 30-kDa protein (unpublished data) which thus is distinct from that (60 kDa) described by Yamaguchi et al. [24]. The ScFv-phage was designed to bind to H. pylori. The natural hosts for ¢lamentous phages, such as M13, fd and f1, are E. coli cells having hair-like F-pili. Such phages

alone do not produce a lytic infection, but rather induce a state in which the infected bacterium produces and secretes phage particles without undergoing lysis [15,25]. Passive immunization with antibodies may prevent a variety of diseases. Due to its site of colonization, deeply embedded under the mucus layer of the gastric mucosa, H. pylori is well protected from conventional antibodies. A phage with the ability to infect, replicate and to lyse the bacterium may have a longer life and may be more e¡ective to infect new bacteria. The present ScFv-phage may be modi¢ed to be capable of lytic replication in H. pylori. In the lytic bacteriophage T4 the host range was shown to expand by duplication of a small domain of the tail ¢ber adhesion protein [26]. Thus, alternative ways to vary the host speci¢city in a natural phage are available besides the present phage display of antigen binding fragments. In humans, the e¡ectiveness of phage therapy is documented in many reports from Eastern Europe [16]. In experimental systems with animals, phage therapy has been shown to be more e¡ective than treatment with antibiotics in experimental E. coli infections in mice [27], and severe experimentally induced E. coli diarrhea in calves was cured by a single dose of phage particles [28]. Bacterial infections, including infections by H. pylori, and the accelerating development of antibiotic resistance in various bacteria, are global problems of great medical concern. Genetically designed bacteriophages, as indicated in the present investigation, have the potential to become attractive alternatives to conventional antibiotics to control bacterial infections. This new ¢eld of research needs to be explored thoroughly in order to meet the present and future medical demands. Acknowledgements We thank Dr. Hans-Ju«rg Monstein for valuable discussions. This research was supported by the Swedish Medical Research Council (4X-4965), Lion Research Foundation, and Astra Ha«ssle AB, Mo«lndal, Sweden.

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