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0021-972X/04/$15.

00/0 The Journal of Clinical Endocrinology & Metabolism 89(4):1512–1525


Printed in U.S.A. Copyright © 2004 by The Endocrine Society
doi: 10.1210/jc.2002-021444

CLINICAL REVIEW 167


Procalcitonin and the Calcitonin Gene Family of
Peptides in Inflammation, Infection, and Sepsis: A
Journey from Calcitonin Back to Its Precursors

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K. L. BECKER, E. S. NYLÉN, J. C. WHITE, B. MÜLLER, AND R. H. SNIDER, JR.
Veterans Affairs Medical Center and George Washington University (K.L.B., E.S.N., J.C.W., R.H.S.), Washington, D.C.
20422; and University Hospitals (B.M.), CH-4031 Basel, Switzerland

Calcitonin (CT) is a hormone that received its name be- Immature and mature CT
cause of its secretion in response to induced hypercalcemia It had been found that immunoreactive CT was present in
and its hypocalcemic effect (1). It was shown to originate multiple heterogeneous forms in MTC tissue as well as in the
from the thyroid gland (2). More specifically, the hormone serum of patients with this tumor (20 –24). Consequently, it
was revealed to be located within the thyroidal parafollicular became apparent that when this peptide was measured with
cells, interspersed within and about the follicular epithelium antisera recognizant to different epitopes the values varied
(3–5). Subsequently termed C cells, they occur primarily in according to the antiserum and the immunochemical heter-
the central region of each lobe of the human thyroid gland ogeneity (25). The phenomenon of heterogeneity was then
(6, 7). These cells, which have CT-containing secretion gran- further clarified by a series of studies which demonstrated
ules, are neuroendocrine. Embryologically, they originate that CT is biosynthesized as part of a larger prohormone,
from the neural crest and migrate to the ultimobranchial procalcitonin (ProCT) (Fig. 1) (21, 23, 25–27).
glands (8). In mammals, the ultimobranchial glands fuse The term “mature” hormone has been used to indicate a
with the thyroid gland. bioactive hormonal peptide that has been derived from a
It was the demonstration that medullary thyroid cancer larger precursor prohormone. This prohormone may be less
(MTC) was a malignancy of the C cells (5, 9) that eventually active, inactive, or characterized by an activity that differs
led to the isolation of human CT from this tumor and the entirely from the mature hormone. Not uncommonly, much
determination of its structure (10, 11). Simultaneously, the of the bioactivity of a mature hormone may be linked to an
amino acid sequence of porcine CT was determined (12). amidation that occurs at its carboxyl end. Within ProCT, CT
Later, the development of immunoassays of serum CT in is in a nonamidated, immature 33-amino acid form, termi-
humans led to the observation that the level of this hor- nating with a glycine (28). It then undergoes posttransla-
mone was increased in the serum of patients with MTC tional processing that results in production of several addi-
(13–15) and to the demonstration that these levels were tional free peptides as well as mature CT (29 –31).
further augmented after iv calcium and/or pentagastrin All species of mature CT contain 32 amino acids, with a
administration (13, 16, 17). These findings had a great disulfide bridge at the amino terminal end (between amino
impact on the clinical diagnosis, the evaluation of efficacy acid positions 1 and 7) and a proline at the carboxyterminal
of surgical extirpation, and the follow-up monitoring of end; hence, for the purpose of clarity in this manuscript, the
MTC. Although RET germline mutation testing has re- term CT(1–32) will be used specifically to refer to this pep-
placed CT for the purpose of determining the presence of tide. Among the various species of CT(1–32), the amino acid
carriers of this tumor associated with multiple endocrine sequence of the peptide tends to be well conserved within the
neoplasia type 2 (18, 19), the measurement of serum CT has amino acid ring structure at the amino terminus, but there are
become and has remained the classical clinical marker for differences elsewhere within the molecule (32–34). At the
MTC. carboxyl terminus of the CT(1–32), the proline is amidated
(35, 36). Importantly, both the ring structure and this ami-
Abbreviations: CCP-I, 21-Amino acid CT carboxyterminus peptide I; dated proline are essential for the full expression of the
CGRP, CT gene-related peptide; CT, calcitonin; CTpr, CT precursors; known bioactions of this hormone.
LPS, lipopolysaccharide; MTC, medullary thyroid cancer; NO, nitric The accurate quantification of the free CT(1–32) peptide
oxide; NProCT, 57-amino acid sequence at the amino terminus of ProCT; requires the selective detection of the amidated carboxyl
PNE, pulmonary neuroendocrine (cell); ProCT, procalcitonin; SCLC,
small cell cancer of the lung. terminal portion of the molecule, thus excluding the nonami-
JCEM is published monthly by The Endocrine Society (http://www.
dated 33-amino acid immature CT, which is found within
endo-society.org), the foremost professional society serving the en- some of the larger molecular weight precursors. Such com-
docrine community. mercially available assays were not developed until the late

1512
Becker et al. • Clincal Review J Clin Endocrinol Metab, April 2004, 89(4):1512–1525 1513

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FIG. 1. Schematic representation of ProCT and the other CT precursors (CTpr) derived from this prohormone (i.e. NProCT, CT-CCP-I, and
CCP-I). The mean concentrations of these peptides in normal serum is indicated. Note that there is appreciably more free NProCT in the serum
than CT(1–32). In sepsis, the principal elevations involve the intact ProCT, free NProCT, and free conjoined CT-CCP-I peptide. Sequencing
reveals that in sepsis, the ProCT may lack the first two amino acids of the aminoterminus of the molecule, presumably due to enzymatic
hydrolytic aminoterminal truncation (110), and perhaps other cleavage forms are present as well (111). The comparative extent to which any
one of these peptides is increased varies among patients. Levels of the free CCP-I peptide also increase but to a lesser extent. In sepsis, serum
CT(1–32) concentrations are undetectable, normal, or only slightly to moderately elevated (data from Ref. 30).

