Expanding the Clinical Indications for α(1)-Antitrypsin Therapy.
α(1)-Antitrypsin (AAT) is a 52-kDa circulating serine protease inhibitor. Production of AAT by the liver maintains 0.9-1.75 mg/mL circulating levels. During acute-phase responses, circulating AAT levels increase more than fourfold. In individuals with one of several inherited mutations in AAT, low circulating levels increase the risk for lung, liver and pancreatic destructive diseases, particularly emphysema. These individuals are treated with lifelong weekly infusions of human plasma-derived AAT. An increasing amount of evidence appears to suggest that AAT possesses not only the ability to inhibit serine proteases, such as elastase and proteinase-3 (PR-3), but also to exert antiinflammatory and tissue-protective effects independent of protease inhibition. AAT modifies dendritic cell maturation and promotes T regulatory cell differentiation, induces interleukin (IL)-1 receptor antagonist and IL-10 release, protects various cell types from cell death, inhibits caspases-1 and -3 activity and inhibits IL-1 production and activity. Importantly, unlike classic immunosuppressants, AAT allows undeterred isolated T-lymphocyte responses. On the basis of preclinical and clinical studies, AAT therapy for nondeficient individuals may interfere with disease progression in type 1 and type 2 diabetes, acute myocardial infarction, rheumatoid arthritis, inflammatory bowel disease, cystic fibrosis, transplant rejection, graft versus host disease and multiple sclerosis. AAT also appears to be antibacterial and an inhibitor of viral infections, such as influenza and human immunodeficiency virus (HIV), and is currently evaluated in clinical trials for type 1 diabetes, cystic fibrosis and graft versus host disease. Thus, AAT therapy appears to have advanced from replacement therapy, to a safe and potential treatment for a broad spectrum of inflammatory and immune-mediated diseases.
By use of transgenic sheep that carry
the genetic sequence for human α
antitrypsin (AAT) under the control of
mammary gland promoter, biologically
active human AAT has been generated in
milk and purified and introduced to hu-
mans by intravenous infusion (1). By
using this method, it is estimated that a
population of 4,500 sheep would be able
to provide in a single year 5,000 kg
(~11,000 lb) of human AAT. Unfortunately,
the reaction of individuals who partici-
pated in the infusion trial was one of rapid
onset of fever due to the mounting of
human anti-sheep anti bodies against
residual sheep α
-antichymo trypsin. The
trial was discontinued. The original goal
of this endeavor was to provide sufficient
AAT to treat an increasing number of pa-
tients who are diagnosed with low circu-
lating levels of AAT. The current source of
AAT for augmentation therapy is human
plasma– derived affinity-purified AAT.
Yet, whereas the purpose of augmentation
therapy is to avoid the progression of
lung emphysema, efficacy studies that as-
sess this goal are incomplete (reviewed in
Parallel studies that examine various
attributes of human AAT depict the mol-
ecule as more than just an antiprotease.
AAT appears to effectively interfere with
inflammatory responses and protect
from cell death in an impressive variety
of in vivo (Table 1) and in vitro (Table 2)
experimental models (3). A noteworthy
example includes the blockade of in-
flammatory cytokine release from
human peripheral blood mononuclear
cells (PBMC) (4). Specifically, AAT de-
creases the production of important in-
flammatory cytokines such as tumor
necrosis factor (TNF)-α and interleukin
(IL)-1β, two prototypical upstream medi-
ators of inflammation. AAT also lowers
the levels of the chemokines IL-8 and
monocyte chemotactic protein (MCP)-1,
two major chemokines in the trafficking
of inflammatory cells. Whereas the activ-
ity of proinflammatory cytokines ap-
pears to consistently diminish in the
presence of elevated AAT, the release of
antiinflammatory mediators increases.
The endogenous inhibitor of IL-1 activ-
ity, IL-1 receptor antagonist, is upregu-
lated by AAT in human blood cells (5).
Similarly, IL-10 levels have been shown
to increase by AAT in various experi-
mental conditions (4,6–9). When examin-
ing the cellular targets of AAT, one finds
that these primarily include members of
the innate immune system, such as
macrophages and neutrophils, as well as
MOL MED 18:957-970, 2012 | LEWIS | 957
Expanding the Clinical Indications for α
Eli C Lewis
Ben-Gurion University of the Negev, Faculty of Health Sciences, Beer-Sheva, Israel
-Antitrypsin (AAT) is a 52-kDa circulating serine protease inhibitor. Production of AAT by the liver maintains 0.9–1.75 mg/mL cir-
culating levels. During acute-phase responses, circulating AAT levels increase more than fourfold. In individuals with one of several
inherited mutations in AAT, low circulating levels increase the risk for lung, liver and pancreatic destructive diseases, particularly
emphysema. These individuals are treated with lifelong weekly infusions of human plasma–derived AAT. An increasing amount of
evidence appears to suggest that AAT possesses not only the ability to inhibit serine proteases, such as elastase and proteinase-
3 (PR-3), but also to exert antiinflammatory and tissue-protective effects independent of protease inhibition. AAT modifies dendritic
cell maturation and promotes T regulatory cell differentiation, induces interleukin (IL)-1 receptor antagonist and IL-10 release, pro-
tects various cell types from cell death, inhibits caspases-1 and -3 activity and inhibits IL-1 production and activity. Importantly, un-
like classic immunosuppressants, AAT allows undeterred isolated T-lymphocyte responses. On the basis of preclinical and clinical
studies, AAT therapy for nondeficient individuals may interfere with disease progression in type 1 and type 2 diabetes, acute my-
ocardial infarction, rheumatoid arthritis, inflammatory bowel disease, cystic fibrosis, transplant rejection, graft versus host disease
and multiple sclerosis. AAT also appears to be antibacterial and an inhibitor of viral infections, such as influenza and human im-
munodeficiency virus (HIV), and is currently evaluated in clinical trials for type 1 diabetes, cystic fibrosis and graft versus host dis-
ease. Thus, AAT therapy appears to have advanced from replacement therapy, to a safe and potential treatment for a broad
spectrum of inflammatory and immune-mediated diseases.
Online address: http://www.molmed.org
Address correspondence to Eli C Lewis, Ben-Gurion University of the Negev, Faculty of
Health Sciences, PO Box 151, Beer-Sheva, Israel. Phone and Fax: +972-8-647-9981; E-mail:
Submitted June 1, 2011; Accepted for publication May 16, 2012; Epub (www.molmed.org)
ahead of print May 16, 2012.
B lymphocytes and dendritic cells. In
contrast, responses of purified T lym-
phocytes are consistently unaffected by
AAT (7,8,10–13), allowing for a variety
of responses to IL-2, as well as to
concanav alin A and anti-CD3/CD28
stimulation, to persist. This cell-specific
discretion, together with the ability to
protect tissues from injury, sets AAT in a
unique niche among modulators of the
immune system, a separate entity to
other antiinflammatory agents and clas-
As an acute-phase protein, AAT rises
in the circulation approximately fourfold
and remains elevated for a week to 10 d.
