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November 2005
Feature Story
Gamma-heavy chain disease
The heavy chain diseases represent a proliferative process of lymphoplasma
cells involving B cells and are characterized by the production of incomplete
heavy chains devoid of light chains. They have been described for the three
main immunoglobulin classes. The most frequent is a-heavy chain disease. The
incidence of m-heavy chain disease is rare, and the incidence of g-heavy chain
disease is intermediate. Since the first description of g-heavy chain disease
in 1964, approximately 100 cases have been reported in the literature. Several
reports have called attention to the heterogeneity of g-heavy chain disease,
even though the disease resembles a lymphoma-like illness, with lymphadenopathy,
spleno megaly, and hepatomegaly in association with a lymphoplasmacytic proliferation
similar to that of Waldenström macroglobulinemia. The authors reported
on 23 patients from a single institution (eight previously published, 15 new
patients) and emphasized the diversity of the clinical manifestations and hematopathologic
features of g-heavy chain disease. The study involved 15 women and eight men
and the median age at diagnosis was 68 years (range, 42 to 87 years). Sixteen
patients had an associated lymphoplasma cell proliferative disorder, three had
a lymphoplasma cell proliferative disorder and an autoimmune disorder, three
had an autoimmune disorder only, and one had no underlying disease. The lymphoplasma
cell proliferative disorder was disseminated in 10 patients and localized in
six. Patients with localized lymphoplasma cell proliferative disorder included
three with plasmacytoma (one tongue, one submandibular area, and one thyroid),
two with lymphoplasma cell proliferative disorder involving bone marrow only,
and one with amyloid of the skin. At diagnosis, lymphadenopathy was present
in eight patients, splenomegaly in seven, and hepatomegaly in one. The authors
documented a monoclonal spike on serum protein electrophoresis in 19 patients.
Gamma-heavy chain was documented by immunofixation in the serum of all patients;
two had an additional immunoglobulin M-l. Gamma-heavy chain was present in the
urine in 19 of 22 patients. Sixteen patients were treated for lymphoplasma cell
proliferative disorder or autoimmune disorder (14 chemotherapy, one splenectomy,
and one thyroidectomy followed by radiation therapy). Treatment was not considered
necessary for five patients, and two patients were too sick for treatment. Of
the 16 patients treated, six had a complete clinical response (in two, g-heavy
chain disappeared; in two, it persisted; and in two, no serologic followup was
available); in 10 patients, clinical disease persisted (in three, g-heavy chain
disappeared; in six, it persisted; and for one, no serologic followup was available).
Of seven patients not treated, two died within five months; one died after 15
months; two had no clinical disease at latest followup, although g-heavy chain
persisted; and two had no change in clinical and serologic status. The median
duration of followup was 33 months (range, one to 26 months), and the median
survival was 7.4 years.
Wahner-Roedler DL, Witzig TE, Loehrer LL, et al. g-heavy chain disease:
review of 23 cases. Medicine. 2003;236–250.
Reprints: Dr. D.L. Wahner-Roedler, Division of Area General Internal Medicine,
Mayo Clinic, 200 First St. SW, Rochester, MN 55905
Urine antigen tests for pneumococcal pneumonia
The development of tests that can rapidly and reliably identify the causative
agent in community-acquired pneumonia has been encouraged. Urine is a suitable
medium for microbiological assays aimed at respiratory pathogens because it
is not influenced by respiratory flora and can be easily collected from most
patients. An in-house serotype-specific latex agglutination test comprising
22 solution reagents for 23 different serotypes was developed in the streptococcus
unit at Statens Serum Institut, Copenhagen, Denmark. The pneumococcal C polysaccharide
can be detected in urine using the commercial Binax Now immunochromatographic
test (ICT) for Streptococcus pneumoniae. This quick test has been recommended
for diagnostic use by the Infectious Diseases Society of America. So far, however,
no prospective study with nonconcentrated urine has evaluated the clinical utility
of dividing visible ICT results lines according to color intensity. The authors’
study compared the performances of the latex agglutination test and ICT, with
visible ICT result lines being divided into two groups according to color intensity,
by use of nonconcentrated urine samples from adult patients with community-acquired
