Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2948-2954
Evidence That Amyloidogenic Light Chains Undergo Antigen-Driven
Selection
By
Vittorio Perfetti,
Paola Ubbiali,
Maurizio Colli Vignarelli,
Marta Diegoli,
Roberta Fasani,
Monica Stoppini,
Antonella Lisa,
Palma Mangione,
Laura Obici,
Eloisa Arbustini, and
Giampaolo Merlini
From the Research Laboratories of Biotechnology and Organ
Transplantation, Clinical Immunology Unit, the Department of Internal
Medicine, Section of Internal Medicine and Medical
Oncology, and the Institutes of Human
Pathology, Biochemistry, and
CNR-IGBE, University of Pavia-IRCCS Policlinico S. Matteo,
Pavia, Italy.
 |
ABSTRACT |
AL amyloidosis is characterized by fibrillar tissue deposits
(amyloid) composed of monoclonal light chains secreted by small numbers
of indolent bone marrow plasma cells whose ontogenesis is unknown. To
address this issue and to provide insights into the processes that
accompanied pathogenic light chain formation, we isolated the complete
variable (V) regions of 14 light (VL) and 3 heavy (VH) chains secreted
by amyloid clones at diagnosis (10 Bence Jones and 4 with complete Igs,
9 
and 5 
) by using an inverse polymerase chain reaction-based
approach free of primer-induced biases. Amyloid V regions were found to
be highly mutated compared with the closest germline genes in the
databases or those isolated from the patients' DNA, and mutations were
not associated with intraclonal diversification. Apparently high usage
of the III family germline gene V III.1 was observed (4 of 9 light chains). Analysis of the nature and distribution of somatic
mutations in amyloid V regions showed that there was statistical
evidence of antigen selection in 8 of 14 clones (7 in VL and 1 in VH).
These results indicate that a substantial proportion of the amyloid clones developed from B cells selected for improved antigen binding properties and that pathogenic light chains show evidence of this selection.
| |
INTRODUCTION |
IN AL AMYLOIDOSIS, monoclonal light
chains accumulate in fibrillar tissue deposits (amyloid), leading to
progressive dysfunction of target organs.1 Light chains,
most frequently of the class, are secreted by small numbers of
indolent bone marrow plasma cells (PC).2 The natural
history of the amyloidogenic clone is at present unknown. Information
might be obtained from an analysis of the nature (silent [S] or amino
acid replacing [R]) and distribution of somatic mutations in Ig
variable (V) regions.3 For example, evidence of clustering
of R mutations in the antigen-binding loops (CDR), together with their
scarcity in the framework (FR) regions (conserved areas with structural
importance), is consistent with a role for antigen4 in
selecting and expanding the B cell that will eventually give rise to
the amyloidogenic PC.
To provide insights into the processes that accompanied the formation
of amyloidogenic light chains and PC, we isolated the complete
nucleotide sequences of the Ig V regions of 14 light (VL) and 3 heavy
chains (VH) secreted by amyloid clones using an inverse polymerase
chain reaction (PCR)-based strategy that uses only primers specific for
constant regions, so as to avoid biases for certain
sequences.5 We focused on light chains and principally on
Bence Jones (BJ) proteins for the following reasons: (1) the primary
structure of VL is implicated in amyloid deposition6; (2)
secretion of free light chains only is a classic feature of AL
amyloidosis (up to 40% of the cases)7; (3) gene usage at
the germline level is unknown in AL amyloidosis; and (4) a study on
multiple myeloma recently stressed the importance of VL in the
characterization of the role of antigenic selection on the B
cell.8
| |
MATERIALS AND METHODS |
Patients and bone marrow studies.
Fourteen patients with biopsy-proven light chain amyloidosis were
studied at diagnosis. Bone marrow aspirates were obtained after
informed oral consent was given. Analysis of bone marrow PC light chain
/ ratios7 and labeling indexes9 were
performed with immunofluorescence procedures. Monoclonal components
were detected by immunofixation of serum and urine using
anti-isotype-specific rabbit antisera (Dako, Glostrup, Denmark).
Inverse-PCR amplification; cloning and sequencing of bone marrow
monoclonal light and heavy chains.