These levels are reproduced clinically by
so-called replacement or augmentation
therapy, using affinity-purified human
AAT (14). The standard protocol involves
lifelong weekly infusions that result in a
typical profile of systemic AAT: a spike
that reaches fourfold the normal values
in the first half of the week and a decline
958 | LEWIS | MOL MED 18:957-970, 2012
EXPANDING THE INDICATIONS FOR
Table 1. Selected
biological activities of AAT.
model Source and dose of AAT Outcomes Reference
Modulation of adaptive immunity
Islet allograft immune response Aralast, 60 mg/kg; matrigel- Graft survival prolonged, immune cell infiltration reduced, 10
embedded islets intragraft insulin content increased, intragraft VEGF
transcript levels elevated
Aralast, 60 mg/kg Grafts accepted, immune tolerance achieved, Tregs 8
Plasmid-derived hAAT, localized at graft sites, systemic and local IL-1Ra elevated
450 μg/mL plasma levels 148
Islet autoimmune response Aralast, 60 mg/kg; adeno- Islet function preserved, immune tolerance achieved, 7,84,159
associated delivery of auto- and alloreactive grafts accepted
Cell allograft immune response Aralast, 60 mg/kg Day 1–5 immune cell infiltration reduced, including 10
macrophages, neutrophils, T cells and NK cells
CIA Prolastin, 60 mg/kg Delayed disease onset, lower disease score 114,115
EAE Mice transgenic for hAAT, Decreased disease incidence, lower disease score, 6
constitutive 0.2 μg/mL increased Treg proportions in lymphoid compartments
GVHD (MHC disparate Aralast, 1–4 mg/dose Attenuation (posttreatment) and prevention 11,12
bone-marrow transplantation) (pretreatment) of GVHD, reduced expansion of
alloreactive T cells, enhanced recovery of Tregs,
reduced serum levels of proinflammatory cytokines
and superior survival
ThG-elicited peritoneal Aralast, 60 mg/kg Infiltrating macrophages and neutrophils diminished 10
Acute myocardial infarction Aralast, 60 mg/kg Myocardial leukocyte infiltration diminished 125
EAE Tissue-specific transgenic hAAT, Decreased spinal leukocytic infiltration 6
0.2 μg/mL plasma levels
Systemic LPS challenge (mice) hAAT plasmid–derived, Antiinflammatory serum cytokine profile, for example, 8
450 μg/mL plasma levels elevated IL-1Ra and IL-10 and greater levels of foxp3 Tregs
Lung LPS challenge (rabbits) hAAT Shanghai Biological Lung function and arterial blood gases improved, 3
Production Institute, bronchoalveolar neutrophil elastase, TNF-α and IL-8
120 mg/kg reduced
Toxic β-cell injury Aralast, 60 mg/kg 48-h cell death reduced, insulin release preserved 10,98
Acute myocardial injury after Aralast, 60 mg/kg Reduced infarct size, decreased caspase-1 tissue levels, 125
LAD occlusion and reduced post-infarct remodeling
CIA, Collagen-induced arthritis; EAE, experimental autoimmune encephalomyelitis; LAD, left anterior descending; ThG, thioglycolate.
toward background levels before the
next weekly infusion is afforded. Admin-
istration of AAT under these parameters
reveals both excellent patient safety and
patient compliance (15).
The attempts to generate transgenic
sheep for producing human AAT and
other methods for AAT mass production
represent a longstanding effort to gener-
ate much AAT for individuals who suffer
from genetic AAT deficiency. However,
the case for human AAT therapy outside
this particular indication appears to be
stronger than ever. In this review, an up-
date is provided on recent findings that
relate to the ability of AAT to protect tis-
sues as well as block unwanted inflamma-
tory processes and modify the immune
system in a beneficial and safe manner.
According to the published studies pre-
sented above, one can readily appreciate
MOL MED 18:957-970, 2012 | LEWIS | 959
Table 2. Selected
biological activities of AAT.
assay Cellular targets Source and concentration Outcomes Reference
Glucose-stimulated insulin Mouse islets Aralast, 0.25–0.5 mg/mL Cytokine-dampened insulin 10
secretion release restored
Human islets Aralast, 0.5 mg/mL Impure islet culture insulin 160
β-cell lines Prolastin, 0.125–1 mg/mL Insulin release improved 82
Collagen I production during Skin fibroblasts Human AAT (Calbiochem), Production increased 161
wound healing 0.1 μg/mL
Proinflammatory cytokine toxicity Primary mouse islets Aralast, 0.25–0.5 mg/mL LDH release diminished 10
Rat INS-1 cell line Prolastin, 0.5 mg/mL Cell death reduced 82
β-cell–specific toxin Murine MIN-6 cell line Prolastin, 0.5 mg/mL Apoptosis reduced 98
Caspase-3–induced apoptosis Primary lung alveolar hAAT (Calbiochem), Apoptosis reduced 60
endothelial cells 0.05 mg/mL
LPS and ischemia-induced injury Adult cardiac myocyte Aralast, 4.0 mg/mL Cell death reduced 125
cell line HL-1
Immune cell cytokine production
LPS stimulation Human PBMC Prolastin, 0.5 mg/mL Reduced proinflammatory 162
Human PBMC Aralast, 8.0 mg/mL Reduced proinflammatory 4
stimulation cytokine release
Steady-state Nonstimulated murine Prolastin, 0.5 mg/mL Steady-state BAFF production 114
B cells decreased
LPS stimulation Human neutrophils Human neuronal cell line– Reduced TNF-α release 163
derived recombinant hAAT,
0.5 mg/mL; prolastin,
Mixed lymphocyte reaction Human PBMC Aralast, 0.1–0.5 mg/mL Reduced IL-32 11
Mixed lymphocyte–DC reaction Murine OT-II T cells Aralast, 0.5 mg/mL Reduced IL-6, elevated IL-2 9
and OVA-loaded DC and elevated IL-10
Immune cells not directly targeted
immunization Mouse splenocytes Prolastin, 0.5 mg/mL Intact T-cell clumping, 114
proliferation, response to
IL-2 and IFNγ release
Concanavalin A stimulation Mouse splenocytes Aralast, 0.5 mg/mL 10
CD3/CD28 stimulation Purified mouse T cells Aralast, 0.5 mg/mL 7,12
BAFF, B-cell activating factor; DC, dendritic cell;
that AAT has a potential benefit for an
impressive broad spectrum of human
diseases, reigniting the requirement for
yet greater supplies of human plasma–
derived AAT, human AAT generated by
recombinant techniques or, at a minimum,
biologically active AAT fragments.
AN OVERVIEW OF α
AAT is a 52-kDa glycoprotein that
earned its name by virtue of being the
major serum trypsin inhibitor, occupy-
ing the α-1 globulin fraction of electrical
current–separated serum proteins (16).
AAT belongs to the 1,500-member fam-
ily of serine protease inhibitors (SER-
PINs), thus also termed SERPINA1.
