Neutrophil-specific granule deficiency (SGD) is a rare disorder characterized by recurrent pyogenic infections, defective neutrophil chemotaxis and bactericidal activity, and lack of neutrophil secondary granule proteins. CCAAT/enhancer binding protein (C/EBP)ε, a member of the leucine zipper family of transcription factors, is expressed primarily in myeloid cells, and its knockout mouse model possesses distinctive defects, including a lack of neutrophil secondary granule proteins. Sequence analysis of the genomic DNA of a patient with SGD revealed a five-basepair deletion in the second exon of the C/EBPε locus. The predicted frame shift results in a truncation of the 32-kD major C/EBPε isoform, with loss of the dimerization domain, DNA binding region, and transcriptional activity. The multiple functional defects observed in these early neutrophil progenitor cells, a consequence of C/EBPε deficiency, define SGD as a defect in myelopoiesis and establish the requirement for C/EBPε for the promyelocyte–myelocyte transition in myeloid differentiation.

Neutrophil-specific granule deficiency (SGD) is a rare congenital disorder marked by frequent and severe bacterial infections. The five reported cases consistently describe pleiotropic characteristics, including lack of secondary granule proteins and defensins, abnormalities in neutrophil migration and disaggregation, atypical nuclear morphology, and impaired bactericidal activity (111). More recent work has revealed additional granule abnormalities in the eosinophils of SGD patients, with absence of eosinophil-specific granule contents, including eosinophil cationic protein, eosinophil-derived neurotoxin, and major basic protein (12). Platelet disorders and associated bleeding diatheses, including the neutrophilic phagocytosis of platelets (13) and the absence of platelet–high-molecular-mass von Willebrand factor multimers stored in platelet α granules (14), have also been reported in SGD patients. In contrast to these seemingly genetically unrelated manifestations, these patients express normal levels of salivary lactoferrin (8, 15, 16), a characteristic specific granule marker absent in neutrophils in SGD, suggesting that the responsible defect involves myeloid-specific transcriptional regulation.

CCAAT/enhancer binding proteins (C/EBPs) comprise a family of transcription factors that are key regulators of cellular differentiation and function in a variety of tissues (17). The prototypic C/EBP is a modular protein consisting of one or more activation domains, a dimerization basic zipper domain and a DNA binding region (18). C/EBPs are least conserved in their activation domains and vary from dominant negative repressors to strong activators.

C/EBPε, the newest member of the family, is expressed exclusively in cells of myeloid and T cell lineage (1922). The human C/EBPε gene encodes four mRNA isoforms with varying splice patterns, driven from two alternative promoters, and from which are translated three protein isoforms (23). Analogous to what has been shown for C/EBPα and C/EBPβ (24, 25), in vitro transfection data suggest that the full length, 32-kD isoform of C/EBPε (C/EBPε32) possesses the fully active transcriptional activation domain, whereas the short, 14.2-kD isoform (C/EBPε14) lacks transcriptional activity (23).

Nearly 60% of C/EBPε knockout mice (26) succumb to low pathogenicity bacterial infections by 4–6 mo of age. Neutrophils from C/EBPε knockout mice have morphological features similar to human SGD neutrophils, including bilobed nuclei, absent specific and tertiary granule contents, and defective chemotaxis and bactericidal activity (27). The striking phenotypic similarities between SGD defects and the C/EBPε knockout model prompted a search for a C/EBPε knockout mutation in an SGD patient's genomic locus.

Materials And Methods

Patient.

Material from a previously described (5, 6) male patient lacking neutrophil-specific granules was studied. Research was conducted with informed consent under the guidelines of a National Institutes of Health (NIH) Internal Review Board– approved protocol, no. 92-I-99. The patient died from complications of pneumonia at age 20.

DNA, RNA, and Protein Extraction.

Peripheral blood neutrophils were isolated as described (28), cryopreserved with dimethylformamide (Sigma Chemical Co.), and maintained at −140°C. Cell proteins were extracted as described (29). DNA extraction from cryopreserved fibroblasts proceeded as described (30). RNA was extracted from patient bone marrow aspirate using RNAzol reagent (Teltest) as per manufacturer's protocol. Normal human bone marrow RNA was purchased from Clontech.

PCR Amplification of Genomic Sequence.