1980s; they use a double-antibody method: one antibody ever, these mice exhibited an increased calcemic response
reacts selectively with the amidated region and the other and a greater bone resorption in response to exogenous PTH,
with a different portion of the molecule (most commonly the perhaps due to the absence of an otherwise inhibiting effect
midportion). Thus, these assays do not cross-react with im- of CT(1–32) on bone resorption. Surprisingly, these knockout
mature CT (37–39). In this regard, it is important to empha- mice manifested a markedly increased bone formation; also,
size that most CT studies in the literature relating to phys- in contrast to wild-type mice that lose bone mass after ovari-
iopharmacologic manipulations as well as such influences as ectomy, they maintained their bone mass. These findings
age, gender, pregnancy, and hormonal milieu were not doc- suggest that the CT/CGRP-I gene product may somehow
umented with these specific assays. regulate bone formation, either directly or indirectly. Further
studies of these interesting observations are needed to de-
Physiologic actions of CT termine whether this action is related to CT(1–32), CGRP-I,
Hundreds of studies of the possible role of CT(1–32) have or both acting conjointly, and also whether it is species spe-
been performed. The great bulk of in vitro and in vivo inves- cific. Additional studies should also determine whether the
tigations have involved laboratory animals, some with prior induced knockout results in a compensatory overexpression
parathyroidectomy and some without. Often the species of of the gene that gives rise to CGRP-II, which, as a result, may
CT(1–32) used in these experiments were other than human conceivably modulate or modify the resultant phenotype.
(e.g. salmon, porcine, eel), the amino acid sequences of which The classic and best-studied action of CT(1–32), which
differ. Furthermore, pharmacologic, not physiologic, doses appears to occur generally throughout the mammalian spe-
often were employed. As a result, many actions have been cies, is the action on the osteoclast (34, 47, 48). Acutely, this
incorrectly imputed to this peptide. Known or alleged bio- hormone alters the osteoclast sensitivity to ambient calcium
logic actions of CT(1–32) have been reviewed elsewhere (31, and induces quiescence of osteoclast motility and a retraction
33, 40, 41). of the pseudopods that is associated with a cessation of
Although seemingly relevant effects have been observed membrane ruffling. The peptide also inhibits the elaboration
in blood, bone, kidneys, and the respiratory, gastrointestinal, by the osteoclast of acid phosphatase, carbonic anhydrase II,
embryogenic, and central nervous systems (40, 42– 45), the focal adhesion kinase, and osteopontin. Possible anabolic
function of CT(1–32) in humans remains enigmatic (41). The effects of CT(1–32) on the osteoblast have been reported (49)
hormone is not confined to the thyroid gland, and it is im- but require further documentation. The overall impact of the
possible to extirpate all cells producing this peptide (see osteoclastic inhibition is to decrease bone resorption (50).
below). However, the recent development of a knockout Nevertheless, neither the diminution of serum CT(1–32) oc-
mouse in which the coding sequences for both CT(1–32) and curring subsequent to thyroidectomy nor the marked excess
CT gene-related peptide (CGRP)-I were deleted have pro- of serum levels of CT(1–32) that occurs in patients with MTC,
vided important information (46). In these animals, no birth appear to be associated with alterations in serum calcium or
defects or difficulty with procreation were noted, and serum noticeable decreases or increases of bone mass (51). Perhaps
levels of basal calcium-related values were normal. How- the major function of CT(1–32) is to combat acute hypercal-
1514 J Clin Endocrinol Metab, April 2004, 89(4):1512–1525 Becker et al. • Clincal Review

cemia in emergency situations and/or to conserve calcium ProCT and CT precursors


stores during growth, pregnancy, and lactation. CT(1–32) is biosynthesized from the polypeptide precur-
The osteoclast has a CT receptor, as do other cells else- sor, ProCT. This 116-amino acid prohormone is comprised of
where, e.g. monocytes, kidney, brain, pituitary, placenta, three constituent peptides: a 57-amino acid sequence at the
prostate, testis, lung, and liver (52–55). The CT(1–32) receptor amino terminus (NProCT); the centrally positioned imma-
has been cloned (56 –58). In the human, there is a polymor- ture CT that contains a terminal glycine; and a 21-amino acid
phism of this receptor (59), which may clinically influence CT carboxyterminus peptide I (CCP-I) (Fig. 1) (28). Subse-
bone density and quality (60), and these different isoforms quent enzymatic posttranslational processing yields several
may also have other functional implications. Stimulation of peptides (31, 73, 74); in addition to CT(1–32), the serum of
the CT(1–32) receptor induces increased cAMP and an in- normal persons contains intact ProCT, free NProCT, free
creased cytosolic free calcium concentration, accompanied CCP-I, and the free conjoined CT-CCP-I peptide. Interest-

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by activation of the MAPK pathway (61). ingly, the normal molar concentration of circulating NProCT
is 2-fold higher than that of CT(1–32) (4.15 fmol/ml vs. 1.84
fmol/ml) (30). Because these peptides are relevant to and
Therapeutic effects of CT(1–32) precede the biosynthesis of CT(1–32), they have been given
Although the usage of CT(1–32) as a drug has greatly the collective appellation CT precursors (CTpr) (75, 76). The
diminished, it continues to have a therapeutic role, princi- extent to which CTpr may have physiologic functions is
pally in osteoporosis and Paget disease. The commercially under study (see below).
used drug is the amidated, synthetic salmon preparation; in
the United States, human CT(1–32) has become an orphan CTpr in MTC
drug. For osteoporosis, it is commonly agreed that bisphos-
phonate therapy is more effective than CT(1–32) and is An appreciable number of conditions are associated with
currently the preferable therapy (62). However, salmon increased serum levels of CTpr (Table 1). Although it has
been known for many years that normal thyroid gland, MTC
CT(1–32) has been well demonstrated to increase bone den-
tissue, and the serum of MTC patients contain large amounts
sity and decrease fracture rate, especially in the vertebrae
of ProCT as well as its component peptides (22, 77), the
(63– 65); adverse effects are minimal. For osteoporosis, the
potential clinical utility of CTpr as a serum marker for MTC
nasal or sc route can be used. Bisphosphonates also have
has very rarely received attention. In this respect, initially it
replaced CT(1–32) for the therapy of Paget disease (66, 67); was found that the carboxyterminal region of ProCT (cor-
nevertheless, sc salmon CT(1–32) still may be useful in the responding to CCP-I, also termed katacalcin) was secreted
occasional patient who cannot tolerate high doses of bisphos- into the medium of MTC cultures as well as into the serum
phonates (68). In the United States, the nasal CT(1–32) prep- of patients with this tumor in a calcium-dependent manner
aration is not approved for Paget disease. As an acute or (78). Subsequently an assay for CCP-I was evaluated as a
subacute therapy for hypercalcemia, the sc or im adminis- serum marker for MTC (79). It was later reported that the
tration of salmon CT(1–32) is not a reliable procedure; the NProCT cleavage product of ProCT also was a potential
treatment of choice is adequate hydration and iv bisphos- marker for this tumor (80). Recently, in a study of MTC
phonates (69). Various possible antinociceptive effects of patients (81), a sensitive and rapid (3-h) two-antibody sand-
CT(1–32) have been described (70). For example, in patients wich assay that quantifies both intact ProCT and CT-CCP-I
with painful osteolytic metastases, symptomatic therapy (82) was compared with an assay that is specific for CT(1–32).
may be beneficial (71); however, the limited evidence in the CTpr were found to be universally present in the serum of
literature does not support its use for this purpose (72). patients with MTC. They were increased whenever CT(1–32)

