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December 2002
Overexpression
of the BAFF gene in Sjögren’s syndrome
Symptoms of dry mouth and dry eyes caused by malfunctioning exocrine
glands, such as salivary and lacrimal glands, are characteristic
of the chronic inflammatory disorder Sjögren’s syndrome. This autoimmune
disease is characterized by large mononuclear cell infiltrates in
exocrine glands, B-cell hyperreactivity, and various serum autoantibodies.
Sjögren’s syndrome can develop alone or in association with other
autoimmune diseases. Abnormal B-cell activity is a predominant feature
of the condition, which is manifested by polyclonal B-cell activation
and elevated secretion of autoantibodies, such as rheumatoid factors,
anti-Ro (SS-A), anti-La (SS-B), and anti-α-fodrin autoantibodies.
However, the role of B cells and autoantibodies in the pathogenesis
of the condition remains unclear. The gene BAFF is a powerful modulator
of B-cell biology expressed by monocytes/macrophages and dendritic
cells. Expression of BAFF is B-cell specific. Overproduction of
BAFF is known to be associated with the development of certain autoimmune
diseases. Transgenic (Tg) mice with BAFF mutation have an elevated
number of B cells in the periphery, secrete various autoantibodies,
and develop a systemic lupus erythematosus-like condition. Excess
BAFF-mediated survival signals might compromise the ability of autoreactive
B cells to respond to censoring death signals; therefore, abnormal
BAFF production may be a key event in autoimmunity. Serum BAFF levels
are significantly higher in patients with SLE and rheumatoid arthritis
than in healthy individuals. The authors described the findings
in BAFF Tg mice. As the mice age, they develop a condition secondary
to their lupus-like disease and some perceived similarities to Sjögren’s
syndrome in humans. The animals show severe sialadenitis, decreased
saliva production, and destruction of submaxillary glands. Sjögren’s
syndrome in humans also correlates with elevated levels of circulating
BAFF and dramatic upregulation of BAFF expression in inflamed salivary
glands. Marginal zone B cells, one of the B cell subsets in the
spleen of BAFF Tg mice, is a potential reservoir of autoreactive
B cells. B cells with a marginal zone-like phenotypic pattern infiltrate
the salivary glands of the BAFF Tg mice. The authors concluded that
altered B cell differentiation and tolerance induced by excess BAFF
may be a central component of the pathogenesis of Sjögren’s syndrome.
Groom J, Kalled SL, Cutler AH, et al. Association
of BAFF/BLyS overexpression and altered B cell differentiation with
Sjögren’s syndrome. J Clin Invest. 2002; 109: 59-68.
Reprints: Fabienne Mackay, The Garvan Institute of Medical Research,
384 Victoria St., Darlinghurst NSW 2010, Australia; f.mackay@garvan.unsw.edu.au
Storage artifact
of clinical hematology specimens
Dyserythropoiesis occurs in a variety of conditions, including megaloblastic
anemias, iron deficiency, thalassemias, congenital anemias, infections,
toxic exposure, and preleukemic states. Observation of dyserythropoiesis
using the French-American-British classification scheme is important
in bone marrow interpretation. Nonetheless, dyserythropoiesis may
be spurious and an artifact of storage in clinical specimens. One
must be aware of this artifact to avoid erroneous diagnosis of myelodysplastic
syndrome. The authors documented the occurrence and severity of
dyserythropoiesis as an artifact of storage of clinical hematology
specimens. They studied bone marrow aspirates collected in EDTA
obtained from seven patients without myelodysplasia. These specimens
were stored at room temperature (20 to 24°C) or refrigerated temperature
(1 to 6°C) and examined for dyserythropoiesis at zero, one, two,
and three days of storage. The initial specimens showed few abnormalities,
but nuclear aberrations occurred in about one percent of the erythroid
population. When stored at room temperature, dyserythropoietic changes
increased significantly with each day of storage. Nuclear and cytoplasmic
aberrations also occurred. The cytoplasmic changes were more extensive
than the nuclear abnormalities. The mean percentage of erythroblasts
with cytoplasmic vacuoles increased by day of storage as follows:
day zero, ≈1.1 percent; day one, ≈22.2 percent; day
two, ≈29.4 percent; day three, ≈35.6 percent. On day
one, nuclear shaped changes increased to ≈6.21 percent, on
day two to ≈11.36 percent, and on day three to ≈12.85
percent. The authors found that after one day of storage at room
temperature, sufficient dysplastic changes occur to cause difficulty
in diagnosing myelodysplastic syndrome. These changes appear to
be inhibited significantly by refrigerated storage.
Wang LJ, Glasser L. Spurious dyserythropoiesis.
Am J Clin Pathol. 2002;117:57-59.
Reprints: Dr. Lewis Glasser, Dept. of Pathology, Rhode Island Hospital,
593 Eddy St., Providence, RI 02903
Maternal serum assays
for activin A and inhibin A in trisomy 18 pregnancies
The triple screen, involving assays for maternal serum α-fetoprotein,
total human chorionic gonadotropin, and unconjugated oestriol, detects
60 percent of trisomy 18 pregnancies (false-positive rate, 0.2 to
0.7 percent). Levels of maternal serum pregnancy-associated plasma
protein-A (PAPP-A) and free β-hCG are reduced in such pregnancies.