PCR reaction was performed using Platinum taq DNA polymerase (Life Technologies) per manufacturer's instructions and cycled as follows: 96°C for 12 min, followed by a three-step cycle—94°C for 30 s, 60°C for 30 s, and 72°C for 2 min—for 35–40 cycles. PCR products were gel purified and recovered using Gene Clean (Bio101). Products were sequenced with an ABI Prism Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Corp.). Primers were chosen from a published sequence available from EMBL/GenBank/DDBJ under accession no. U48865. Primer sets (upstream, downstream): B, 5′-AGC GGC CAT GCA AAA GGA AAG ACA, 5′-TCC ACC TAC CCC CAA GAG AAA GTT (bp 667–1186); C, 5′-CCC ACG GGA CCT ACT ACG A, 5′-GGG CTG GCC TGC TCT TAC (bp 1818–2343); F, 5′-CTC CCC GGC TGG CCC CTT ACA C, 5′-GCC AAC AGT CCC AAC ACC CAG TCA (bp 3133–3615); G, 5′-GGA GGT GGG GCT ACA AAA GAA ACT, 5′-TCA GGG AGG GGC AGG ACA (bp 1143–1553); H, 5′-ACA GGA GTG GGT GAC AGA GGA GAC, 5′-GGG CCG AAG GTA TGT GGA GGG TAG (bp 1563–2104); I, 5′-CCA TGC CCC CTC CTC TTG TTT CTC, 5′-ACT GCC TTC TTG CCC TTG TGT AA (bp 2594–3171); K, 5′-AAC TTT CTC TTG GGG GTA GGT GGA, 5′-TCG TAG TAG GTC CCG TGG (bp 1163– 1837). Homozygosity was determined by hybridization of the PCR product fragment C to a [32P]γdATP-endlabeled internal oligonucleotide (H. downstream; sequence above). Labeled oligo was mixed with hybridization buffer (75 mM NaCl, 5 mM EDTA) in a ratio of 1:10, and 10 μl was added to 30 μl PCR product. Hybridization was cycled in a thermal cycler: 95°C for 5 min and 55°C for 10 min. Reactions were immediately placed on ice. Products were resolved on 4–20% Tris/borate/EDTA polyacrylamide gel (Novex) at 250 V.

RNA Blotting Assay.

10 μg of total RNA isolated from the patient's bone marrow and 0.25, 0.5, and 1 μg of control polyadenylated (pA)-mRNA was electrophoretically separated, blotted, hybridized, and washed as described (27). The membrane was stripped by boiling and stored at −20°C.

Immunoblotting.

Protein quantitation was performed using a BCA Protein Assay kit (Pierce Chemical Co.) according to the manufacturer's instructions. 10–100 μg protein extracts were electrophoretically separated, transferred to nitrocellulose, and incubated with primary antibody as described (29). Primary antibody was generated in rabbits by Research Genetics, Inc., using a synthetic peptide encoded in exon 2 of C/EBPε, downstream of the SGD deletion (DPRAVAVKEEPRGPEGSR). The membranes were washed, incubated with anti–rabbit horseradish peroxidase conjugate antibody (ECL Western blot kit; Amersham Pharmacia Biotech, Inc.), and developed according to the manufacturer's instructions. Membranes were stripped and reblotted with anti–mouse human β actin antibody (Boehringer Mannheim) to control for protein loading.

In Vitro Mutagenesis Assay.

The patient's mutation was introduced into the pCMV-C/EBPε32 expression vector using a Stratagene QuikChange site-directed mutagenesis kit per manufacturer's instructions using a complementary oligonucleotide (PAGE purified; purchased from Genosys Biotech) containing the deletion (5′-CCA CTA CTT GCC GCC CTC GGC CCT TTG CCT ACC). Presence of the mutation and maintenance of the vector sequences was verified by sequencing and restriction enzyme digestion, respectively.

Transient Transfections.

HeLa cells were maintained in DMEM (BioWhittaker) supplemented with 10% heat-inactivated FBS (Life Technologies, Inc.) and penicillin/streptomycin at 37°C and 5% CO2. Cells were plated in 6-well plates and transfected within 24 h, at 30–50% confluency. Transfections, using the Mammalian Transfection System (Stratagene), were performed using 5 μg reporter plasmid (G-CSF receptor promoter-luc); 1, 2, or 5 μg inducer plasmid (pCMV-C/EBPα, pCMV-C/EBPε32 isoform, pCMV-C/EBPε14 isoform, or pCMV-C/EBPε32-SGD, described above); and 0.5 μg pCMVβ, as described (23, 31). The DNA content of transfections was normalized, and transfection was performed according to the manufacturer's instructions, with 300 μl transfection solution applied to the cells. Samples were harvested 24 h after transfection.