pneumonia and controls. ICT was considered to be strongly positive for result
lines at least as intense as the control line and weakly positive for less intense
result lines. When 215 adult patients with community-acquired pneumonia were
tested, strong ICT, weak ICT, and latex agglutination positivity were found
in 28, 24, and 16 patients, respectively. Of these patients, 13 (46 percent),
six (25 percent), and 13 (81 percent), respectively, had pneumococcal bacteremia,
and 27 (96 percent), 17 (71 percent), and 15 (94 percent), respectively, had
S. pneumoniae isolated from blood, sputum, and/or nasopharynx. Among 108 controls
tested, two (1.9 percent) were weakly ICT positive. When weak positivity was
considered negative, the sensitivity of ICT decreased from 79 percent (19 of
24) to 54 percent (13 of 24), while the specificity increased from 83 percent
(158 of 191) to 92 percent (176 of 191); no controls were false positive. The
sensitivity and specificity of latex agglutination were 54 percent (13 of 24)
and 98 percent (188 of 191), respectively. Eight of nine latex agglutination
serotypes corresponded to culture serotypes. The authors concluded that using
nonconcentrated urine and dividing ICT-positive results into strongly and weakly
positive results is a suitable way of performing ICT. While weak ICT positivity
should be interpreted with caution, strong ICT positivity and latex agglutination
positivity should be considered supportive of pneumococcal etiology in adult
community-acquired pneumonia. As such, these assays might have implications
for antibiotic use in this type of pneumonia. Latex agglutination shows potential
for pneumococcal serotyping, although further evaluation is required.
Strålin K, Kaltoft MS, Konradsen HB, et al. Comparison of two urinary
antigen tests for establishment of pneumococcal etiology of adult community-acquired
pneumonia. J Clin Microbiol. 2004;42:3620–3625.
Reprints: Kristoffer Strålin, Dept. of Infectious Diseases, Örebro
University Hospital, SE-70185 Örebro, Sweden; kristoffer.stralin@ore broll.se
Fibrinogen degradation products and cerebral hemorrhage
Intracranial hemorrhage after cerebral thrombolysis is the major complication
of this type of treatment, especially with intravenous streptokinase, but also
with intra-arterial prourokinase and intravenous rt-PA. In double-blind studies
with intravenous rt-PA (NINDS, ECASS I and II, ATLANTIS), predictive factors
of these bleedings included severity at entry, brain edema, early ischemic changes
on CT, age, and aspirin use. Phase IV studies mentioned the role of protocol
violations and hyperglycemia. However, coagulation factors have not been taken
into account in all these trials. In 2000, after a prospective study of coagulation
parameters in the Lyon intravenous rt-PA trial, the authors described a biological
syndrome predictive of cerebral bleeding related to an early coagulopathy and
characterized by an increase of fibrin(ogen) degradation products (FDP) two
hours after the beginning of thrombolysis. In the present study of 157 patients,
they showed that this early coagulopathy is exclusively predictive of early
parenchymal hematomas at less than 24 hours and is the major and single factor
of their occurrence. The authors included consecutive patients in the Lyon rt-PA
protocol. Early hematomas (within 24 hours) diagnosed on an anatomoradiological
basis (symptomatic and not symptomatic) were considered for the study. Fibrinogen
and fibrin(ogen) degradation products were assessed at entry and at two and
24 hours after the beginning of thrombolysis. Of the 157 patients, 11 had early
parenchymal hematomas (seven percent), 31 had early hemorrhagic infarcts (19.7
percent), and 115 had no bleeding (73.2 percent). In logistic regression, FDP
at two hours was the single predictor of parenchymal hematomas. Early parenchymal
hematomas were indicative of a poor prognosis at three months (P=0.001). The
authors concluded that early parenchymal hematomas appear as both “malignant”
and exclusively related to an explosive increase of FDP at two hours—that
is, an early fibrinogen degradation coagulopathy (EFDC). All patients scheduled
to rt-PA thrombolysis should have an assay of FDP two hours after the beginning
of thrombolysis. Patients with an established EFDC (FDP greater than 200 mg/L)
should be monitored specifically, and they should not receive an antithrombotic
drug during the first 72 hours. Patients with FDP greater than 100 mg should
share the same monitoring.
Trouillas P, Derex L, Philippeau F, et al. Early fibrinogen degradation
coagulopathy is predictive of parenchymal he ma to mas in cerebral rt-PA thrombolysis.