Other acronyms may include A1AT and
A1PI. Nevertheless, as discussed below,
some nonserine proteases are also inhib-
ited by AAT, and, in addition, some ac-
tivities of AAT may be unrelated to pro-
tease inhibition altogether, an important
aspect to consider as far as its widely ac-
cepted mechanism of action and release
criteria upon purification for clinical
Circulating AAT is controlled mainly
by the liver. Hepatocytes are responsible
for the steady-state constitutive circulat-
ing levels of 0.9–1.75 mg/mL AAT, al-
though these ranges might slightly vary
in the literature. Hepatocytes are also re-
sponsible for IL-1/IL-6–inducible AAT
production during inflammation. Levels
of AAT also increase in the circulation
during normal pregnancy (17) and in the
process of aging (18). The half-life of
AAT in the circulation is 3–5 d (16). In
contrast, lung type II alveolar epithelial
cells are primarily responsible for inter-
stitial AAT in the lung (19). In addition
to hepatocytes and alveolar epithelial
cells, AAT is expressed by monocytes
and macrophages, neutrophils, endothe-
lial cells, human intestinal paneth cells,
endometrial cells and other types of ep-
ithelial cells, as well as by human pan-
creatic islet α and δ cells. This list of cell
sources likely does not contribute to sys-
temic AAT levels but rather to local,
inflammation-driven and hypoxia-driven
inducible AAT levels.
Disorders in the levels of circulating
AAT allow for the degradation of lung
tissue that leads to the characteristic man-
ifestation of pulmonary emphysema (20).
The decline in circulating AAT levels
below 0.5 mg/mL represents a severe
form of deficiency, affecting 1 in
1,600–5,000 Caucasian individuals of
western European origin (21). A com-
monly encountered mistaken concept is
that individuals with genetic AAT defi-
ciency do not synthesize AAT and that
administration of AAT to individuals
with normal AAT levels might be prob-
lematic. Yet the molecule is readily pro-
duced by hepatocytes and some amounts
do appear in the circulation in patients
with AAT deficiency. However, most of
the protein forms aggregates inside the
producing hepatocytes, resulting in endo-
plasmic reticulum stress and cellular
damage that can progress into hepatocyte
autophagy and liver organ injury (22). In-
deed, AAT deficiency represents the most
common inherited condition that leads to
liver transplantation in infants, children
and adults. The presence of a null varia-
tion in humans has been reported in two
families, yet, intriguingly, its recently de-
veloped animal counterpart in the form
of a genetically engineered knockout
mouse for AAT proves nonviable (23).
Unlike the AAT knockout mouse, the
phenotype of the elastase knockout
mouse is near normal (24). The reason for
nonsustainable life in the AAT knockout
mouse remains unknown.
Molecular Profile of AAT
AAT is comprised of 394 amino acids.
The prototypical antiproteolytic function
of AAT is contained within a nine–amino
acid reactive center loop (RCL) that stems
out of the globular structure and is com-
prised of a primary sequence that forms
“the perfect bait” for a highly specific se-
quence-directed set of proteases. Elastase,
for example, will bind to the RCL in an at-
tempt to cleave a targeted peptidic bond
between amino acids 358 and 359, only to
remain irreversibly bound to its cleaved
product. As a consequence, cleaved AAT
undergoes refolding and a binding site
becomes exposed that exhibits high affin-
ity to a receptor for the newly formed
AAT:elastase complex, termed
SERPIN:enzyme complex (SEC) receptor
(25). Cells that express the SEC receptor
will internalize the inactive complex, as
readily occurs in hepatocytes. Interest-
ingly, the domain that is required for AAT
to bind to the SEC receptor after an en-
counter with a serine protease is a pen-
tapeptide with the sequence FVFLM, lo-
cated at positions 370–374 (26). These five
amino acids are located within a
36–amino acid C-terminal stretch, span-
ning amino acids 359–394. The so-called
C-36 peptide was shown to attract neu-
trophils (27), as well as activate mono-
cytes (28,29) and display atherogenic
properties (30,31). The sequence is highly
conserved among SERPINs and, intrigu-
ingly, is also mostly hydrophobic. This lat-
ter trait allows the C-36 peptide to engage
with hydrophobic lipid molecules, such as
cholesterol. The remainder portion of
AAT outside the RCL and the C-36 pep-
tide appears to have no reported binding
partners, yet is strikingly conserved
within members of the SERPIN family.
Molecular Targets of AAT Closely
Relate to Inflammation
Protease inhibition by AAT reduces in-
flammation. For example, AAT binds to
and inactivates elastase, trypsin and
proteinase-3 (PR-3), the activity of which
includes proteolytic cleavage of a specific
cassette of membrane protein receptors
called protease-activated receptors
(PARs). In the presence of inactivated pro-
teases, such as in the presence of excess
AAT, PARs lack their primary trigger for
activation. Once cleaved, PARs undergo a
conformational change and initiate an in-
tracellular signaling cascade. The role of
PAR-1 through PAR-4 during inflamma-
tory responses, as well as during innate
and adaptive immunity, appears essential
(32). For example, PAR-1 awaits an
N-terminal cleavage event to expose a
self-binding site within one of its loops,
which will cause rapid increase in intra-
cellular calcium levels and subsequent
MCP-1 release. Similarly, PAR-2 has its
960 | LEWIS | MOL MED 18:957-970, 2012
EXPANDING THE INDICATIONS FOR
N-terminal self-binding site unavailable
until cleaved by serine proteases; the re-
ceptor then facilitates release of IL-1β,
IL-6, IL-8 and TNF-α and increases neu-
trophil motility. Dendritic cells mature on
activation of PAR-2; accordingly, bone
marrow cells from mice that lack PAR-2
fail to mature under in vitro protocols (33).
Protease- activated receptors are also im-
plicated in cardiovascular diseases (34),
implying that intervening with their acti-
vation by AAT may interfere with rele-
vant pathological pathways. PARs are
also implicated in inflammatory gastro -
intestinal and mucosal disorders (32),
since they are expressed in gut epithelial
cells, mast cells, nerve cells and smooth
muscle cells. In patients with inflamma-
tory bowel disease, PAR-1 is expressed at
high levels and PAR-1 antagonism ame-
liorates inflammation in the respective an-
imal model (35). Each of the four PARs is
expressed by cells of the central nervous
system, and their activating proteases can
be produced locally or enter through a
breached blood-brain barrier, such that
might be encountered in central nervous
system–related pathologies (35). In an ani-
mal model for multiple sclerosis, a role for
PAR-2 was established; accordingly, mice
that lack PAR-2 displayed diminished dis-
ease progression (36). That said, a role for
AAT in controlling protease-related
events outside the cells does not preclude
the presence of other aspects of AAT
activity—some intracellular and some
altogether unrelated to the activity of
Some targets for AAT inhibition are,
surprisingly enough, not serine pro-
teases. Aggrecanase-1 (ADAMTS-4) is in-
volved in the pathogenesis of rheuma-
toid arthritis and was recently shown to
be inhibited by AAT (37). AAT also tar-
gets the metalloproteinase MMP-9
(gelatinase B) (38), an important IL-1–
inducible protease that is suspected of
contributing to the progression of cardio-
vascular disease, as well as to rheuma-
toid arthritis, chronic obstructive pul-
monary disease (COPD) and multiple
sclerosis (39). Inhibition of calpain-1 by
AAT was recently reported (40). Calpain-
1 processes the precursor of IL-1α, a
proinflammatory intracellular IL-1 fam-
ily member that is constitutively ex-
pressed in nearly all epithelial cells. It
was recently established that blockade of
calpain-1 by AAT results in neutrophil
polarization and random migration (40).