And fetal nuchal translucency thickness algorithms have been developed
that, along with the PAPP-A and free β-hCG measurements, identify
90 percent of cases (false-positive rate, one percent). Controversy
surrounds the value of adding inhibin A measurement to second trimester
screening protocols for trisomy 21. Limited data suggest that inhibin
A cannot discriminate trisomy 18 aneuploidy in the second trimester
of pregnancy. Likewise, activin has been found in various small
studies to be a poor discriminator of trisomy 21. The authors of
this study evaluated whether maternal serum levels of inhibin A
and total activin A are altered in the first trimester of pregnancies
involving trisomy 18. They studied 45 cases of trisomy 18 and 493
control pregnancies. The pregnancies were examined at 10 to 14 weeks
of gestation. The authors measured serum inhibin A, total activin
A, free β-hCG, and PAPP-A. The median values in the trisomy
18 pregnancies were 0.74 MoM for inhibin A, 1.23 MoM for activin
A, 0.38 MoM for free β-hCG, and 0.16 MoM for PAPP-A. The levels
of inhibin and activin deviated from normal only slightly in comparison
with those for free β-hCG and PAPP-A. The authors concluded
that adding these two assays to the screening for trisomy 18 is
not likely to further improve the sensitivity of screening.
Spencer K, Liao AW, Ong CYT, et al. Maternal serum
activin A and inhibin A in trisomy 18 pregnancies at 10-14 weeks.
Prenat Diagn. 2001;21:571-574.
Reprints: Kevin Spencer, Endocrine Unit, Clinical Biochemistry
Dept., Harold Wood Hospital, Gubbins Lane, Romford, Essex RM3 OBE,
United Kingdom; kevinspencer1@cs.com
A prospective longitudinal
study of gene polymorphisms in rheumatoid arthritis
HLA and non-HLA genes contribute to the pathogenesis of rheumatoid
arthritis. The presence and dose effect of HLA-DRB1 alleles encoding
the shared epitope affect the course and outcome of the disease.
Estimates of the HLA component of overall genetic risk for RA are
less than 50 percent. Identifying new susceptibility and severity
genes for RA is an important challenge being addressed via genome-wide
screening or candidate gene approach. Matrix metalloproteinases
(MMP) have been implicated in connective tissue destruction and
remodeling associated with various chronic disease conditions. The
expression of these metalloproteinases is regulated at the transcription
level by a variety of factors. Collagenase-1 (MMP-1) can degrade
the interstitial collagens types I, II, and III. It is produced
by synoviocytes and chondrocytes. The level of MMP-1 is elevated
in the plasma and synovial fluid of patients with RA. The authors
undertook a prospective longitudinal study to test the hypothesis
of an association between the MMP-1 gene polymorphism and susceptibility
to and severity of RA. They used the candidate gene approach in
a cohort of 103 patients diagnosed early with RA and followed for
four years. They also investigated the association between HLA-DRB1
gene polymorphism and the severity of RA. The authors used a radiographic
damage score to quantify disease severity at baseline and after
four years of followup in each patient. They used fluorescent-based
polymerase chain reaction to analyze MMP-1 polymorphism genotyping.
And they used PCR sequence-specific oligonucleotide probes for HLA-DRB1
genotyping. These assays were also performed on 133 healthy control
subjects. The authors found that the MMP-1 allele and genotype frequencies
did not differ between RA patients and controls. Radiographic damage
or its progression over four years of followup did not differ across
MMP-1 genotypes. The radiographic damage score and its progression
over four years did, however, differ across HLA-DRB1 genotypes.
The HLA-DRB1 shared epitope +/+ genotype was associated with the
highest radiographic damage score, while the shared epitope -/-
genotype was associated with the lowest. The authors concluded that
their results do not support the hypothesis of an association between
the MMP-1 gene promoter polymorphism and susceptibility to or severity
of RA.
Constantin A, Lauwers-Cancès V, Navaux F, et al.
Collagenase-1 (MMP-1) and HLA-DRB1 gene polymorphisms in rheumatoid
arthritis: a prospective longitudinal study. J Rheum. 2002;29:15-20.
Reprints: A. Cantagrel, Service de Rhumatologie, CHU Rangueil,
31403 Toulouse Cedex 4, France; cantagre@cict.fr
Karyotype followup
on maternal serum screening for Down syndrome
Several fetal chromosome anomalies, in addition to Down syndrome,
are found in populations that are subjected to maternal serum screening,
including trisomy 18, Turner Syndrome, and other aneuploidies. It
is not clear which aneuploidies are concentrated in the group at
increased risk for Down syndrome and which are present by chance.