A study of 157 cases. Stroke. 2004;35: 1323–1328.
Reprints: Dr. Paul Trouillas, Cerebrovascular Unit, Hôpital Neurologique,
59 Boulevard Pinel, 69003 Lyon, France; paul.trouillas @chu-lyon.fr
Role of magnesium levels in preeclampsia
The exact incidence of gestational hypertension-preeclampsia in the United
States is unknown. Estimates indicate that five percent to eight percent of
all pregnant women will have preeclampsia, defined as hypertension and proteinuria
beginning during the second half of gestation. Pre eclampsia may also be associated
with increased neuromuscular irritability and seizures. Interestingly, neuromuscular
excitability, vasoconstriction, elevated blood pressure, and increased vascular
sensitivity to pressor agents are also characteristic of magnesium (Mg) depletion.
The therapeutic use of intravenous Mg sulfate is universal, at least in the
United States, for women with mild preeclampsia to prevent eclampsia seizures.
Its effectiveness was confirmed in a recent meta-analysis showing that parental
Mg more than halves the risk of eclampsia. However, the role of Mg deficiency
in the pathophysiology of pre eclamp sia has not been clearly established, and
dietary Mg supplementation does not seem to prevent subsequent incidences of
preeclampsia. The authors’ group developed the use of 31P nuclear magnetic
resonance (NMR) spectroscopic techniques to noninvasively measure intracellular-free
magnesium (Mgi) content in a variety of clinical disease states, including hypertension,
where Mgi levels were closely and inversely related to the height of blood pressure.
The authors used 31P NMR spectroscopy to noninvasively measure in situ intracellular-free
magnesium levels in the brain and skeletal muscle of fasting nonpregnant women
(n=12) and third trimester women with uncomplicated pregnancies (n=11) and preeclampsia
(n=7). Compared with the nonpregnant controls (brain, 519±59 µmol/L;
muscle, 604±34 µmol/L), brain and skeletal muscle intracellular
magnesium levels were significantly lower in the normal pregnant (brain, 342±23
µmol/L; muscle, 482±40 µmol/L; P=0.05 for both tissues) and
preeclamptic women (brain, 229±17 µmol/L; muscle, 433±46
µmol/L; P=0.05 for both tissues). Brain intra-cellular magnesium levels
were further reduced in the preeclamptic women compared with normal pregnant
subjects (P=0.05). For all of the pregnant women, blood pressure was significantly
and inversely related to the concomitantly measured intra-cellular magnesium
level in the brain (systolic, r=–0.59, P=0.01; diastolic, r=–0.52,
P=0.02) but not in muscle. The authors concluded that cellular magnesium depletion
is characteristic of normal pregnancy and may contribute to the pathophysiology
of preeclampsia. Furthermore, the influence of central nervous system factors
on blood pressure may be mediated, at least in part, by ambient intra-cellular
magnesium levels.
Resnick LM, Barbagallo M, Bardicef M, et al. Cellular-free magnesium depletion
in brain and muscle of normal and preeclamptic pregnancy. A nuclear magnetic
resonancy spectroscopic study. Hypertension. 2004;44: 322–326.
Reprints: Dr. Mario Barbagallo, chair of geriatrics, University of Palermo,
Via F Scaduto 6/c, 90144 Palermo, Italy; mabar@ unipa.it
MHC locus in islet cell transplantation
Type I diabetes is a multifactorial autoimmune disease directed against antigen
of the insulin-producing b-cells. Macrophage, dendritic-cell (DC), B-cell, and
T-cell populations contribute to disease pathogenesis. The non-obese diabetic
(NOD) mouse develops a spontaneous form of diabetes with many etiologic and
pathogenic similarities to the human disease. More than 20 diabetes-susceptibility
(insulin-dependent diabetes [Idd]) loci have been identified, but the Idd-1
locus encoding the major histocompatibility complex (MHC) genes remains the
most significant risk factor. The mechanisms by which MHC genes mediate susceptibility
to autoimmunity remain poorly understood. The authors’ previous studies
demonstrated indefinite survival of b2mnull syngeneic islet grafts in diabetic
NOD mice, suggesting a primary role for MHC class I molecules on islet cells
in causing autoimmune destruction or recurrent diabetes in transplanted islets.