Binding Targets That Are Unrelated to
Binding of IL-8. AAT directly binds to
the major neutrophil chemoattractant
IL-8 (41). Blockade of IL-8 may provide
benefit during diabetic retinopathy,
sickle cell disease, transfusion-related
acute lung injury, acute respiratory dis-
tress syndrome, renal microvasculopathy,
acute coronary artery syndrome and
stroke. In parallel, neutrophils are tar-
geted by AAT, primarily by the blockade
of granule- and membrane-contained
serine proteases (42). Added to the find-
ing of reduced IL-8 release in the pres-
ence of AAT (40), it is not unexpected
that lack of AAT, as occurs in lungs of in-
dividuals with genetic AAT deficiency,
results in excessive mobilization of neu-
trophils into the parenchyma.
Binding of heat shock proteins. In
general, heat shock proteins (HSPs) fit the
definition of dual-function molecules: in-
side the cells, they chaperone proteins for
proper folding, yet when they leak out
from a failing cell membrane, for exam-
ple, during necrosis, they function as im-
mune adjuvants and participate in in-
flammatory responses, whether alone (43)
or in complex with other inflammatory
mediators (44). Elevated levels of HSP70,
for example, were found to be present in
the plasma of type 1 diabetic individuals
but not in plasma from healthy individu-
als (45). Affinity chromatography fol-
lowed by immunoprecipitation and im-
munoblot analyses of HSP-enriched,
plasma-purified fractions revealed that
HSP70 is closely linked to AAT in these
patients. Whether it is its functional role
as a chaperone of aberrant proteins that
has HSP70 clinging to plasma AAT, or
perhaps an inherent ability of HSP70 to
directly engage with AAT, remains to be
confirmed. Nevertheless, the potential in-
jurious properties of extracellular HSP70,
particularly in the context of islet β-cell
biology, may favor its blockade.
Direct association of AAT with lipid
rafts and low-density lipoproteins. AAT
was shown to localize within lipid rafts
in human monocytes (46). In an elegant
study in which cells from AAT-deficient
individuals were investigated, the lipid
raft-bound form of AAT was shown to
originate from the circulation, and not
the cell cytoplasm, an important aspect
when considering a clinically relevant
administration route. Interestingly, a di-
rect association was demonstrated be-
tween AAT and cholesterol; the associa-
tion of AAT with monocytic membranes
is enhanced by free cholesterol, and AAT
appears to deplete lipid raft cholesterol
as well as inhibit oxidized low-density
lipoprotein (LDL) uptake. In addition,
studies point at a direct relationship be-
tween AAT and apolipoprotein B on LDL
particles (47,48), and AAT-LDL com-
plexes have been detected in human ath-
erosclerotic lesions (48). In support of a
therapeutic implication for such an asso-
ciation, a protective role for AAT in ath-
erosclerosis was demonstrated in the
Lipid Coronary Angiography Trial that
evaluated male participants after coro-
nary bypass surgery (49); the authors of
the trial concluded that low AAT levels
are associated with increased occurrence
Binding to human immunodeficiency
virus entry proteins. Initially described
by Shapiro et al. (50), infection of whole
human blood in vitro with human immu-
nodeficiency virus (HIV)-1 does not
occur, whereas whole blood from AAT-
deficient patients is readily infected (50).
This observation suggests that endoge-
nous levels of AAT prevent infection of
HIV-1. In fact, adding exogenous human
AAT to latently-infected cell lines re-
duced production of HIV-1 in vitro (49).
Thus, steady-state levels of AAT appear
to provide a certain degree of HIV-1 in-
fection inhibition. Subsequently, a screen-
ing study of ultra-filtered human plasma
and protein sequencing revealed that, in-
deed, the major plasma-derived inhibitor
MOL MED 18:957-970, 2012 | LEWIS | 961
of HIV-1 is circulating human AAT (51).
Accordingly, it was later demonstrated
that HIV-1 infection is associated with re-
duced serum AAT concentrations (52). A
C-terminal 26-residue peptide fragment
of AAT, the result of internalized and
cleaved AAT, was found to be a direct in-
hibitor of viral infection (53). Recently, a
10-d intravenous monotherapy course
using a peptidic derivative of the
C-terminal of AAT reduced viral RNA
levels in treatment-naive, HIV-1–infected
individuals without causing adverse
effects (54). Such activity of AAT is essen-
tially devoid of serine protease inhibition.
Induction of vascular endothelial
growth factor. In various studies, AAT
has been shown to induce vascular en-
dothelial growth factor (VEGF) produc-
tion (55–57) and to prevent proteolytic
degradation of VEGF (58). In addition,
AAT was shown to facilitate smooth
muscle myocyte migration and prolifer-
ation (59) and reduce endothelial cell
and smooth muscle cell apoptosis
(55,56, 60,61). In fact, the pathological
hallmark of AAT deficiency, pulmonary
emphysema, can be observed in animal
models in which the VEGF signaling
pathway is blocked in the presence of
normal local levels of AAT (62–64), sug-
gesting that AAT deficiency-related em-
physema is the result of insufficient
AAT-induced VEGF, and a subsequent
collapse of the capillary bed. Indeed, the
promoter for AAT contains the hypoxic
response element for HIF-1α, and AAT
is upregulated during hypoxia (65). In-
terestingly, such association cannot be
reproduced by mimics of serine pro-
Thus, whereas AAT is a textbook exam-
ple of actions afforded by its antiprotease
properties, increasing amounts of data are
emerging that support the concept by
which domains within AAT that are inde-
pendent of protease binding can provide
clinically beneficial functions (66).
Cellular Targets of AAT
As early as 1978, it was reported that
AAT does not engage with T lympho-
cytes, but rather brings along diminished
T-cell responsiveness in the context of
particular experimental setups in an in-
direct manner (13). As confirmed in sev-
eral recent reports, AAT indeed does not
interfere with the biological activity of
IL-2 (7,8,10–12), allowing T cells to re-
lease interferon (IFN)-γ and proliferate in
an undeterred manner in the presence of
IL-2. With the benefit of having intact
IL-2 signaling, T regulatory cells (Tregs)
can readily differentiate, particularly
when considering that levels of IL-6 are
reduced by AAT (4,9,10), a prerequisite
for directing of naive T cells away from
Th17 (67). Additionally, Th17 cells rely
on an intact IL-1 pathway (68), a willing
cytokine-related target of AAT. Thus, the
expansion of Tregs in animal models
that incorporate AAT distinguishes the
therapeutic efficacy of AAT from IL-2–
How are T cells affected by AAT
without being direct cellular targets?