The inverse PCR-based procedure used to isolate the Ig V regions of AL
amyloidosis patients has recently been described in detail.5 Briefly, double-stranded cDNA from
Ficoll-separated bone marrow mononuclear cells was blunt-ended, ligated
upon itself to form a circle with T4 DNA ligase (GIBCO-BRL, Life
Technologies, Grand Island, NY), and PCR-amplified using
oligonucleotides specific for the 5
and 3 of the heavy
and light chain constant region isotypes. Amplimers are oriented toward
the V region (A primers) or toward the 3 end of constant regions
(B primers); consequently, the amplification product consists of
(5
3): namely, the 3 end of the constant
region and the untranslated region, poly-A tail, 5 untranslated
region, leader, V region, and the 5 end of the constant region.
The PCR products obtained from 3 to 4 independent amplification rounds
were pooled, gel-purified, and cloned into plasmid. Several plasmid
inserts were then sequenced from both sides using an automated DNA
sequencer5 and compared with each other. The presence of
the same V region sequence in several clones indicates its monoclonal
origin, because no primer-induced bias can be introduced during
amplification and the fraction of plasmid clones with a given V region
sequence is proportional to the amount of its mRNA in the bone
marrow.5 The accuracy of this method was tested by
amplifying, cloning, and sequencing a plasmid containing a V region
fragment of known sequence; only one mismatch of a total of more than
2,000 bases was noted.5
Data analysis and identification of mutations in the monoclonal V
regions of AL amyloidosis.
To identify the presumed germline gene of monoclonal V regions,
alignment was made with the current releases of EMBL-GenBank and V-BASE
(V BASE Sequence Directory; Tomlinson et al, MRC Centre for Protein
Engineering, Cambridge, UK) sequence directories using the
BLAST10 and DNAPLOT (H.-H. Althaus, University of Cologne, Cologne, Germany) search tools, respectively. A binomial distribution model4 was used to determine the likelihood that the
observed R mutations in a gene segment occurred by chance. The formula predicts the expected number of R mutations and is based on the total
observed mutations (R+S), the R mutations found in the CDR or FR, the
relative lengths of the CDR or FR, and the expected proportion of R
mutations (Rf). Nucleotides in the CDR show greater susceptibility to
generating R mutations, ie, substitutions in these regions are more
likely to produce amino acid replacements.11 For this
reason, specific Rf values were calculated for each germline segment
according to Chang and Casali.11
Isolation of VL germline genes.
After identification of the presumed VL germline gene through database
searching, the patients' own germlines were isolated by means of an
adaption of established procedures.12,13 DNA from the
peripheral blood neutrophils of 5 patients was PCR-amplified using
5 primers complementary to both the leader sequences of the
monoclonal V regions and germline genes VIII.1
(5-ttcctcggcgtccttgctta-3: patients SEM, PAP, DIB, and
DEP) and B3 (primer VIV LEA13: patient QUA) and 3
primers that anneal to regions of the 3 recombination signals
that are highly conserved within gene families.12,13 The
generality of the 3 primers allows coamplification of related germline genes.12,13 After cloning, six plasmid inserts
were sequenced as described above.
Amino acid sequencing of light chains from amyloid fibrils and BJ
proteins.
Amyloid fibrils from patients CAR (IgG) and DEP (IgA) were
extracted from biopsy specimens. Light chains were digested with trypsin and peptides were purified by reverse-phase chromatography following reported procedures.14 PAP and SEM BJ
proteins were isolated from urine. Amino acid sequencing was performed
by adsorptive biphasic column technology using an HPG-1000 A protein
sequenator (Hewlett Packard, Palo Alto, CA) as previously
described.15
| |
RESULTS |
Isolation of VL and VH regions secreted by amyloid clones.
Table 1 reports the characteristics of the
14 patients at diagnosis of AL amyloidosis. Bone marrow PC /
ratios showed expansions of PC with the same isotype as the monoclonal
component. PC in the S-phase of the cell cycle (PC labeling indexes)
were extremely rare, a typical finding in AL amyloidosis.16
There was no evidence of associated multiple myeloma. In 7 cases, it
was possible to observe patients for more than 2 years; the disease
remained stable with only slight modifications in the bone marrow PC
numbers.