Luciferase and β-galactosidase activities were measured using a Dual-Light kit (Tropix, Inc.), according to the manufacturer's protocol, on a Turner 20/20 Luminometer. Samples were read for 15 s after a 3-s delay.

Results And Discussion

Sequencing of PCR products from genomic DNA detected a 5-bp deletion, TGACC, in exon 2 of the patient's C/EBPε sequence. Fig. 1 A shows sequence data from one normal control (top sequence) and the SGD patient (bottom sequence). The mutation predicts a frameshift and a premature termination of the encoded C/EBPε32 isoform (Fig. 1 B). The missense code after the frameshift results in the loss of the critical DNA binding domain and leucine zipper region required for C/EBP dimerization and function. C/EBPε transcripts encoding the shorter 27- and 14-kD isoforms are predicted to be unaffected, based upon the splice donor and acceptor and translational start sites (23).

Homozygosity of the deletion was determined by PCR amplification of the affected region and resolution of the DNA fragments on a 4–20% polyacrylamide gel (Fig. 1 C). DNA from one normal control and the SGD patient were mixed before amplification and electrophoresis (lane 3), showing bands from both affected and normal alleles. In comparison, PCR products from the SGD patient (lane 4) and normal controls (lanes 1 and 2) show only one fragment, indicating homozygosity for their respective alleles.

RNA blot analysis of the SGD patient's bone marrow total RNA showed decreased amounts of C/EBPε transcripts in comparison with control human bone marrow pA-RNA (Fig. 2 A). Hybridization with a [32P]dCTP- labeled actin probe (provided by L. Perera, National Cancer Institute, NIH) showed that 10 μg of SGD patient bone marrow total RNA was equivalent to 1 μg of normal bone marrow pA-mRNA and verified the stability and quality of the patient's RNA preparation. Specific loss of C/EBPε transcripts in the SGD patient is likely due to mRNA instability secondary to the frameshift and the premature termination codon, as seen in other similar gene mutations (32, 33). Residual C/EBPε message is likely comprised by C/EBPε14 and C/EBPε27 transcripts, which are unaffected by the 5-bp deletion and similar in size to the C/EBPε32 transcript. Transcripts of C/EBPα were present in normal amounts. C/EBPα has a more proximal role in the myelopoietic pathway and specifically induces expression of C/EBPε (31, 34, 35). As expected, message for lactoferrin was not detected in the SGD patient's bone marrow RNA.

As predicted from the C/EBPε transcript maps (Fig. 1 B), immunoblotting detected C/EBPε27 and C/EBPε14 isoforms, but not C/EBPε32, in neutrophils from the SGD patient (Fig. 2 B). All three isoforms were seen in the normal control. The antibody used is specific for a peptide sequence immediately downstream of the 5-bp mutation and should not bind the C/EBPε32-SGD protein.

Transient transfection assays in HeLa cells, using the G-CSF receptor promoter driving the luciferase gene (31), compared the transactivation potentials of the inducer genes C/EBPα, C/EBPε32, C/EBPε14, and C/EBPε32-SGD (Fig. 3). C/EBPε32 has been shown to transactivate the G-CSF receptor promoter, whereas the C/EBPε14 isoform lacks transactivating function (23). Transient transfection of these plasmid constructs showed a significant loss of transactivation with the C/EBPε32-SGD isoform (P = 0.02, Mann-Whitney U test). The demonstrated in vitro data, as well as the in vivo SGD phenotype, mark the full length, 32-kD isoform as the major transactivator encoded in the C/EBPε locus.

The temporal link between granule protein production and myeloid lineage differentiation is well described: primary granule proteins are synthesized in myeloblasts and promyelocytes, secondary granules are produced in myelocytes and metamyelocytes, and tertiary granule proteins are generated in band and segmented neutrophils (36).