TABLE 1. Clinical conditions in which serum CTprs are increaseda

Sepsis with or without documentation of bacterial infection; sepsis-related conditions, such as pancreatitis, severe burns, polytrauma,
heatstroke, marked systemic bacterial infection as may occur in pneumonia or pyelonephritis; occasional patients with marked systemic
viremia or fungal infection; and severe malaria
Medullary thyroid cancer
Aspirational or inhalational pneumonitis
Adult respiratory disease syndrome (ARDS)
Pulmonary neuroendocrine hyperplasia as occurs in chronic obstructive pulmonary disease or smoking-related chronic bronchitis
Small cell cancer of the lung
Non-small cell lung cancer (probably due to admixture of malignant small cells, and/or to smoking-related pulmonary neuroendocrine cell
hyperplasia)
Carcinoid tumor
Other neuroendocrine tumors (pheochromocytoma, pancreatic islet cell tumor)
Other, seemingly non-neuroendocrine malignancies, such as breast cancer
a
In these listed conditions, using a sensitive assay (76), CTpr may be increased to enormous levels in the serum, e.g. ranging from 1,000
to 100,000 pg/ml in patients with sepsis, or, only minimally increased, e.g. 200 pg/ml in pulmonary neuroendocrine hyperplasia [normal, less
than 76 pg/ml (12 fmol/ml)]. In sepsis and septic-related conditions, CT(1–32) usually is normal or only minimally increased. In contrast, the
high serum CTpr that occurs in medullary thyroid cancer, small cell carcinoma of the lung, carcinoid, and some other neuroendocrine tumors
is nearly always accompanied by a marked increase of serum CT(1–32).
Becker et al. • Clincal Review J Clin Endocrinol Metab, April 2004, 89(4):1512–1525 1515

was increased and, on a pg/ml basis, exceeded CT(1–32) of macrophages, altered capillary function, transudation of
levels by about 10-fold. Also, both markers responded to serum into the tissues, and the release of various humoral
pentagastrin. Whether CTpr are more sensitive markers for substances (Table 2).
the presence of MTC or might be more useful prognostically There are a variety of distinct conditions that manifest
remains to be determined. systemic inflammation (e.g. severe burns, pneumonitis or
other marked local infections, bacteremia, endotoxemia,
CTpr in several other clinical disorders trauma, heat stroke, and pancreatitis). Typically any of these
Other neuroendocrine tumors, such as small cell cancer of conditions may lead to a clinical syndrome that has been
the lung (SCLC), carcinoid, pheochromocytoma, and pan- termed the systemic inflammatory response syndrome,
creatic islet tumors may exhibit increased serum CTpr. In which is defined by varying combinations of fever or hypo-
contrast to MTC, in these conditions, the serum CTpr/ thermia, tachypnea, tachycardia, and polymorphonucleocys-
tosis or leukopenia (94, 95). Associated with these manifes-