V regions from bone marrow cDNA were inverse-PCR amplified and cloned
into plasmid, and multiple inserts were sequenced. The number of
identical/sequenced plasmid inserts for each patient is included in
Tables 2 (VL) and 3 (VH). In
each case it was possible to identify a single, identical, repeated V
region. The other sequences were different from one another. A minimum
of 3 (Table 2; SET IgG) and an average of 6.6 repeated plasmid
clones were sequenced for each patient. Even when 9 repeated clones
were sequenced from the same patient (Table 2; QUA BJ, CAR IgG,
DEP IgA), no significative nucleotide substitution was observed. We
detected only 11 mismatches in more than 42,000 sequenced basepairs
(112 plasmid clones), a result that is fully compatible with the
estimated error of the cloning procedure.5 No clones
presented more than one mismatch with the predominant sequence.
Nucleotide changes were present mainly in the FR (9/11) and none in the
CDR3; the latter, subject to the greatest in vivo variation, were
isolated to single plasmid inserts, ie, they were not found to occur
more than once and were distributed in different patients. Patient SET
had only 3 of 10 identical plasmid inserts (Table 2); this result is
compatible with a small amyloid PC clone, as documented by the
slight deviation in the bone marrow PC / ratio (Table 1).
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Table 2.
The VL and JL Germline Segments Used by Amyloidogenic
Light Chains and the Number of Identical/Sequenced Plasmid Inserts
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Table 3.
The VH and JH Germline Segments Used by Three
Amyloidogenic Clones and the Number of Identical/Sequenced Plasmid
Inserts
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Cloned VL regions correspond to the light chains isolated from
fibrils and urine.
In 4 cases, we compared the derived amino acid sequences of cloned VL
regions with partial protein sequences of light chains extracted from
amyloid deposits (residues: CAR = 1 to 30; DEP = 2 to 40 and 62 to 87) and of BJ proteins (residues: PAP = 5 to 21; SEM = 2 to
38). Protein-derived sequences confirmed identity in both germline
coded and somatically mutated residues (see "Light and heavy chains
from amyloid clones are highly mutated. Analysis of somatic
mutations"), thus demonstrating that the correct monoclonal VL
regions had been identified.
Gene usage by amyloid clones.
For the most part, and amyloid light chains used genes that
belong to the most numerous gene families: VIII (5 of 9 light chains) and VI (3 of 5; Table 2). The germline gene
VIII.1,17 also known as DPL2312 and
3r,18 was rearranged in 4 of 9 cases. By contrast, light chains used various germline genes. There was almost constant
usage of J2/3 (8 of 9 light chains;
Fig 1A), the most commonly rearranged J
segment in general,17,19 whereas 2 of 5 light chains
used J3 (Fig 1B), which is not frequently employed in peripheral
blood -positive lymphocytes (<10% of functional
rearrangements).20
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| Fig 1.
Formation of amyloidogenic light chain CDR3. The most
likely mechanism is reported. Sequences are compared with the closest VL and JL segments. (A) light chains; (B) light chains. R mutations, upper case letters; S mutations, lower case letters. Nontemplate coded nucleotides (N) are shown in bold; P nucleotides are
in parentheses. *Nucleotides apparently removed at recombination.
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VH region analysis is reported in Table 3. Two heavy chains used
members of VHIII gene family and one used a member of VHI. The JH4b
segment was employed in all cases (Fig 2).
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| Fig 2.
Formation of heavy chain CDR3 from amyloid clones. The
most likely mechanism is shown. Sequences are compared with the closest DH and JH segments. R mutations, upper case letters; S mutations, lower
case letters.
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CDR3 formation mechanism.
Figure 1 illustrates the most likely mechanism of amyloid light chain
CDR3 formation. Trimming of VL and/or JL segments occurred in 9 of 14 cases, and in 5 instances (~36%), nongermline nucleotides were
found at junctions. Both templated (P; Fig 1, nucleotides in
parentheses)21 and nontemplated (N) nucleotides were
apparently observed. Figure 2 shows the CDR3 formation of VH. In this
case, too, trimming of JH and N nucleotides were found.
Light and heavy chains from amyloid clones are highly mutated.
Analysis of somatic mutations.