Previous work suggested that C/EBPε functions at the terminal stages of myeloid differentiation (23, 26). However, the total absence of patient neutrophil secondary granules and the selective loss of primary granule defensins marks an early myelopoietic block at the promyelocyte transition (Fig. 4). Further evidence for this conclusion comes from in vitro differentiation experiments using C/EBPε-deficient stem cells, which do not proceed beyond the promyelocyte stage (26). Other functional defects seen in mouse and human C/EBPε-deficient neutrophils, such as loss of tertiary granule gelatinase (27) and abnormalities in chemotaxis and cytokine expression (6, 27), may occur secondary to the block at the promyelocyte or later stage.

Functional analysis of the previously developed C/EBPε knockout mouse model (26, 27) was critical for the interpretation of the C/EBPε mutation in SGD. The apparent multiplicity of C/EBPε target genes at different cell stages suggests that C/EBPε transactivates a set of early cell stage– specific genes, inducing normal promyelocyte differentiation and granule development. Additional evidence supporting these conclusions comes from recent observations suggesting that C/EBPε is induced by and transduces the G-CSF signal in neutrophils early in myelopoiesis (37). Absence of secondary granules, defensins, eosinophil cationic protein, eosinophil-derived neurotoxin (12), and platelet α granule high-molecular-mass von Willebrand factor (14) in SGD demonstrates a critical role for C/EBPε in the development of granules and their contents in multiple myeloid lineages.

Acknowledgments

We are grateful to Dr. Helene Rosenberg for providing SGD patient bone marrow RNA and Dr. Mitchell Horwitz for providing normal peripheral blood CD34+ selected cells and expertise.