To address the role of MHC class I/II within islets, the authors created and
used NOD. b2mnull. CIITAnull mice. They reported that the lack of MHC class
II expression does not impact islet graft survival, which does not abrogate
the benefit of class I deletion and confirms that MHC class I has an essential
role in causing recurrent diabetes. Furthermore, the Th cytokine profile is
mixed and similar regardless of MHC expression on islet grafts. These findings
clarify the respective roles for MHC class I and class II as well as CD8+ and
CD4+ T cells in islet destruction. Donor islets from NOD mice deficient in one
or both of b2-microglobulin and class II transactivator genes were transplanted
into diabetic NOD mice. Immunohistochemistry was performed to identify the phenotype
of infiltrating cells and to assess graft insulin production. The presence of
cytokines in the grafts was assayed by reverse transcription polymerase chain
reaction. MHC class II-null islets demonstrated rates of rejection comparable
with control wild-type (wt) islets. In contrast, MHC class I- and II-null islets
demonstrated indefinite survival (more than 100 days). Infiltrates of both failed,
and surviving grafts were composed of cytotoxic lymphocytes, helper T cells,
and macrophages. Grafts also showed the presence of Th1- and Th2-type cytokines
(interleukin [IL]-2, IL-4, IL-10, and interferon-g), independent of graft status.
The authors concluded that MHC class I molecules are of primary importance in
the pathogenesis of diabetes recurrence post-islet transplantation. Conversely,
MHC class II expression is not a necessary mechanistic component of transplant
destruction. In addition, these results implicate MHC class I-restricted cytotoxic
lymphocytes, but not MHC class II-restricted T cells, in disease recurrence.
Young HY, Zucker P, Flavell RA, et al. Characterization of the role of
major histocompatibility complex in type I diabetes recurrence after islet transplantation.
Transplantation. 2004;78:509–515.
Reprints: Dr. Bhagirath Singh, Dept. of Microbiology and Immunology, Dental
Sciences Bldg., University of Western Ontario, London, Ontario, N6A 5C1, Canada;
bsingh@uwo.ca
Glucose, insulin, and gene transcription
Pancreatic islet b-cells can be regulated by multiple stimuli, including nutrients
and growth factors. b-cell proliferation and function are controlled by plasma
glucose concentration and growth factors acting via multiple intracellular signaling
pathways. Changes in gene expression that result from the activation of these
signaling pathways are likely responsible for the adaptation of b-cells to physiological
and pathological states. However, much is yet unknown about the changes in gene
expression and the molecular mechanisms mediating these b-cell responses to
nutrients and growth factors. Several immediate early genes have been shown
to be rapidly induced by glucose-activated depolarization in islet b-cells.
Current studies address aspects of glucose-regulated transcription: the number
and characteristics of these genes, if depolarization is the major mechanism,
and if glucose-stimulated insulin secretion is responsible because insulin,
per se, can activate transcription. The authors of this study assessed by endocrine
pancreas cDNA microarrays the expression profiles of glucose-responsive insulinoma
cells 45 minutes after adding glucose, KC1 to induce depolarization, or insulin.
Glucose activated more than 90 genes, representing diverse gene ontology functions,
and most were not previously known to be glucose responsive. KC1 activated 80
percent of these same glucose-regulated genes and, along with the effects of
pretreatment with diazoxide, suggested that glucose signaling is mediated primarily
via depolarization. More than 150 genes were activated by insulin, and 71 percent
were also regulated by glucose. Preincubation with a phosphatidylinositol (PI)
3-kinase inhibitor resulted in almost total inhibition of depolarization and
insulin-activated transcriptional responses. Thus, through gene expression profiling,
these data demonstrate that glucose and insulin rapidly activate a PI 3-kinase
pathway, resulting in transcription of a common set of genes. This is consistent
with glucose activation of gene transcription directly or indirectly through
a paracrine/autocrine effect via insulin release. These results illustrate that
expression gene profiling can contribute to the elucidation of important b-cell
biological functions.
Ohsugi M, Cras-Méneur C, Zhou Y, et al. Glucose and insulin treatment
of insulinoma cells results in transcriptional regulation of a common set of
genes. Diabetes. 2004;53:1496–1508.