To test amyloid V regions for the presence of somatic mutations, their
nucleotide sequences were compared with the closest germline genes in
the databases. In 4 cases (DEP, PAP, SEM, and DIB) and 1 case
(QUA), the corresponding germline segment was also looked for in the
patients' neutrophil DNA and a gene identical to the sequences
published was found (data not shown; sequences available from GenBank
[accession nos. AF026934-38]).
The deduced amino acid sequences of the amyloid VL regions are depicted
in Fig 3; their nucleotide differences as
compared with the corresponding germline gene are summarized in Table
2. VL sequences deviated substantially from the closest germline genes,
with a median percentage of mutation of 5.7% and a wide range (4.0%
to 10.2%). The mutation rate was apparently similar in and light chains, BJ proteins, and light chains that are part of a complete
Ig. By contrast, the portion coding for the leader peptide (which is
deleted from the mature V region) showed no, or rare, nucleotide
differences from the germline gene sequence (the median mutation rate
decreases from 5.7% to 5.0% [range, 3.4% to 8.8%] when the leader
is included in the comparison), thus substantiating correct
identification of the corresponding germline gene.
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| Fig 3.
Deduced amino acid sequences of the VL regions of
amyloidogenic light chains. Comparisons are made with the most
homologous germline gene. (A) light chains; (B) light chains. R
mutations, upper case letters; S mutations, lower case letters; R
mutations in JL are shown in bold, S mutations are underlined.
*Germline segment isolated from patient DNA. A continuous line
indicates the amino acid sequence that was also found in the monoclonal proteins isolated from amyloid fibrils (DEP and CAR) and urine (PAP and
SEM). Protein sequencing of the N-terminal portion of light chains
showed the absence of the first amino acid in DEP and SEM and of 4 residues in PAP. The absence of the first residue occurs in many III
light chain protein sequences,22 whereas multiple amino
acids were missing in an amyloid light chain recently
reported.23
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The derived amino acid sequences of the VH regions of the amyloid
clones are shown in Fig 4, and the results
of detailed analysis of somatic mutations and gene usage are
recapitulated in Table 3. These findings were similar to those reported
for light chains, with a wide range in somatic mutation (from 3.7% to
10.5% of nucleotide substitutions).
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| Fig 4.
Deduced amino acid sequences of the VH regions secreted
by amyloid clones. Comparisons are made with the most homologous
germline gene. R mutations, upper case letters; S mutations, lower case letters; R mutations in JH are shown in bold; S mutations are underlined.
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Somatic mutations were also present in JL and JH segments (Figs 1 and
2).
Nucleotide sequences of the V regions were submitted to the
EMBL-GenBank databases (accession nos. Z66542,
AF026918-33).
Replacement mutations and antigen selection in amyloid Igs.
Clonal expansion takes place in the germinal center cells that acquired
mutations that improve antigen binding while preserving the correct
folding of the Ig V region. Ig subjected to several rounds of antigenic
selection are expected to exhibit clustering of R mutations in the
antigen-binding loops (positive selective pressure for amino acid
changes), whereas, on the contrary, R mutations would be less frequent
in the FR (negative selection for amino acid
substitutions).4 A binomial model tests whether the
observed distribution of R mutations follows this pattern.4 The results of this statistical analysis are included in Tables 2 (VL)
and 3 (VH). Consistent with selection by Ag, 13 of 14 VL and 2 of 3 VH
segments displayed higher numbers of R mutations in the CDR than those
theoretically expected (Tables 2 and 3). In accordance with the
preservation of amino acids in the FR regions, all but one of the VL
and VH segments showed lower numbers of R mutations than those expected
for a randomly mutating gene segment (Tables 2 and 3). This difference
was statistically significant in 7 of 14 VL and 1 of 3 sequenced VH
segments (P 
.01 in 5 VL; P .05 in 2 VL and 1 VH;
Tables 2 and 3). That 7 of 14 amyloidogenic light chains originated
from selected somatic mutations accumulated during an antigenic
response is further substantiated by the significant rarity of FR-R
mutations (P .01 in 4 and P .05 in 3 VL; Table 2).
In the remaining 7 amyloid VL, the observed R mutations in the CDR and
FR almost reached significativeness in 3 (Table 2; SEM BJ, CAR
IgG, and DEP IgA), whereas the others demonstrated CDR-R and FR-R
mutations close to the numbers predicted by chance.