References

References
1
Spitznagel
JK
,
Cooper
MR
,
McCall
AE
,
DeChatelet
LR
,
Welsh
IRH
Selective deficiency of granules associated with lysozyme and lactoferrin in human polymorphs with reduced microbicidal capacity
J Clin Invest
1972
51
93a
. (Abstr.)
2
Strauss
RG
,
Bove
KE
,
Jones
JF
,
Mauer
AM
,
Fulginiti
VA
An anomaly of neutrophil morphology with impaired function
N Engl J Med
1974
290
478
484
[PubMed]
3
Parmley
RT
,
Ogawa
M
,
Darby
CP
,
Spicer
SS
Congenital neutropenia: neutrophil proliferation with abnormal maturation
Blood
1975
46
723
734
[PubMed]
4
Komiyama
A
,
Morosawa
H
,
Nakahata
T
,
Miyagawa
Y
,
Akabane
T
Abnormal neutrophil maturation in a neutrophil defect with morphologic abnormality and impaired function
J Pediatr
1979
94
19
25
[PubMed]
5
Breton-Gorius
J
,
Mason
DY
,
Bruiot
D
,
Vilde
JL
,
Griscelli
C
Lactoferrin deficiency as a consequence of a lack of specific granule in neutrophils from a patient with recurrent infections
Am J Pathol
1980
99
413
419
[PubMed]
6
Gallin
JI
,
Fletcher
MP
,
Seligmann
BE
,
Hoffstein
S
,
Cehrs
K
,
Mounessa
N
Human neutrophil-specific granule deficiency: a model to assess the role of neutrophil-specific granules in the evolution of the inflammatory response
Blood
1982
59
1317
1329
[PubMed]
7
Boxer
LA
,
Coates
TD
,
Haak
RA
,
Wolach
JB
,
Hoffstein
S
,
Baehner
RL
Lactoferrin deficiency associated with altered granulocyte function
N Engl J Med
1982
307
404
410
[PubMed]
8
Ambruso
DR
,
Sasada
M
,
Nishiyama
H
,
Kubo
A
,
Komiyama
A
,
Allen
RH
Defective bactericidal activity and absence of specific granules in neutrophils from a patient with recurrent bacterial infections
J Clin Immunol
1984
4
23
30
[PubMed]
9
Borregaard
N
,
Boxer
LA
,
Smolen
JE
,
Tauber
AI
Anomolous neutrophil granule distribution in a patient with lactoferrin deficiency
Am J Hematol
1985
18
255
260
[PubMed]
10
Ganz
T
,
Metcalf
JA
,
Gallin
JI
,
Boxer
LA
,
Lehrer
RI
Microbicidal/cytotoxic proteins of neutrophils are deficient in two disorders: Chediak-Higashi Syndrome and “specific” granule deficiency
J Clin Invest
1988
82
552
556
[PubMed]
11
Tamura
A
,
Agematsu
K
,
Mori
T
,
Kawai
H
,
Kuratsuji
T
,
Shimane
M
,
Tani
K
,
Asano
S
,
Komiyama
A
A marked decrease in defensin mRNA in the only case of congenital neutrophil-specific granule deficiency reported in Japan
Int J Hematol
1994
59
137
142
[PubMed]
12
Rosenberg
HF
,
Gallin
JI
Neutrophil-specific granule deficiency includes eosinophils
Blood
1993
82
268
273
[PubMed]
13
Sakura
T
,
Murakami
H
,
Matsushima
T
,
Tamura
J
,
Sawamura
M
,
Tsuchiya
J
Ultrastructure of neutrophilic phagosome of autologous platelet in vivo in specific granule deficiency
Am J Hematol
1993
43
149
150
[PubMed]
14
Parker
RI
,
McKeown
LP
,
Gallin
JI
,
Gralnick
HR
Absence of the largest platelet-von Willebrand multimers in a patient with lactoferrin deficiency and a bleeding tendency
Thromb Haemost
1992
67
320
324
[PubMed]
15
Lomax
KJ
,
Gallin
JI
,
Rotrosen
D
,
Raphael
GD
,
Kaliner
MA
,
Benz
EJ
,
Boxer
LA
,
Malech
HL
Selective defect in myeloid cell lactoferrin gene expression in neutrophil-specific granule deficiency
J Clin Invest
1989
83
514
519
[PubMed]
16
Raphael
GD
,
Davis
JL
,
Fox
PC
,
Malech
HL
,
Gallin
JI
,
Baraniuk
JN
,
Kaliner
MA
Glandular secretion of lactoferrin in a patient with neutrophil lactoferrin deficiency
J Allergy Clin Immunol
1989
84
914
919
[PubMed]
17
Lekstrom-Himes
JA
,
Xanthopoulos
KG
Biological role of the CCAAT/enhancer-binding protein family of transcription factors
J Biol Chem
1998
273
28545
28548
[PubMed]
18
Williams
SC
,
Cantwell
CA
,
Johnson
PF
A family of C/EBP-related proteins capable of forming covalently linked leucine zipper dimers in vitro
Genes Dev
1991
5
1553
1567
[PubMed]
19
Antonson
P
,
Stellan
B
,
Yamanaka
R
,
Xanthopoulos
KG
A novel human CCAAT/enhancer binding protein gene, C/EBPε, is expressed in cells of lymphoid and myeloid lineages and is localized on chromosome 14q11.2 close to the T-cell receptor α/δ locus
Genomics
1996
35
30
38
[PubMed]
20
Chumakov
AM
,
Grillier
I
,
Chumakova
E
,
Chih
D
,
Slater
J
,
Koeffler
HP
Cloning of the novel human myeloid-cell-specific C/EBPε transcription factor