Reprints: Dr. M. Alan Permutt, Division of Endocrinology, Metabolism, and
Lipid Research, Washington University School of Medicine, 660 S. Euclid Ave.,
Campus Box 8127, St. Louis, MO 63110; apermutt@ im.wustl.edu
A new biomarker for West Nile virus infection
West Nile virus is an emerging and significant public health problem. Since
1999, the United States has experienced annual epidemics of disease in humans
and animals caused by West Nile virus (WNV) over an expanding geographic range.
Outbreaks of the disease with neurological manifestations have also been reported
in Eastern Europe, North Africa, and Israel since the mid-1990s. More specific
rapid diagnostic assays must be developed to limit the impact of the virus,
which is a member of the Japanese encephalitis virus group of the genus Flavivirus,
family Flaviviridae. Other members include Japanese encephalitis virus, found
throughout Asia; St. Louis encephalitis virus, found in the Americas; and Murray
Valley encephalitis virus, found in Australia and New Guinea. These viruses
have a similar ecology and are antigenically related to WNV. Their co-circulation
in several regions of the world has complicated the specific diagnosis of infections
by these viruses in humans and other vertebrate hosts. Cross-reactions in patients
ultimately diagnosed with probable dengue virus infections have also been reported
in evaluations of WNV testing assays. The authors previously reported the identification
of WNV-specific neutralizing epitopes within structural domain III of the WNV
envelope (E) protein. Earlier investigations with other flaviviruses have also
reported the presence of virus-specific epitopes within this region of the E
protein, and other authors have suggested the utility of domain III from dengue
virus types 1 to 4 or Japanese encephalitis virus as antigens for specific serological
diagnosis of infections with those flaviviruses. These observations led the
authors to investigate the utility of a recombinant, bacterially expressed domain
III (r-EIII) antigen derived from the envelope protein of a North American WNV
strain for discriminating WNV from other Japanese encephalitis virus group infections.
The authors concluded from their study that more extensive testing and evaluation
of this antigen are needed to develop reliable diagnostic assays and to establish
parameters to discriminate between positive, negative, and equivocal results.
However, the authors’ initial results also suggested that the WNV r-EIII
antigen provides good sensitivity and specificity compared to whole virus antigens
and has potential for applications directed at specific, rapid screening of
samples for the presence of anti-WNV antibody.
Beasley DWC, Holbrook MR, Travassos da Rosa APA, et al. Use of a recombinant
envelope protein subunit antigen for specific serological diagnosis of West
Nile virus infection. J Clin Microbiol. 2004;42:2759–2765.
Reprints: Alan D.T. Barrett, Dept. of Pathology, UTMB, 301 University Blvd.,
Galveston, TX 77555-0609; abarrett@utmb.edu
An assay for smoking damage to blood cells
The single-cell gel electro pho resis assay, also called the comet assay, is
a rapid method for detecting DNA damage in individual cells. The assay is simpler
and faster than other conventional genotoxicity techniques, such as cytogenetics.
The parameter often used to evaluate DNA damage is the “tail moment”
of the electropherogram, which is defined as the product of the tail length
and the fraction of the total DNA content in the tail of the electropherogram.
In this study, groups of seven CF1 male mice were given i.p. injections of relatively
low doses of methyl methanesulfonate (25 mg/kg body weight), a direct acting
genotoxic agent, or cyclophosphamide (50 mg/kg body weight), which requires
metabolic activation. Three, six, eight, 12, 16, 20, and 65 hours after treatment,
5 µL of blood was collected from each animal and processed for the alkaline
single-cell gel electrophoresis assay. On the basis of an analysis of tail moment,
the results showed that this assay can detect DNA damage induced by both kinds
of alkylating mutagens. The authors then conducted a preliminary study to assess
the status of DNA damage in a young (19 to 23 years old), healthy population
of male smokers (n=6) and nonsmokers (n=6) using the comet assay in whole blood
cells. They observed a significant difference between the two groups, showing
that the method can detect DNA damage in the smoking group despite the short
time that the volunteers had been smoking.
Ellahuene MF, Pérez-Alzola LP, Farfán-Urzua M, et al. Preliminary
evaluation of DNA damage related with the smoking habit measured by the comet
assay in whole blood cells. Cancer Epidemiol Biomarkers Prev. 2004;13:1223–1229.
Reprints: Manuel Ellahuene, Centro Nacional del Medio Ambiente (CENMA),
Av. Larrain 9975, La Reina, Santiago, Chile; mellahuene@ cenma.cl
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