In the case of DEP IgA, significant clustering in the CDR was
present in VH rather than in VL (Table 3). Therefore, our analysis
showed that 8 of 14 (57%) amyloidogenic PC clones manifested statistical evidence of antigenic selection.
Overall, the frequency of CDR-R mutations (8.8 × 10
2 CDR-R/base) found in the 14 amyloidogenic light
chains was about twice that expected (4.7 × 102 CDR-Rexp/base; P < 1 × 104, 
2 test). In accordance with
structural preservation of FR regions, the rate of R mutations in these
areas (2.8 × 102 FR-R/base) was 1.6 times
lower than expected (4.5 × 102 FR-Rexp/base;
P < 3 × 104).
| |
DISCUSSION |
Our results indicate that many amyloid clones develop from B cells
whose Ig were subjected to antigenic selection and that pathogenic
light chains manifest evidence of this process.
V regions varied substantially from their germline counterparts and the
differences were attributed to somatic mutations. Isolation of the
corresponding VL germline gene from the DNA of patients' neutrophils
ruled out possible genetic factors such as allelic variants or
better-matching new germline genes in 5 cases. Furthermore, a major
contribution of allelic polymorphism or undiscovered germline genes to
nucleotide changes has been reported to be highly unlikely in
V24,25 and most probably in V as well.18
In addition, the discrepancy between the monoclonal V sequences and
their germline counterparts was too great (median of 5.7% in VL and
from 3.7% to 10.5% in VH) to be attributed exclusively to
polymorphism. That somatic hypermutations occurred on amyloid V regions
is further substantiated by the presence of deviations from the
germline sequence in JL and JH segments.
Sequencing of multiple plasmid clones containing PCR-amplified VH and
VL regions showed the identity of repeated sequences. Somatic mutations
were therefore homogeneous; there was no significant in vivo
intraclonal variation in the V regions expressed by these amyloid
clones at diagnosis. This is consistent with a clonal cell that is no
longer under the influence of the hypermutation process in the germinal
center.26 This result was expected in clones secreting free
light chains only (the lack of heavy chains prevents antigen receptor
formation and, consequently, Ig hypermutation), but homogeneity of
somatic mutations was also observed in the few cases in which complete
Ig were available. Recently, a study documented a modest degree of
intraclonal diversification in a minority of patients with monoclonal
gammopathy of undetermined significance.27 Although we
cannot exclude the possibility that this phenomenon may also occur in
some amyloid clones, our results suggest that this finding, if present,
must be rare in amyloidosis, in which BJ clones can constitute up to
approximately 40% of all cases.7
Our data show frequent usage of the most numerous germline gene
families, namely VI and VIII. At the level of germline genes, we
found that the VIII.1 alone (of a total of 30 functional V genes)18 accounted for 4 of 9 amyloid light chains and
for 4 of 5 amyloid light chains of the III family. By contrast, such a bias was not observed in amyloid light chains (Table 2) or in
myeloma light chains.8 Sequencing of the coding
portions of VIII.1 germline gene from these four unrelated amyloid
patients showed their identity to nonamyloid subjects.12,17
Usage restriction of germline genes appears to be a feature of the
normal humoral response.3,19 This has been shown for
VH28 and V genes,13,29 whereas V are only
now being investigated. Overrepresentation of VIII.1 in AL
amyloidosis might therefore be apparent, due to its preferential
expression in light chains in general or to limited patient
sampling. VIII.1 is the closest gene to the J-C cluster, being
only 14 kb away,17 and this proximity may predispose it to
rearrangement. However, only 1 of 6 myeloma light chains used this
V gene segment,8 and analysis of a compilation of
antibodies to various complex antigens from hybridomas/B-cell clones
showed involvement of VIII.1 in just 2 of 26 light chain
rearrangements.19 Taken together, this evidence suggests
that V gene usage in AL amyloidosis might be unique and that the
high frequency of VIII.1 expression may reflect some intrinsic
amyloidogenic properties of this gene. More nucleotide sequencing of
amyloid and nonamyloid light chains is needed to test the
association between VIII.1 and amyloidosis.