Mol Cell Biol
1997
17
1375
1386
[PubMed]
21
Koike
M
,
Chumakov
AM
,
Takeuchi
S
,
Tasaka
T
,
Yang
R
,
Nakamaki
T
,
Tsuruoka
N
,
Koeffler
HP
C/EBP-ε: chromosomal mapping and mutational analysis of the gene in leukemia and preleukemia
Leuk Res
1997
21
833
839
[PubMed]
22
Williams
SC
,
Du
Y
,
Schwartz
RC
,
Weiler
SR
,
Ortiz
M
,
Keller
JR
,
Johnson
PF
C/EBPε is a myeloid-specific activator of cytokine, chemokine and macrophage-colony-stimulating factor receptor genes
J Biol Chem
1998
22
13493
13501
[PubMed]
23
Yamanaka
R
,
Kim
G-D
,
Rodomska
HS
,
Lekstrom-Himes
J
,
Smith
LT
,
Antonson
P
,
Tenen
DG
,
Xanthopoulos
KG
CCAAT/enhancer binding protein ε is preferentially up-regulated during granulocytic differentiation and its functional versatility is determined by alternative use of promoters and differential splicing
Proc Natl Acad Sci USA
1997
94
6462
6467
[PubMed]
24
Ossipow
V
,
Descombes
P
,
Schibler
U
CCAAT/enhancer-binding protein mRNA is translated into multiple proteins with different transcription activation potentials
Proc Natl Acad Sci USA
1993
90
8219
8223
[PubMed]
25
Descombes
P
,
Schibler
U
A liver-enriched transcriptional activator protein, LAP, and a transcriptional inhibitory protein, LIP, are translated from the same mRNA
Cell
1991
67
569
579
[PubMed]
26
Yamanaka
R
,
Barlow
C
,
Lekstrom-Himes
J
,
Castilia
L
,
Liu
P
,
Eckhaus
M
,
Decker
T
,
Wynshaw-Boris
A
,
Xanthopoulos
KG
Impaired granulopoiesis, myelodysplasia, and early lethality in C/EBPε deficient mice
Proc Natl Acad Sci USA
1997
94
13187
13192
[PubMed]
27
Lekstrom-Himes
JA
,
Xanthopoulos
KG
C/EBPε is critical for effective neutrophil-mediated response to inflammatory challenge
Blood
1999
93
3096
3105
[PubMed]
28
Kuhns
DB
,
Young
HA
,
Gallin
EK
,
Gallin
JI
Ca2+-dependent production and release of IL-8 in human neutrophils
J Immunol
1998
161
4332
4339
[PubMed]
29
Dorman
SE
,
Holland
SM
Mutation in the signal-transducing chain of the interferon γ receptor and susceptibility to mycobacterial infection
J Clin Invest
1998
101
2364
2369
[PubMed]
30
Laird
PW
,
Zijderveld
A
,
Linders
K
,
Rudnicki
MA
,
Jaenisch
R
,
Berns
A
Simplified mammalian DNA isolation procedure
Nucleic Acids Res
1991
19
4293
[PubMed]
31
Smith
LT
,
Hohaus
S
,
Gonzalez
DA
,
Dziennis
SE
,
Tenen
DG
PU.1 (Spi-1) and C/EBP alpha regulate the granulocyte colony-stimulating factor receptor promoter in myeloid cells
Blood
1996
88
1234
1247
[PubMed]
32
Hovnanian
A
,
Rochat
A
,
Bodemer
C
,
Petit
E
,
Rivers
CA
,
Prost
C
,
Fraitag
S
,
Christiano
AM
,
Uitto
J
,
Lathrop
M
et al
Characterization of 18 new mutations in COL7A1 in recessive dystrophic epidermolysis bullosa provides evidence for distinct molecular mechanisms underlying defective anchoring fibril formation
Am J Hum Genet
1997
61
599
610
[PubMed]
33
Christiano
AM
,
Amano
S
,
Eichenfield
LF
,
Burgeson
RE
,
Uitto
J
Premature termination codon mutations in the type VII collagen gene in recessive dystrophic epidermolysis bullosa result in nonsense-mediated mRNA decay and absence of functional protein
J Invest Dermatol
1997
109
390
394
[PubMed]
34
Zhang
D-E
,
Zhang
P
,
Wang
N-D
,
Hetherington
CJ
,
Darlington
GJ
,
Tenen
DG
Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein alpha-deficient mice
Proc Natl Acad Sci USA
1997
94
569
574
[PubMed]
35
Radomska
HS
,
Huettner
CS
,
Zhang
P
,
Cheng
T
,
Scadden
DT
,
Tenen
DG
CCAAT/enhancer binding protein alpha is a regulatory switch sufficient for induction of granulocytic development from bipotential myeloid progenitors
Mol Cell Biol
1998
18
4301
4314
[PubMed]
36
Borregaard
N
,
Sehested
M
,
Nielsen
BS
,
Sengelov
H
,
Kjeldsen
L
Biosynthesis of granule proteins in normal human bone marrow cells. Gelatinase is a marker of terminal neutrophil differentiation
Blood
1995
85
812
817
[PubMed]
37
Nakajima
H
,
Cleveland
JL
,
Nagata
S
,
Ihle
JN
Granulocyte colony-stimulating factor regulates myeloid differentiation through CCAAT enhancer binding protein ε
Blood
1998
92
712a
[PubMed]

Author notes

Address correspondence to John I. Gallin, Bldg. 10, Rm. 2C146, 10 Center Dr. MSC 1504, Bethesda, MD 20892-1504. Phone: 301-496-4114; Fax: 301-402-0244; E-mail: jgallin@cc.nih.gov