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CT(1–32) ratio is further increased, probably because these
lesions lack sufficient posttranslational enzymatic capability tations, there is a variable local as well as systemic increase
(30, 83, 84). In the case of SCLC and carcinoid tumor, the of many cytokines and other hormonal messenger molecules
occurrence of increased serum CTpr is very frequent. Using [e.g. TNF␣, IL-1␤, IL-6, interferon-␥, arachidonic acid deriv-
atives, cortisol] (Table 2). Some of these substances, acting in
an immunoassay that is reactive to the midportion of CT [and
a hemocrine and/or paracrine manner, are protective to the
hence detects the presence of the ProCT and free CT-CCP-I
host; some may be harmful, and some may be either bene-
molecules as well as CT(1–32)], there was found to be an
ficial or damaging, depending on their concentrations, their
association between the levels obtained and the clinical
timing, or the ambient humoral milieu. Alternatively, the
course of the tumor (85). Similarly, levels usually decreased
increases of some of these substances may be epiphenomenal
concomitantly with radiotherapy and/or chemotherapy.
and exert no relevant bioeffects.
Also, decreased levels corresponded to clinical remissions.
The clinical term, sepsis, has been used to indicate a sys-
Interestingly, SCLC is thought to originate from the same
temic inflammatory response syndrome in which bacteria or
precursor cell as does the normal pulmonary neuroendocrine
microbial products have been shown or suspected to be the
(PNE) cell, a cell that contains large amounts immunoreac-
etiology. In some cases, infection cannot be documented
tive CT (42, 86, 87). However, lung cancers other than SCLC
because microbial culture does not reveal a pathogenic mi-
may contain and secrete immunoreactive CT. In such pa-
crobe. It is likely that in some of these culture-negative cases,
tients, these peptides may originate from the tumor, admixed
the sepsis is indeed due to microbes, but the methods of
SCLC cells, or adjacent noncancerous PNE cells that are
detecting them are not completely reliable and, therefore, the
known to become hyperplastic in response to chronic ciga-
pathogen may not be identified. Moreover, in other cases,
rette smoking (88, 89). Heterogeneity studies that have been
toxic byproducts of the pathogen may be responsible for the
performed in such cancer patients also reveal a large CTpr/
syndrome. For example, the translocation through the gut
CT(1–32) ratio (83).
wall of toxins [e.g. endotoxin (lipopolysaccharide, LPS)] from
Some noncancerous conditions that are associated with
bacteria normally inhabiting the gastrointestinal tract may be
increased levels of serum CTpr appear to be explicable, all or
the cause of the sepsis (96, 97).
in part, on the basis of hypersecretion or hyperplasia of the
In sepsis, the patient is not ill principally because of the
PNE cells [i.e. chronic bronchitis (e.g. smoking, occupational,
initial injury or infection but because of a humoral and/or
cystic fibrosis), chronic obstructive pulmonary disease, acute
cellular overreaction of the host. The unrestrained or unbal-
inhalational burn injury, acute chemical pneumonitis, and
anced cytokine and humoral response in this illness may
tuberculosis] (84, 90). Furthermore, the kidney plays an im-
progress sufficiently to cause multiple organ failure, char-
portant role in the metabolism of CT(1–32) (91), and renal
acterized by varying degrees of severe occurrences, such as
disease often is associated with increased serum immuno-
myocardial insufficiency, hypoperfusion, shock, coagulopa-
reactive CT levels, much of it consisting of CT-precursor
thy, respiratory failure, hypoxemia, renal failure, and coma.
peptides (24, 92, 93).
Sepsis is the 11th leading cause of death in the United States.
Lastly, as emphasized in the present review, extraordinary
Approximately half of the fatalities in intensive care units
increases of serum CTpr occur in patients with severe in-
result from this condition. The incidence of sepsis is increas-
flammation, systemic infection, and sepsis. Indeed, CTpr
ing (currently approximately 750,000 cases per year), and the
serum levels can be used as markers for the presence and
mortality remains unchanged (approximately 30%, with
severity of these conditions. Furthermore, in these condi-
rates up to 75% in septic shock) (98).
tions, high levels of CTpr appear to play a harmful role, and
their immunoneutralization offers the potential of effective
therapy. CTpr as markers of inflammation, systemic infection, and sepsis.
An initial publication in 1983 first called attention to in-
CTpr in inflammation, systemic infection, and sepsis creased serum levels of immunoreactive CT in patients with
the staphylococcal toxic shock syndrome, a severe form of
Pathophysiology. Inflammation, a highly complex phenome- sepsis (99). The assay used an antiserum that was not selec-
non that may be beneficial and/or detrimental to the host, is tive for the amidated carboxyl terminal portion of CT(1–32)
a reaction to a large variety of injuries. Inflammation can be and hence would also bind to the immature CT within the
local or systemic. It is characterized by vasodilation, attrac- ProCT and CT-CCP-I molecules. Gel filtration studies dem-
tion of polymorphonuclear cells and lymphocytes, activation onstrated that this immunoreactive CT was of large molec-
1516 J Clin Endocrinol Metab, April 2004, 89(4):1512–1525 Becker et al. • Clincal Review

TABLE 2. Schematic illustration of events and humoral factors following exposure to a variety of insults that trigger an inflammatory
responsea

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a
Several of these factors have multiple and/or variable roles, and some may be either anti- or proinflammatory. In addition, many other
humoral factors have been identified. For further documentation and elaboration, see Refs. 183–186. ADM, adrenomedullin; AP-1, activator
protein-1; AVP, arginine vasopressin; CRP, C-reactive protein; HMG-1, high mobility group-1; IFN, interferon; IL-ra, IL receptor antagonist;
LTs, leukotrienes; MIF, monocyte migration inhibitory factor; MOF, multiple organ failure; NF-␬B, nuclear factor-␬ B; PAF, platelet activating
factor; PAMPS, pathogen-associated molecular patterns; PGs, prostaglandins; PTX, pertussis toxin; RANTES, regulation on activation normal
T-expressed and secreted; ROI, reactive oxygen intermediates; TBs, thromboxanes; TNFr, TNF receptor.

ular weight, now known to correspond to ProCT and CT- and the routine laboratory tests (e.g. an abnormal leukocyte
CCP-I. This paper provided the inspiration for multiple count, elevated serum C-reactive protein, or positive bacte-
subsequent studies of CTpr in inflammation, systemic infec- riologic studies) may be nonspecific or may not occur at all.
tion, and sepsis. Furthermore, the classical associated proinflammatory cyto-
The traditional clinical signs of severe infection (e.g. fever kines of severe inflammation, systemic infections, and sepsis
or hypothermia, tachycardia, tachypnea, or hypotension) (i.e. TNF␣, IL-1␤, or IL-6) commonly are increased in the
Becker et al. • Clincal Review J Clin Endocrinol Metab, April 2004, 89(4):1512–1525 1517

serum only transiently or intermittently. In contrast, serum present in the sera of sepsis patients, there appears to be
levels of CTpr are very frequently increased, sometimes at- universal agreement that both ProCT and multiple fragments
tain levels several thousand-fold normal, and these high of ProCT are present. Interestingly, in all these sepsis and
levels often persist for long periods of time. Moreover, the sepsis-like conditions, CT(1–32) remains undetectable or nor-
levels often correlate positively with the severity of the con- mal or slightly to moderately elevated (30, 100, 102) (see
dition and mortality. Indeed, after the first systematic study below).
of sepsis due to severe bacterial infection (100), many clinical There are four assays that have been created to measure
studies have documented the considerable utility of serum serum CTpr. Currently none of these assays are commer-
CTpr to identify and follow the course of this illness and cially available in the United States. The authors developed
sepsis-like conditions (Fig. 2) (100 –102). a single-antibody RIA for NProCT, which quantitates ProCT
Thus, serum CTpr have been shown to be extremely useful and the free NProCT peptide (as well as ProCGRP and
markers in sepsis, whether blood cultures are positive or NProCGRP) (76, 101). This sensitive assay [10 pg/ml (1.6