The authors used the databases of the South Australian State Neonatal
Screening Programme, South Australian Maternal Serum Antenatal Screening
(SAMSAS) Programme, and other sources to create a complete list
of abnormal karyotypes present in the pregnancies screened at increased
risk of fetal Down syndrome. There were 65,328 pregnancies screened
by the SAMSAS Programme from 1991 through 1997. Of these, 5.25 percent
were declared at increased risk of fetal Down syndrome, and 79.8
percent, including 16 with early fetal loss, had fetal or neonatal
karyotypes determined. Overall, 129 instances of abnormal karyotype
were found, including 84 Down syndrome, four trisomy 18, three trisomy
13, two triploidy, six female sex chromosome aneuploidy and five
male sex chromosome aneuploidy, 19 inherited balanced rearrangements,
four mosaic or de novo balanced abnormalities, and two unbalanced
karyotypes. Of these, only the karyotype for Down syndrome occurred
in the pregnancies at increased risk for fetal Down syndrome with
a frequency greater than that expected for the general pregnant
population.
Ryall RG, Callen D, Cocciolone R, et al. Karyotypes
found in the population declared at risk of Down syndrome following
maternal serum screening. Prenat Diagn. 2001;21:553-557.
Reprints: R.G. Ryall, Dept. of Chemical Pathology, Women’s & Children’s
Hospital, North Adelaide, South Australia 5006, Australia; ryallr@wch.sa.gov.au
New modification
of the Lancefield classification for group A streptococci
The Lancefield classification system for typing group A streptococci
was developed in 1928. It is based on variable antigenic properties
of protein M on the surface of the organisms. The authors reported
on an exchange of strains between six international group A streptococci
(GAS) reference laboratories that resulted in 22 additional sequence
types designated emm103 to emm124 being added to the
M protein gene sequence types. Continued expansion of the serology-based
Lancefield classification scheme for GAS has become difficult during
the past 20 years. Using a new sequence-based methodology that adheres
to previously established strain criteria, while being predictive
of known M protein serotypes, has permitted types emm94 to
emm102 to be added to the Lancefield scheme. Continued expansion
through the addition of emm103 to emm124 is now proposed.
As with emm94 to emm102, each of these new emm
types was represented by multiple independent isolates recovered
from serious disease manifestations. Each of these was nontypeable
with all M protein typing sera stocks available to international
GAS reference laboratories. And each demonstrated antiphagocytic
properties in vitro by multiplying in normal human blood.
Facklam RF, Martin DR, Lovgren M, et al. Extension
of the Lancefield classification for group A streptococci by addition
of 22 new M protein gene sequence types from clinical isolates: emm103
to emm124. Clin Infect Dis. 2002;34:28-38.
Reprints: Dr. Bernard Beall, Centers for Disease Control and Prevention,
Mail Stop C02, 1600 Clifton Rd. NE, Atlanta, GA 30333; bbeall@cdc.gov
An assay for determining
anti-acetylcholine receptor antibodies
The antigenic target in the autoimmune disorder myasthenia gravis
is the nicotinic acetylcholine receptor (nAChR) of skeletal muscles.
Diagnosing and monitoring this disease involves detecting and quantifying
anti-acetylcholine receptor antibodies in serum. The most commonly
used assay for this purpose is the immunoprecipitation assay using
iodine125 labeled α-bungarotoxin (125I-α-BuTx).
This is a valid, specific, and widely used assay; however, it is
highly desirable to reduce the use of radioactivity in clinical
laboratories. High-sensitivity detection of lanthanides uses time-resolved
fluorescence methods. The authors developed a nonradioactive immunoassay
using Eu3+-labeled α-cobratoxin and time-resolved
fluorescence. They derivatized α-CTx with a diethylenetriaminepentaacetate
moiety and formed a complex with Eu3+. This complex was
purified by high-pressure liquid chromatography and the fractions
tested for receptor binding. The Eu3+-labeled α-CTx
competed with 125I-α-BuTx for binding to nicotinic
acetylcholine receptors and saturated the binding sites of human
acetylcholine receptors. The results of the immunoassays performed
with Eu3+-labeled α-CTx were similar to those obtained
with 125I-α-BuTx, with a slightly higher limit of
detection (0.3 nmol/L versus 0.1 nmol/L for the isotopic assay).
None of 34 negative sera tested gave a value greater than 0.3 nmol/L.
Thirty-five positive myasthenic sera were compared using the two
assays, and 32 tested positive with the Eu3+ assay. Linear
regression analysis yielded the equation y=1.035x-0.013
nmol/L; Sy/x=0.172 nmol/L; and r2=0.977. The
authors concluded that this new time-resolved fluorescence assay
is comparable to the widely used isotopic assay.
Ricny J, Simkova L, Vincent A. Determination of
anti-acetylcholine receptor antibodies in myasthenic patients by
use of time-resolved fluorescence. Clin Chem. 2002;48:549-554.
Jan Ricny, Institute of Physiology, Academy of
Sciences of Czech Republic. Videoska 1083, 142 20 Prague, Czech
Republic; ricny@biomed.cas.cz
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