Innate cells appear to undergo marked
changes in the presence of added AAT.
Neutrophils, in particular, are disarmed
(40,42,69). In fact, the local shortage of
AAT in lungs of patients with genetic
AAT deficiency are readily associated
with an influx of neutrophils with injuri-
ous consequences (20). Dendritic cells
and macrophages are modified; dendritic
cells become semi-mature (9), a state as-
sociated with reduced co-stimulatory
abilities, excess IL-10 production and fa-
cilitation of antigen-specific Treg expan-
sion (8). Interestingly, Ozeri et al. (9)
demonstrated that AAT promotes semi-
mature IL-10–producing and readily mi-
grating dendritic cells, allowing the cells
to reach the draining lymph nodes and
exert their tolerogenic functions. In this
study, an intriguing uncoupling of in-
flammation-mediated elevation in the
dendritic cell chemoattractant receptor
(CCR7) was observed, whereby AAT ap-
pears to have allowed for persistent
CCR7 surface expression but had down-
regulated other dendritic cell inflamma-
tory markers (9). Indeed, in the whole an-
imal and, specifically, in lymph nodes
during an antigenic event, IL-10 produc-
tion rises as a consequence of AAT ther-
apy (6–9). Moreover, ex vivo AAT-treated
graft-derived dendritic cells were shown
to communicate a tolerogenic profile
across to the host dendritic cells, causing
the host cells to increase IL-10 production
(9). B cells represent a lymphocyte with a
biological response profile that is closer
to the innate system than to the adaptive
immune system; indeed, B-cell activation
is, in part, inhibited by AAT (70).
These cell types are the topic of ongo-
ing research with particular attention to-
ward a tissue-protective antiinflamma-
tory tolerogenic function, such that
would drive Tregs to predominate with-
out the T cells ever engaging directly
AAT DEFICIENCY POINTS TO POTENTIAL
INDICATIONS FOR AAT THERAPY
AAT deficiency is largely underdiag-
nosed. An asymptomatic drop in levels
of AAT to as low as 85% of normal circu-
lating levels can readily be detected in
healthy individuals by random evalua-
tion (71). Moreover, serum AAT concen-
trations may not be representative of the
functional capacity of the antiprotease
aspect of AAT, since inactivated forms
might falsely ascribe to normal “im-
munogenic” levels of the molecule, as
detected by Western blot or enzyme-
linked immunosorbent assay (72). It is
also suggested that failure to elevate
AAT under physiological conditions
might present as a novel relative func-
tional deficiency. Systemic conditions
that have been associated with AAT
deficiency include panniculitis, vasculi-
tis, pancreatitis, glomerulonephritis,
bronchiectasis and asthma; all are charac-
terized by excessive inflammation (73).
In more recent literature, polymorphism
studies revealed several unexpected dis-
eases that might be associated with mod-
erate AAT deficiency (non-M polymor-
phisms), such as fibromyalgia, mood
disorders and intense creative energy
(74,75). Although frank deficiency in
AAT renders the patient eligible for AAT
augmentation therapy, none of the condi-
tions above are included as labeled indi-
cations for AAT.
962 | LEWIS | MOL MED 18:957-970, 2012
EXPANDING THE INDICATIONS FOR
ANTIINFLAMMATORY THERAPIES POINT
TO NOVEL INDICATIONS FOR AAT
The role of inflammation in the patho-
genesis of several diseases cannot be
overstated. Yet, some illnesses have not
been associated with inflammation and,
subsequently, appear to have attracted
therapeutic approaches using antiinflam-
matory agents. Blockade of inflammatory
pathways by these agents in clinical tri-
als has provided proof of concept for the
potential benefit that would be gained by
incorporation of AAT into treatment
Type 2 Diabetes
Type 2 diabetes is associated with the
failure of insulin to communicate intra-
cellular signals upon engagement with
its receptor. The current therapeutic ap-
proach primarily centers around diet and
exercise, plus manipulation of liver cells
to halt endogenous glucose production
and attempts to enhance insulin sensitiv-
ity and insulin release; these approaches
are joined by few and recent agents that
also target inflammation (76). The ration-
ale for the latter approach is that insulin
signaling is negatively affected by active
inflammatory signals. Under physiologi-
cal conditions, inflammation evolves into
local and systemic insulin resistance, cre-
ating a temporary and desired rise in
circulating glucose. Blockade of inflam-
matory pathways might thus salvage in-
sulin responsiveness. Indeed, a reduction
in the levels of glycated hemoglobin
) has been reported in a clinical
trial that examined the effect of a deriva-
tive of aspirin, salsalate (77). In a similar
manner, blockade of IL-1 by using the
IL-1 receptor antagonist anakinra (78),
and also by using an antibody to IL-1β
(79), provides consistent outcomes. In
addition, it has become widely accepted
that islet β-cell injury is inherent to dis-
ease pathogenesis. Chronic high glucose
and fatty acid levels exert direct β-cell
toxic effects (80), and IL-1β has been
shown to be highly toxic for β cells (81).
Protection of islets from inflammatory
cytokine-mediated injury has been
widely reported in both mouse and
human islets (7,8,10,82–86). Support for
an apparent association between the lack
of protection by AAT and type 2 diabetes
may be found in a recent report that de-
scribes low circulating AAT levels in 50%
of type 2 diabetic patients (87). It remains
to be determined whether insulin signal-
ing and systemic glucose control can im-
prove in the presence of added AAT, and
whether AAT can ameliorate β-cell injury
in the case of chronic elevated glucose or
fatty acid levels and can thus benefit
type 2 diabetic patients.
Type 1 Diabetes
Formerly termed “juvenile diabetes,”
type 1A autoimmune diabetes harbors an
elaborate antigen-specific attack on
β cells. Thus, protection of islets from in-
jury would appear to be the obvious tar-
get in type 1 diabetes, yet many trials
have been primarily directed at the
T-cell–mediated autoimmune arm of dis-
ease. In the advent of an apparent failure
of recent clinical trials that incorporate re-
moval of T cells using anti-CD3 antibod-
ies (88), the fact that blockade of inflam-
mation appears as effective, if not more
effective, in the ideal context than im-
munosuppression is of great importance.
A factor absent in classic immunosup-
pression that predominates in antiinflam-
matory approaches may be a metabolic
one, such that facilitates insulin receptor
responsiveness. IL-1 is a target in this
context, both as a relevant provocateur of
inflammation and also as a direct and po-
tent β-cell toxic agent (89). In a recent
clinical trial, IL-1 blockade was evaluated
in 15 children within 1 wk of type 1 dia-
betes diagnosis using daily anakinra for
28 d. As a consequence, compared with
controls, insulin requirement was re-
duced for the duration of 4 months (90).