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negative, and also in sepsis-like conditions such as severe fmol/ml)] detects CTpr in healthy nonsmoking persons [the
burns, pancreatitis, pneumonitis, inhalational injury, bacte- upper limit of normal is 76 pg/ml (12 fmol/ml)]. There are
rial meningitis, heat stroke, and severe mechanical trauma two two-site rapid assays. In Europe, a commercially avail-
after extensive surgery, and also in some infections of non- able assay (LUMItest PCT, B.R.A.H.M.S. Diagnostica GmbH,
bacterial causation (e.g. severe malaria or systemic fungal Henningsdorf/Berlin, Germany), shortly to be available in
infections) (100 –108). This phenomenon also occurs in pa- the United States, measures both ProCT and the conjoined
tients with a prior thyroidectomy (100, 109). CT-CCP-I by means of a luminometer (100). This assay,
Molecular sizing by gel filtration and HPLC studies in which has been used in many clinical studies, is inaccurate
sepsis and in the above septic-like conditions have demon- at levels less than 300 pg/ml (24 fmol/ml). This company
strated that serum components corresponding to ProCT, manufactures another more sensitive double-antibody kit,
NProCT, CT-CCP-I, and the CCP-I peptide are all elevated which can reliably detect levels as low as 20 pg/ml (1.6
to varying degrees (30) (Fig. 1). The individual components fmol/ml) (82). The sensitive assays, including a recently de-
were quantified with antisera specific for CT(1–32), NProCT, veloped tracer technology Kryptor assay (B.R.A.H.M.S.), the
CCP-I, and also a two-site assay that used antibodies specific latter of which is commercially available in Europe, are es-
to the CT and CCP-I regions of ProCT. Using capillary zone pecially useful for the detection of early forms of infection
electrophoresis spectrometry and Edman sequence analysis, and for follow-up determinations (112).
the serum ProCT in sepsis has been reported to lack the first
two amino acids of the molecule (Ala-Pro), consequent to an Ubiquity of expression of the calcitonin-I gene in response to sepsis.
aminoterminal truncation by the dipeptidyl peptidase IV Normally, immunoreactive CT is found predominantly in
enzyme (EC3.4.14.5) (110). In another study, immunoreactive some neuroendocrine cells such as the thyroid C cells and
ProCT and its cleavage products were extracted from pooled PNE cells, and CGRP is found predominantly in brain and
septic serum using octadecylsilyl columns and characterized neurologic tissues. However, immunoreactive CT is found in
by HPLC and Western blot analysis of electrophoresis-sizing many tissues throughout the body (113). Furthermore, low
gels. The peptides were identified using antisera specific for levels of CT gene mRNAs have been reported to occur in liver
CT, NProCT, and CCP-I (111). These investigators found (55) and also several other tissues (114). In septic hamsters,
ProCT and multiple fragments of ProCT that appeared to substantially increased levels of immunoreactive CT (appar-
differ in molecular size from those found in the serum of ently mostly ProCT as shown by gel filtration chromatography
patients with MTC. Thus, whereas there is some uncertainty and HPLC) were found in liver, lung, kidney, pancreas, brain,
as to the absolute identities of some of the fragments of ProCT heart, and small intestine (115). In this investigation, quantita-

FIG. 2. Receiver operating-curve analysis of se-


rum CTpr for the diagnosis of sepsis in an inten-
sive care unit, as compared with values for cir-
culating C-reactive protein, IL-6, lactate, and
pentraxin-3 (PTX-3). The sensitivity of CTpr for
the diagnosis of sepsis was 89%, specificity 94%,
negative predictive value 90%, and positive pre-
dictive value 94% (as assessed by the commer-
cially available LUMItest PCT). However, the
other non-CTpr markers were considerably less
sensitive and less specific and had relatively poor
negative or positive predictive values (modified
from Ref. 102).
1518 J Clin Endocrinol Metab, April 2004, 89(4):1512–1525 Becker et al. • Clincal Review

tive analysis of CT mRNA expression was determined by the nating from this superfamily of genes: one of the genes yields
CT/␤-actin ratio of the septic tissues in relation to the CT/␤- a very slightly different form of CGRP (CGRP-II), another
actin ratio of the respective control tissues (Taq-Man technol- gene gives rise to amylin, and another gives rise to ad-
ogy). Among the tissues studied, the relative increase of CT renomedullin. Furthermore, there is a gene that gives rise to
mRNA, compared with controls, was, in descending order, a CT receptor-stimulating peptide with some homology to
adrenal, spleen, spinal cord, brain, liver, pancreas, colon, lung, CGRP (31, 124, 125).
fat, testes, and stomach (115). This phenomenon of marked Similar to the case for CT-mRNA, in septic hamster tissues
increase of CT mRNA in extrathyroidal tissues also occurs in the but not in similar healthy control tissues, there also is a
septic human (116). Of course, when evaluating the impact of tissue-wide expression of CGRP mRNA (126). Here, as well,
each increase, consideration must be given to the total weight CGRP mRNAs are more specifically up-regulated than are
of each organ. Thus, for example, the demonstration of CT the mRNAs of classical cytokines (e.g. IL-6 and TNF␣). A
mRNA in fat of septic individuals assumes marked pathophys- similar phenomenon occurs for adrenomedullin. In contrast,