Light chain CDR3 showed trimming of V and J segments and insertion of P
and N nucleotides, thus showing, besides mutations, intense
exonucleasic and transferasic activity during both and amyloidogenic light chain rearrangements (Fig 1). Therefore, junctional
variation (due to truncations or insertions) appears to be a general
phenomenon of antibody diversification that involves 29,30 as well as light chains.
According to the type and distribution of somatic mutations and the
binomial distribution model of probabilities, there was statistical
evidence of antigenic selection in 8 of 14 clones (~57%). VL alone
was sufficient to show selection in 7. Analogously to light chains that
are part of a complete Ig, BJ proteins were highly mutated and showed
nucleotide changes compatible with antigen-driven selection; because
this process can only operate on B lymphocytes with surface Ig
receptors, which are composed of both light and heavy chains, selection
of the BJ clone most likely occurred before the heavy chain was lost.
Sahota et al8 analyzed myeloma VH and VL regions and found
that 10 of 15 cases (~67%) showed evidence of clonal selection (VL
contributed to 4 cases and VH to the other 6), a result that is quite
similar to what was found in our study (8 of 14 amyloid clones with
significant concentration of R mutations in the CDR). The mutation rate
of amyloid light chains is also comparable to that observed in myeloma,
with a median of 5.7% for amyloidogenic VL and 5.8%8 and
8.2%31 for myeloma VL and VH, respectively. A similar
degree of somatic mutations was also found in the VH regions of
patients with monoclonal gammopathy of undetermined significance, but
evidence of clone selection is still not clear in this
condition.27 These results suggest that
myeloma8,27,31-33 and AL amyloidosis progenitors similarly
undergo somatic hypermutation and antigenic selection and, together
with the apparent absence of significant intraclonal diversification,
indicate that the transformation generating the expanded amyloidogenic
marrow PC population probably occurs very late, after completion of
B-cell maturation and selection.
Despite numbers of R mutations in the CDR generally higher than those
predicted by chance (Tables 2 and 3), clustering of CDR-R mutations was
not significant in about 40% of amyloid clones. However, it should be
kept in mind that this type of analysis is limited to the study of the
V segment, which comprises only part of the antigen binding loops in
the mature V region: CDR1 and CDR2 and, in light chains, the 5
portion of CDR3. Therefore, the contribution of CDR3, which is often
essential for optimal antigen recognition,34 could not be
addressed fully in this study.
Light chain deposition disease, another condition characterized by
monoclonal light chain tissue deposition, though most frequently of the
type and lacking the characteristic birefringence of amyloid
deposits, has been studied using biochemical and genetic approaches.35 Similarly to our findings in AL amyloidosis,
sequencing data36 in this latter condition also suggest
possible overrepresentation of a germline gene, B3, the only member of
the VIV subgroup. R substitutions, likely caused by somatic
hypermutation, were also found here preferentially in the CDR of two
VIV light chains.37,38
Dimers of free light chains may function as primitive antibodies,
because they can structurally mimic the combining site,34 and it has been proposed that the initial event leading to amyloid formation might be an antigen-antibody interaction involving amyloid light chains and tissue components and that this phenomenon may account
for the heterogeneity of organ involvement typically observed in AL
amyloidosis.39 According to mutation analysis, a
substantial proportion of amyloidogenic light chains have genetic
features that are not incompatible with this hypothesis; evidence is
shown that many amyloid forming light chains were synthesized by clones selected during antibody response to a T-cell-dependent antigen and
may therefore possess the capacity to interact with specific ligands,
albeit to a lesser extent than intact Ig.
| |
FOOTNOTES |
Submitted October 6, 1997;
accepted December 9, 1997.
Supported by AIRC, Italian Ministry of Health (project no.
261RFM92/02), CNR target projects ACRO (projects no. 94.01322.PF39 and
96.000626.PF39), Fondazione Ferrata-Storti, and IRCCS Policlinico S. Matteo.
Address reprint requests to Giampaolo Merlini, MD, Internal Medicine
and Medical Oncology, Research Laboratory of Biotechnology, University
Hospital-IRCCS Policlinico S. Matteo, P.le Golgi 2, 27100 Pavia, Italy.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr Angelo Corti for his helpful discussions and Dr
Alessandra Cobianchi, Simona Casarini, and Irene Zorzoli for their
technical assistance.
| |
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