Nonfunctional circulating AAT has
been shown to exist in most individuals
with type 1 diabetes (72,91–95). The loss-
of-function relates, most probably, to ex-
cessive nonenzymatic glycation. More-
over, in the autoimmune animal model
for type 1 diabetes, the nonobese diabetic
(NOD) mouse, AAT levels were half the
levels found in the majority of other wild-
type mouse strains (96). Indeed, preclini-
cal data show a consistent benefit with
AAT in the protection of islets and modi-
fication of immune systems across multi-
ple models of diabetes (7,8,10,84, 96–98).
Furthermore, after NOD mice revert to
normoglycemia by a 14-d course of AAT,
grafted autoreactive β cells are accepted in
the animals in the absence of subsequent
requirement for exogenous AAT (7).
The fact that AAT is endogenously
produced under inflammatory stimuli by
both mouse and human islet cells
(99,100) strengthens its relevance for type
1 diabetes. Protection of grafted human
islets for individuals with type 1 diabetes
may thus incorporate AAT as both an
islet protector and a tolerogenic agent,
addressing several of the current multi-
ple goals for successful long-term human
islet transplantation outcomes. As for re-
cently diagnosed type 1 diabetic patients,
three clinical trials are currently running
that study AAT in youngsters with type
1 diabetes (National Institutes of Health
clinical trial registry NCT01304537,
NCT01319331 and NCT01183468), all in-
corporating as part of their inclusion cri-
teria that residual islet function be meas-
urable before initiation of treatment with
weekly intravenous infusions of AAT.
Inflammation in COPD, both acute
and chronic, is key in disease progres-
sion. Whereas environmental assaults,
such as cigarette smoke, dust or pollu-
tion directly contribute to an inflamma-
tory flare, the process of airway inflam-
mation ensues long after the trigger is
gone. An association between COPD and
AAT may not require that a genetic defi-
ciency in AAT be present in patient back-
ground, since evidence for protease/
antiprotease imbalance can be found also
in COPD patients with normal AAT lev-
els (101). Pharmacologically speaking,
lungs present with a practical benefit: in-
haled approaches have gained advances
in the introduction of antiinflammatory
agents. Inhaled nonsteroidal antiinflam-
matory drugs, for example, create signifi-
MOL MED 18:957-970, 2012 | LEWIS | 963
cant improvement in disease parameters
(102). The effects of inhaled AAT in mice
are consistent with protection from in-
flammation and tissue damage caused by
cigarette smoke (69). With the recent de-
velopment and approval of an inhaled
preparation of AAT (103), one can expect
local lung concentrations of AAT to be
achieved at low doses, sparing both sys-
temic delivery of AAT and cost.
Even in the absence of demonstrable
infection, inflammation is evident in
lungs of cystic fibrosis patients (104).
Antiinflammatory agents have proven
effective, at most, yet at times result in
adverse effects such as in the case of
chronic oral corticosteroids. Given by
inhalation, some agents display a wider
therapeutic index (102). As such, in-
haled AAT has proven effective in re-
ducing inflammation in cystic fibrosis
patients in multiple reports (studies
compiled in ). The effect of AAT on
neutrophils, both as an inhibitor of IL-8
function (42) and also as an avid in-
hibitor of injurious neutrophil serine
proteases, supports its use in this partic-
ular clinical indication.
Graft versus Host Disease
A limiting factor in the success of
hematopoietic stem cell transplantation,
graft versus host disease (GVHD) re-
mains difficult to control. Immunosup-
pression, intended to blunt the aggres-
sive immune response against the host
tissues, exposes the individual to oppor-
tunistic infections and impairs graft-
versus-leukemia responses. There is a
need to develop immune modulating
agents that can allow T-cell–mediated
graft-versus-leukemia responses while
sparing the recipient from an injurious
inflammatory and immunological as-
sault. Given the ability of AAT to modify
dendritic cells toward a tolerogenic phe-
notype and to facilitate the expansion of
Tregs (6,8,9), together with its potent an-
tiinflammatory profile and outstanding
safety record, AAT is an attractive candi-
date to address acute GVHD. Several
lines of evidence support this approach.
Of particular interest, a recent report de-
scribes an elevated loss of AAT during
intestinal GVHD as a measure of GVHD
severity in children (106). In addition, ev-
idence points to a significant involve-
ment of IL-1 during the progression of
GVHD, albeit mostly in advanced stages
of the pathology (107); in 16 of 17 pa-
tients with steroid-resistant GVHD, a 7-d
continuous intravenous infusion of re-
combinant IL-1 receptor antagonist re-
duced the severity of the disease (108).
However, the sole blockade of IL-1 dur-
ing the development of GVHD was less
effective (109). Because cell injury ap-
pears to fuel GVHD (110), the ability of
AAT to provide tissue protection may re-
sult in a synergistic advantage to cyto -
kine modification. Indeed, in several
animal models for GVHD, AAT pro-
vided clear benefit in immune profile,
animal weight and cohort survival
(11,12). Its role in blocking serine pro-
tease PR-3–related IL-32 activation fur-
ther denotes a possible mechanism for
protection during GVHD, particularly in
light of the recent report of elevated
blood IL-32 mRNA transcripts in 10
acute GVHD patients compared with 5
GVHD-free allogeneic hematopoietic cell
transplant recipients (11).
The progression of rheumatoid arthri-
tis (RA) involves the maintenance of an
inappropriate inflammatory process by
immune cells (111). Biologics include
systemic or local blockers of TNF-α and
IL-1β, and an anti-IL-6 receptor antibody
that neutralizes the effector function of
IL-6 (111). These particular cytokines are
inhibited by AAT at several levels, in-
cluding both their release and function,
suggesting that AAT may serve to pre-
vent the positive inflammatory feedback
loop that appears to perpetuate the dis-
ease. In fact, a relationship between AAT
inactivation and TNF-α concentrations
in the synovial fluid of patients with
rheumatoid arthritis was described (112).
In addition, inhibition of neutrophil elas-
tase has been shown to interfere with
disease progression in respective animal
models (113). Thus, it is not unexpected
that AAT was recently shown to delay
arthritis development in a mouse model,
both in the form of injected human ma-
terial and in the form of adenoviral plas-
mid-derived circulating human AAT
(114,115). In the particular case of IL-1
processing, the role of non–caspase-1 ex-
tracellular processing of the IL-1β pre-
cursor has been shown to incorporate
enzymatic targets of AAT, such as elas-
tase, cathepsin G and PR-3 (107). In this
particular context, gout is another excel-
lent candidate that would benefit from
blockade of IL-1 activities by AAT in
Inflammation is a therapeutic target in
neurological disorders (116). In addition,
the metalloproteinase MMP-9 appears to
take part in the pathogenesis of multiple
sclerosis (MS) and has been shown to be
a target for AAT inhibition (38). Whether
insufficient AAT plays a role during the
disease is not yet established, although
mutation analysis has detected the pres-
ence of inactive alleles of AAT in individ-
uals with MS (117,118). Additionally,
mice that express human AAT in their
circulation are protected from disease
course in the respective mouse model of
multiple sclerosis (6). In that study, the
small proportion of AAT-treated animals
that did display initial signs of pathology
after disease induction overlapped the
timing of the control sick group as far as
initial signs of neuronal damage, but
then exhibited a rapid regression in
symptoms. Another support for the rele-
vance of AAT therapy for MS patients is
the detection of biologically active ele-
vated levels of AAT in the cerebrospinal
fluid of patients with MS (119).