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iological significance when one considers the great bulk of amylin mRNA has not been found to be up-regulated in
adipose tissue (116). nonneuroendocrine septic tissues. Thus, in sepsis several
The extraordinary tissue-wide expression and secretion members of the CALC gene superfamily escape from their
explains the enormous elevation of CTpr in the sera of septic normal tissue-selective expression pattern. In studies in sep-
patients. The substantial suppression of CALC-I gene ex- tic humans, serum levels of CGRP and adrenomedullin are
pression by nonneuroendocrine cells that occurs in normal increased in sepsis, although to levels considerably below
persons is altered in septic patients by a unique stimulus those found for CTpr.
arising from the infectious and/or cytokine insult that then
influences the transcriptional regulation of the gene. Indeed, ProCT as a toxic factor in severe inflammation, systemic
in sepsis the mRNA is more uniformly up-regulated than are infection, and sepsis
the mRNAs of the classical sepsis-related cytokines, TNF␣,
IL-1␤, and IL-6. In a broader sense, in sepsis the entire body Development of animal models of sepsis and correlation of serum
becomes a CTpr-producing endocrine gland. It is because CTpr with sepsis and mortality. In humans, the concentration
this novel form of secretion is intrinsic to the host response of serum CTpr often reflects the severity of sepsis and may
to sepsis, and is reminiscent of the expression of the classical be predictive of mortality. Accordingly, to study this phe-
cytokines in this condition, that CTpr are referred to as hor- nomenon more extensively, a model for sepsis was devel-
mokines (115). oped in hamsters and pigs (127–131).
The lack of a significant increase of CT(1–32) in sepsis
merits further investigation. In part, this is indicative of a Sepsis in the hamster
shift away from the normal, regulated, neuroendocrine path- Severe peritonitis was induced in hamsters by the ip im-
way (characterized by a progressive posttranslational pro- plantation of pellets containing measured quantities of Esch-
cessing, maturation, and secretion via secretory granules) to erichia coli, and serum CTpr was determined at intervals.
a constitutive pathway (characterized by a nonstoring, bulk- According to the dosage of bacteria, the 72-h mortality in-
flow secretion from nonneuroendocrine cells); this latter creased proportionately from zero to approximately 20, 70,
pathway is deficient in the enzymatic processing required to and 100%. Serum CTpr also demonstrated a dose-related
produce CT(1–32) (31, 117). Furthermore, once secreted, increase; at the highest dose of bacteria, serum CTpr levels
ProCT and NProCT are extremely resistant to enzymatic exceeded control values by nearly 200-fold. Thus, CTpr levels
degradation; in contrast, CT(1–32) is extremely labile. Thus, correlated both with the severity of bacterial insult and the
circulating CT(1–32) may not be increased in sepsis because mortality. Gel filtration and HPLC studies revealed that most
of its rapid degradation. Yet another factor may be the in- of the CTpr in these septic animals was in the form of ProCT
fluence of heat shock proteins. These stabilizers of cellular (127).
function are synthesized in response to heat, other stressful
stimuli, and sepsis. In this latter illness, they may serve a Relationship between serum CTpr and cytokines. Using this an-
protective role (118). Heat shock proteins also are found imal model of sepsis, the relationship of CTpr to the proximal
in the blood as well as intracellularly (119, 120). They bind proinflammatory mediators, IL-l␤ and TNF␣, was studied
to CT(1–32) (121), and perhaps this further augments the (128). Whereas serum CTpr remained extremely high
disposal of CT(1–32) or interferes with its immunologic throughout the 24 h of this study, the increases of these
detection. cytokines in the serum were less than 2-fold greater than the
baseline and, importantly, were transient in duration. In
Other members of the CT-gene family of peptides in sepsis: CGRP healthy hamsters, the iv administration of human ProCT
and adrenomedullin. There are five genes in the CT-gene fam- caused no evident adverse effects and no changes in serum
ily of peptides. The gene that was initially discovered gives IL-1␤ and TNF␣ levels. In septic animals, the ProCT injec-
rise to two alternative splice variants: CT mRNA, resulting tions, albeit markedly increasing mortality (see below), only
in ProCT and its components, and CGRP mRNA, giving rise modestly blunted IL-1␤ levels and did not affect TNF␣ val-
to CGRP-I (122, 123). In the healthy, noninfected state, there ues. Interestingly, however, when TNF␣ was injected into
is a preferential synthesis of either CT(1–32) mRNA or healthy animals, there was a 25-fold increase of CTpr levels,
CGRP-I mRNA according to the cellular phenotype. Addi- compared with noninjected controls. Thus, as is the case in
tionally, there are other structurally related peptides origi- the human, the magnitude and duration of the CTpr eleva-
Becker et al. • Clincal Review J Clin Endocrinol Metab, April 2004, 89(4):1512–1525 1519

tion demonstrated its utility as a marker of sepsis in the hour of the experiment (131). In contrast to the parameters
hamster. It also revealed that ProCT does not secondarily for control septic animals, all of which progressively wors-
enhance levels of IL-1␤ or TNF␣ in the systemic blood. In ened and ultimately died, most physiologic and biochemical
contrast, it showed that in healthy animals, TNF␣ can induce parameters of the treated animals improved or stabilized
a sepsis-like elevation of serum CTpr. after infusion of the ProCT-reactive IgG (Fig. 3). Although all
untreated animals had died during the experiment, most of
ProCT as a toxic factor. Based on these clinical and animal
the treated pigs survived until being killed (P ⫽ 0.010). Thus,
studies, it was hypothesized that ProCT per se may be a
in this model, the findings indicate that the immunoneutral-
toxic factor in sepsis and may adversely influence survival.
ization of ProCT was useful in a clinically relevant situation
The iv administration of human ProCT, which appeared
in which sepsis was fully established and far advanced.
not to be overtly injurious to normal hamsters, doubled the
mortality of septic animals (129). In contrast, administra-

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tion of human CT(1–32) to septic hamsters was without
effect. Furthermore, a goat antiserum raised to this hor-
mone, and shown to be completely cross-reactive with
ProCT, was found to increase survival whether adminis-
tered prophylactically or therapeutically.

Sepsis in the pig


To investigate in detail the physiologic and metabolic con-
sequences of sepsis and evaluate how ProCT immunoneu-
tralization might affect these parameters, a larger animal
model of a rapidly fatal porcine polymicrobial peritonitis
was then developed (130, 131). The subsequent sepsis is
similar pathophysiologically to that encountered in human
disease but far more lethal.
Early immunoneutralization. After having determined the
structure of porcine ProCT, and, in rabbits, having produced
an antiserum that was specific to the NProCT portion of this
peptide, sepsis was induced in Yorkshire pigs by the ip
instillation of a suspension of cecal content (l g/kg) plus E.
coli (2 ⫻ 1011 cfu). Simultaneous to the induction of the
peritonitis, experimental pigs received an iv infusion of the
ProCT-reactive purified rabbit IgG, whereas control animals
received non-ProCT-reactive IgG. All animals had physio-
logic data (e.g. urine output, core temperature, arterial pres-
sure, heart rate, cardiac index, and stroke index), and met-
abolic data (e.g. blood urea nitrogen, serum creatinine,
arterial lactate, and pH) collected or recorded hourly until
death or being killed (15 h after ip instillation) (130).
Similarly to what occurs in humans, in this large animal
model of lethal peritonitis, serum CTpr levels were found to
be significantly elevated. Most of the untreated animals died
within 9 h, and none survived for the 15-h duration of the
experiment. However, immune IgG administration resulted
in a significant improvement or a beneficial trend in most of
the measured physiologic and metabolic derangements in-
duced by sepsis. Moreover, most of these treated animals FIG. 3. Top, Time course in septic pigs showing the changes in mean
survived until the time of being killed, in contrast to animals arterial pressure (MAP). Time point 0 h represents commencement of
treated with the non-ProCT-reactive IgG (P ⫽ 0.007). the fourth hour after induction of sepsis and is the time of infusion of
purified nonreactive rabbit antibody to the control group and purified
Immunoneutralization of moribund pigs. In this septic pig antiporcine ProCT rabbit IgG to the treated group. After time point
0, there is a sharp fall in MAP in the control group, but the MAP of
model, the physiologic and metabolic parameters of the un-
the treated group remains near normal levels. *, Statistically signif-
treated animals worsened rapidly, so that the animals were icant data points (P ⬍ 0.017; mean ⫾ SEM). Bottom, Time course
essentially moribund by 4 h after the induction of the peri- showing the changes in serum creatinine. The graph shows very
tonitis. This state is comparable with the syndrome of mul- similar creatinine concentrations before antibody infusion, which is
tiple organ failure that occurs in humans with preterminal followed by a sharp rise in the control group but with concentrations
that remain near normal for the treated group. Other physiologic or
sepsis. Accordingly, to determine whether these gravely ill metabolic parameters also showed benefits. *, Statistically significant
animals might be rescued, an evaluation of iv therapeutic data points (mean ⫾ SEM, P ⬍ 0.037), which were noted at 2, 4, and
immunoneutralization was undertaken during the fourth 6 h, after which all control animals died (from Ref. 131).
1520 J Clin Endocrinol Metab, April 2004, 89(4):1512–1525 Becker et al. • Clincal Review