Inflammatory Bowel Disease
Although several studies suggest an
imbalance of protease activities during
inflammatory bowel disease (IBD), corre-
lation appears to exist between AAT defi-
ciency and IBD only in populations with
a severe type of genetic AAT deficiency
964 | LEWIS | MOL MED 18:957-970, 2012
EXPANDING THE INDICATIONS FOR
(PI*ZZ) (120,121). Nevertheless, consider-
ing that intestinal paneth cells are potent
producers of inducible AAT (122), its in-
volvement in IBD as being produced
extra-hepatically may be relevant.
Ischemic Heart Disease
Approaches to diminish inflammatory
pathways during myocardial injury
show benefit in various parameters of
cardiomyocyte viability and cardiac
function (123). Whereas inflammation
might display a positive role in cardiac
repair and scar formation (124), the par-
ticular ability of AAT to block arms of in-
flammation while preserving tissue in-
tegrity may place AAT as an attractive
antiinflammatory agent in the context of
cardiac damage and repair. In addition,
cells of the innate immune system take
part in the injury that follows myocardial
reperfusion, serving as another target for
AAT inhibition. In a study reported by
Toldo et al. (125), AAT-treated mice had
significantly smaller infarct sizes (–30%
on d 1 and –55% on d 7) compared with
mice treated with albumin. Also, AAT
treatment resulted in >90% reduction in
caspase-1 activity in homogenates of
hearts 24 h after ischemia reperfusion.
Seven days after acute myocardial infarc-
tion, AAT-treated mice exhibited supe-
rior cardiac function. The increase in
caspase-1 activity in cardiomyocytic
HL-1 cells induced by lipopolysaccharide
(LPS) and nigericin or after ischemia was
reduced by >80% and cell death by >50%
in the presence of AAT. As suggested by
Daemen et al. (126), there is demonstra-
ble protection from ischemia reperfusion
injury in renal mouse models by AAT, in-
cluding renal function. The authors of
that study conclude that exogenous ad-
ministration of AAT may provide new
therapeutic means of treatment for ische-
mia reperfusion injury.
AAT rises during normal pregnancy
(17). An intriguing association appears
to exist between AAT inactivity and pre-
term premature rupture of membranes.
The relationship between trypsin activ-
ity in the amniotic fluid and premature
rupture of membranes has been de-
scribed in early reports (127), and AAT
is found at subnormal levels in amniotic
fluid obtained from patients with pre-
term premature rupture of membranes
(128). The source of AAT was identified
as human amniotic epithelial cells.
Screening for levels of AAT is not a stan-
dard blood test in pregnant women, es-
pecially not at late stages of pregnancy.
However, should an association between
AAT and normal pregnancy be estab-
lished, it might be strongly proposed to
incorporate plasma AAT testing in the
third trimester of pregnancy, at which
point most normal pregnancies would
exhibit circulating AAT levels greater
than nonpregnant normal values (128).
SAFETY OF PROLONGED AAT THERAPY
Safety Demonstrated in Prolonged
Clinical Trials with AAT
Although naturally existing, AAT might
be considered a biological once intro-
duced to patients over prolonged periods
of time and in excess (similar to other nat-
urally occurring molecules that are repre-
sented by drugs, such as CTLA-4 and IL-1
receptor antagonist). Yet experience with
humans that receive AAT under protocols
that are comprised of prolonged periods
of time and excess material has proven
safe. With a focus on the United States,
extrapolation of data from population-
based screening studies, evaluations of
patients with COPD and genetic epidemi-
ologic surveys lead to an estimated
60,000–90,000 Americans with the severe
type of genetic AAT deficiency (PI*ZZ).
However, AAT deficiency is highly under-
diagnosed, and of the estimated numbers
above, only about 6,000 will have been di-
agnosed as having AAT deficiency, as of
the year 2009 (129). Administered to these
patients once weekly for the past 3 dec-
ades, serum AAT levels increase to ap-
proximate the concentrations of an acute-
phase response for the first part of the
week and then decline to normal values
in the second half of the week (14). Since
these treated individuals exhibit no ad-
verse effects, the molecule is considered
safe for chronic therapy. In addition, as
suggested by Churg et al. (130), added
benefit is demonstrated when administer-
ing AAT, even in the absence of AAT defi-
ciency (130). Studies demonstrate high pa-
tient compliance, demonstrate no
evidence of compromised host defense,
such as opportunistic infections or reacti-
vation of Mycobacterium tuberculosis, and
actually report reduced frequency of
pneumonia incidents (131). In addition, in
the presence of elevated systemic levels of
AAT in mouse models, tumor angiogene-
sis appears to be compromised (132) and
metastasis appear to lose protease-
dependent migratory capabilities (133).
AAT Is an Antibacterial
SERPINs possess antibacterial func-
tions (134). Findings that relate to the
ability of AAT to inhibit bacterial expan-
sion include binding to the bacterial-
essential furin (135), as well as undergo-
ing S-nitrosylation in the presence of
nitric oxide (136), both actions that are
independent of protease inhibition. The
observation that the virulence of E. coli is
increased by IL-1 (137) suggests that
blockade of IL-1 by AAT may interfere
with bacterial growth, a finding corrobo-
rated by reduced numbers of live S.
pneumoniae in infected mouse lungs (EC
Lewis, unpublished observations). In this
context, notably, the role of AAT in re-
ducing bacterial load involves tissue
modulation, that is, reducing the levels
of tissue-derived bioactive IL-1. Indeed,
the protection from LPS mortality in a
model of 24-h pretreatment with low-
dose systemic IL-1 is attributed to induc-
tion of acute-phase proteins (138). Clini-
cal studies indicate that augmentation
therapy with AAT for genetically defi-
cient patients effectively attenuates mi-
crobial colonization, as well as the fre-
quency and severity of acute COPD
exacerbations (131,139–143). Moreover,
the fact that cystic fibrosis patients bene-
fit from AAT inhalation, even when in-
troduced during active lung infections
(Staphylococcus and Pseudomonas) (144),
further supports the claim that host de-
MOL MED 18:957-970, 2012 | LEWIS | 965
fenses are intact, if not enforced, in the
presence of elevated AAT levels.