Mechanism of toxicity of ProCT in sepsis: hypotheses and early cAMP response to relatively small amounts of CT(1–32)
unanswered questions has been shown to be blocked by other hormones [e.g. PTH
Currently the physiologic actions of ProCT are relatively (147), epinephrine (148)].
unexplored, and it is not known how this polypeptide or its A principal function of the monocyte is its migration to
components might worsen the septic process. Although sites of inflammation, a phenomenon that has been demon-
acute studies have not been done in the human, the experi- strated to be induced by the addition of CT(1–32); in contrast,
ence in noninfected patients with MTC indicates that chron- when patients receive therapeutic doses, this motion is di-
ically high levels of ProCT do not appear to cause any ob- minished, strongly suggesting down-regulation of CT(1–32)
vious ill effects. Nonetheless, although administration of receptors (149).
ProCT to healthy, noninfected hamsters had no apparent ill The immunologic relevance of the monocyte response to
effects, its administration to hamsters that were septic sig- CT(1–32) remains to be fully elucidated. In vivo immunologic
activities of this hormone have been demonstrated in several

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nificantly increased mortality. This indicates that ProCT is
not an initial toxic factor but requires the prior presence of studies (150, 151), and salmon CT(1–32) has been reported to
a proinflammatory stimulus followed by an altered cytokine diminish the local inflammation after various forms of in-
milieu. jury in rats (152). In humans with rheumatoid arthritis, eel
CT(1–32) decreased production of IgG immunoglobulin
Initial studies of actions of CTpr. In addition to the osteoclast, and inhibited IL-1␤ (153). However, as mentioned above,
it is well known that receptors for CT(1–32) are present in CT(1–32) is not appreciably elevated in patients with severe
different cell types, and multiple experimental studies of this inflammation or sepsis, and because of the marked increase
peptide have been performed. However, in the past, the of serum ProCT and its component CTpr in such patients in
effects of the various CTpr have very rarely been evaluated. the serum, more interest is being focused on the effects of
Insofar as ProCT is concerned, one study revealed the pres- these latter peptides on the monocyte.
ence of receptors to this prohormone in newborn rat calvarial
cells (132). Also, the human NProCT peptide portion of CTpr and the monocyte
ProCT has been reported to be mitogenic to human osteo-
In vitro studies of isolated human monocytes have dem-
blastic cells (U-2 OS osteosarcoma) and to induce an increase
onstrated that not only CT(1–32) but also ProCT and the free
in intracellular cAMP (133), although a subsequent study
CCP-I peptide act as chemoattractants, inducing migration,
was not confirmatory (134). In a preliminary and uncon-
which is dosage dependent. This phenomenon is accompa-
firmed report, a large-molecular-weight species of immuno-
nied by an intracellular elevation of cAMP levels. Further-
reactive CT, corresponding to an undetermined portion of
more, paradoxically, these peptides may deactivate the mi-
the ProCT molecule, appeared to suppress prostaglandin-
gratory effects of other unrelated chemoattractants and/or
E2-stimulated osteoclastic bone resorption (135). It is because
modify monocyte surface signals (154, 155). Monocytes and
of the recent awareness of the phenomenon of greatly in-
neutrophils are stimulated by LPS and another proinflam-
creased serum levels of CTpr in severe inflammation and
matory product of bacteria, formyl methionyl leucyl phe-
sepsis that studies of the action of CTpr are now underway
nylalanine peptide, which induces these cells to produce an
by several groups.
important integrin, CD11b, a substance that is involved in
The monocyte. Although the cell that has been most associated chemotaxis. Both NProCT and CGRP decrease this cellular
with the action of CT is the osteoclast, it is well known that CD11b production (156). In an initial study (157), human
this cell is produced from a precursor monocyte/macro- ProCT was added to mononuclear cells of peripheral blood
phage cell line that also gives rise to the mature monocyte harvested from normal humans, and cytokine secretion was
(136 –139). Moreover, monocytes influence osteoclast activity measured in the ambient culture media. When compared
and also directly induce bone resorption (139 –141). Impor- with background cytokine synthesis by unstimulated cells,
tantly, the monocyte plays an essential role in phagocytosis, IL-1␤ secretion was augmented 4-fold, TNF␣ 2-fold, and that
T lymphocyte immune activity, and inflammation; thus, it is of IL-8 2-fold. These preliminary findings suggest that ProCT
greatly involved in the initiation and course of sepsis. The might stimulate cytokine secretion from monocytes in local
awareness that monocytes have CT receptors (142, 143) circulatory pools. As mentioned, TNF␣ is a known stimulus
has led to several experiments investigating the effects of to ProCT secretion, and in sepsis this cytokine might locally
CT(1–32) and CTpr on these cells. induce a yet further local production of this prohormone in
a positive-feedback manner. The clinical impact of all of these
CT(1–32) and the monocyte actions and complex interactions on the monocyte awaits
clarification.
Insofar as CT(1–32) is concerned, after exposure of the
lymphocyte/monocyte/macrophage family to this peptide, CTpr and nitric oxide (NO). The production of the vasodilator,
cAMP increases (144). This cAMP response is inhibited after NO, is elevated in sepsis (158), and this agent has been
exposure of these cells to various mechanical or hormonal proposed as a mediator of the shock that may occur during
stimuli (145). Also, analogous to the finding of down-regu- the course of this illness (159). However, others have re-
lation of the osteoclast CT(1–32) receptor that occurs when ported that NO may have a beneficial role. When ProCT is
hypercalcemic patients receive CT(1–32) therapy (146), a sim- added to cultures of vascular smooth muscle cells of normal
ilar response to an excess of this hormone occurs in vitro rats that had previously been exposed to LPS, TNF␣, and
when the monocyte CT(1–32) receptor is studied (143). The interferon-␥, the prohormone amplified the expression of the
Becker et al. • Clincal Review J Clin Endocrinol Metab, April 2004, 89(4):1512–1525 1521

inducible NO synthase gene as well as NO production (160). teins), thus perhaps modulating the action of the CT gene
The potentially detrimental effects of such an occurrence in products according to ambient circumstances (176 –180).
the septic process requires further evaluation. Conceivably, the much higher serum levels of CTpr may
interfere with receptors or with receptor-activity-modifying
CTpr and hypocalcemia. Hypocalcemia, involving particularly
protein expression. Such an occurrence may block CGRP
but not exclusively the ionized component, is a frequent
and/or andrenomedullin activity and hence impede their
concomitant of critical illness and sepsis (161, 162). Studies
otherwise beneficial effects. Nevertheless, both CGRP and
in the septic rat indicate that this hypocalcemia is accompa-
adrenomedullin have vasodilatory actions, and whether
nied by an increase of intracellular calcium (163), and a
such effects may be beneficial (e.g. by increasing the blood
similar intracellular increase occurs in the septic human
supply to vital organs) or harmful (e.g. by inducing systemic
(164). Interestingly, it is known that calcium infusions may
hypotension) requires additional clarification.
be harmful to septic humans (165, 166), and when septic
Clearly, it is essential to further investigate the means by

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hypocalcemic rats are administered calcium, the fatalities
which ProCT exerts its toxicity in sepsis. ProCT and its com-
increase markedly (167). In a large study of critically ill pa-
ponents may exert actions that differ according to their target
tients, dose-related correlations were noted among the se-
tissues as well as having actions that differ according to the
verity of illness, the degree of hypocalcemia, and the level of
ambient milieu of the host. Multiple in vitro and in vivo
serum CTpr (161). Also, in this condition, serum ionized
investigations will be required.
calcium is known to correlate inversely with levels of TNF␣
and IL-6 (162). The cause of the hypocalcemia is unknown.
CT(1–32) has been demonstrated to augment the intracellular Potential advantages of ProCT as a target for
calcium concentration in several different cell lines (61, 168 – immunoneutralization in the human
170). However, CT(1–32) is rarely appreciably elevated in
The administration of endotoxin results in a form of sys-
sepsis. A direct effect of other CTpr on intracellular calcium
temic inflammation that is associated with cellular activation
has not been demonstrated. In a study in hamsters, admin-
and the release of inflammatory mediators (Table 2); albeit
istration of human ProCT did not cause hypocalcemia; in-
being different from sepsis, it is an experimental model of the
deed, this prohormone was found to completely block the
mediators and the control mechanisms that are relevant to
hypocalcemia normally induced by injection of CT(1–32)
this devastating state. In humans, it was shown that endo-
(171). Thus, further studies are required to define any rela-
toxin caused a rise in serum CTpr that persisted for the 24 h
tionships between the CTpr peptides and serum calcium.
of the experiment, although the duration of this elevation
Influence of other peptides of the CT gene family. Lastly, as dis- was unknown (179). Therefore, an investigation was under-
cussed above, both CGRP and adrenomedullin are increased taken to evaluate the duration of the CTpr elevation (180).
in sepsis. These hormones have been reported to exert an- Serum CTpr were observed to increase by 3 h and attained
tiinflammatory effects (172–175), and these actions could po- peak values at 24 h. Subsequently values very slowly and
tentially be beneficial in severe infections or sepsis (e.g. an- progressively declined. Surprisingly, at 7 d, all volunteers
tibacterial, increased dilatation of coronary arteries, positive still exhibited levels that were above normal (Fig. 4). In two
cardiac inotropic and chronotropic effects). Furthermore, the of the subjects who were studied for a longer period, the
biologic effects of the members of the CT gene family of levels did not normalize until 10 –14 d. In contrast, when
peptides are exerted via the same family of receptors. The subjects were administered identical doses of endotoxin and
physiological profiles of these receptors are modified by cytokines were measured for 24 h, serum levels of the proin-
certain accessory proteins (receptor-activity-modifying pro- flammatory cytokine TNF␣ increased at 1 h, reached a peak

FIG. 4. Exposure of human volunteers to one injection of


endotoxin illustrates the differences in the release and
subsequent decrease of several humoral markers of crit-
ical illness: TNF␣, IL-1 receptor antagonist (IL-1ra),
IL-6, C-reactive protein (CRP), and CTpr (from Ref. 183).
1522 J Clin Endocrinol Metab, April 2004, 89(4):1512–1525 Becker et al. • Clincal Review

at 1.5 h, and had normalized by 24 h. Similar patterns of suggest that this prohormone may possibly be a useful target
secretion occurred for the IL-1 receptor antagonist (IL-1ra), for therapeutic immunoneutralization in the human.
IL-6, and granulocyte colony stimulating factor and also sev-
eral other cytokines, some of which were even more transient Acknowledgments
(181, 182). Thus, these short-lived acute phase cytokine ele-
vations contrast with the extremely prolonged elevation of Received September 16, 2002. Accepted December 24, 2003.
serum CTpr after a systemic inflammatory episode in healthy Address all correspondence and requests for reprints to: Dr. Kenneth
humans; this suggests that CTpr not only have advantages L. Becker, Director of Endocrinology, Veterans Affairs Medical Center,
as excellent markers of sepsis but also may offer a durable 50 Irving Street NW, Washington, D.C. 20422. E-mail: klb1@erols.com.
target for therapeutic immunoneutralization, even several
days after the severe inflammatory illness has commenced. References
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