ADDRESSING AAT FOR EXPANDED
The 8th World Congress on Trauma,
Shock, Inflammation and Sepsis - TSIS
2010, held in Munich, Germany, hosted a
symposium titled “Alpha-1-Antitrypsin
(AAT) as a Novel Therapeutic in Inflam-
matory Diseases.” The symposium re-
viewed several indications for AAT aug-
mentation therapy outside pulmonary
emphysema and provided preclinical evi-
dence for the effects of AAT in type 1 dia-
betes, GVHD, IBD and acute myocardial
infarction. The Alpha-1 Foundation’s 12th
Gordon L. Snider Critical Issues work-
shop, entitled “New Formulations and
Applications of α-1-Antitrypsin,” pro-
vided preliminary findings for AAT ther-
apy in transplant rejection, type 1 dia-
betes, IBD, rheumatoid arthritis, viral
infection and cystic fibrosis. These scien-
tific programs form a bridge between the
vast body of recently published preclini-
cal studies and the potential clinical use
of AAT outside the context of genetic
Importance of Activities Unrelated to
Evidence for functions of AAT that
occur without the requirement of its
RCL to bind to a serine protease is accu-
mulating. Oxidized AAT (erroneously
called “inactive”) cannot inhibit elastase,
yet exerts antiinflammatory activities
(130). Modified forms of AAT cause a
rise in cAMP and induce IL-10 release,
even though they lack protease in-
hibitory activity (145). The concept of a
protease- antiprotease imbalance was re-
cently revisited, pointing at the possibil-
ity that in some disease models, AAT
may function in an unrelated manner
(66). Indirect evidence also exists. The
C1 inhibitor is a SERPIN that displays
antiinflammatory attributes, despite
having targets of inhibition that are
nonoverlapping with those of AAT (146).
Similarly, the SERPIN antithrombin III
exerts antiinflammatory activities, yet
shares little overlap with the serine pro-
teases that are inhibited by AAT (147).
These findings are important because re-
lease criteria for clinical-grade human
AAT are uniformly based on the level of
elastase inhibition per milligram of pro-
tein. Yet it is likely that during the pu-
rification steps from human plasma, the
nonprotease-interacting domain(s) of
AAT are compromised such that the an-
tiinflammatory properties are reduced.
In support of this concept, plasmid-de-
rived human AAT (hAAT) (148) and
transgenic hAAT (6) modulate the im-
mune system at concentrations too low
to afford elastase inhibition by the
equivalent concentration of injected
plasma-derived hAAT. In addition,
knockout mice to metalloproteinase
MMP-12 are protected from emphysema,
despite the presence of elastase (62); in
this study, TNF-α is reduced by the ab-
sence of MMP-12, further suggesting
that the inhibitory effect of AAT on
TNF-α is of benefit for tissue protection,
regardless of the status of elastase.
Moreover, in the same study, the out-
come remained consistent when using
oxidized AAT, which has a 2,000-fold
lower association rate constant for neu-
trophil elastase. Interestingly, whereas
elastase knockout mice do not develop
emphysema, they do appear to have
normal neutrophil development and re-
cruitment. But perhaps even more im-
portantly, they are resistant to lethal
doses of LPS (24).
Sources of AAT
Pooled human plasma affinity puri-
fied AAT. Clinical-grade AAT is fac-
tory-lined for a rare genetic condition,
found among human populations at a
rate anywhere from 1:1,600 in Denmark
to 1:5,000 in the U.S. Defined under
“Medical Needs” in the review “Emerg-
ing drugs for alpha-1-antitrypsin defi-
ciency” (149), COPD is the sole exten-
sion of clinical indications for AAT.
Providing adequate supply of AAT for a
common condition such as type 1 or
type 2 diabetes will be challenging—
even more so for multiple conditions
with varying requirements of duration
Gene therapy. Being a single-gene dis-
ease, AAT deficiency is one of several
well-studied human genes to be experi-
mentally delivered by genetic manipula-
tions (150). Moreover, evidence exists to
show that native gene-derived circulat-
ing human AAT is superior to the
plasma-purified material (148). Gene
therapy may surpass the requirement of
purification of AAT, as well as reduce
the exposure of patients to human-de-
rived material and supply the circulation
with the native molecule. However, gene
therapy still holds the downside of ge-
netic manipulation in humans and the
inability to control circulating AAT lev-
els once introduced. Notably, a plasmid
that contains a significant stretch of the
human gene for AAT, including introns,
exons and a generous expanse of the
original promoter, has been generated
(151) and introduced into animals. In
these studies, the animals display en-
hanced protection of transplanted islets
much like the findings obtained using
the injected material (148).
Recombinant AAT. Recombinant AAT
has been derived from plants, yeast,
fungi, animals, insect cells, bacteria and
mammalian cells and has been manipu-
lated toward humanized systems, mu-
tated at specific amino acids and conju-
gated with polyethylene glycol (PEG)
(152). Bacterial nonglycosylated 44-kDa
recombinant human AAT typically ag-
gregates, is inactive and is also rapidly
cleared from the circulation; the prolon-
gation of its half-life by PEGylation may
provide a solution to its inferiority (153).
Inhaled recombinant AAT has been
shown to ameliorate cigarette smoke–
induced emphysema in mice (69), much
like the injected material (130). Biologi-
cally active N-glycosylated human AAT
was generated in mouse urine directed
by the uromodulin promoter, raising yet
another attractive option for the potential
large-scale production of functional ther-
apeutic human AAT in livestock (154).
Nevertheless, none of the above ap-
proaches appears to appeal more to
966 | LEWIS | MOL MED 18:957-970, 2012
EXPANDING THE INDICATIONS FOR
patients in need than the traditional
plasma-derived affinity-purified form of
This review opened with the descrip-
tion of transgenic livestock that generate
human AAT in milk (1). It is a dual chal-
lenge to introduce AAT into clinical use
at the appropriate effective window of
clinical opportunity, as well as to find a
creative way by which to supply such a
potentially broad demand. Any new
AAT-based treatment agent will also be
required to stand the challenge of eluci-
dating the mechanism of AAT and to de-
fine a “grand unified theory,” should one
mechanism account for the broad spec-
trum of its disease-modifying properties.
The ~52-kDa glycoprotein that is deco-
rated by a 9 amino acid–long protease-
binding region most probably contains
domains in the nonprotease-interacting
conserved areas of the molecule that can
account for the profoundly altered pro-
tease-independent in vivo activities. Since
its first description in 1906 as an in-
hibitor of trypsin (155), a mechanism is
yet to be identified for the protease-
independent properties of AAT that
modify gene expression profiles of piv-
otal beneficial molecules, such as VEGF
and IL-1 receptor antagonist. That elas-
tase activity is not the sole indicator of
AAT activity is rapidly becoming a ac-
Efficacy of AAT augmentation therapy
in conditions other than pulmonary em-
physema recently was addressed in an ev-
idence-based analysis (156). Its use in con-
ditions such as type 1 and type 2 diabetes;
acute myocardial infarction and postin-
farction remodeling; and GVHD, cystic fi-
brosis and IBD is currently being evalu-
ated worldwide in controlled trials.
Conditions such as stroke, Alzheimer’s
disease, MS, vasculitis and organ and cell
transplantations are also promising clini-
cal indications. In light of the reduced
production and activity of IL-1β by AAT,
diseases that are responsive to blockade
of IL-1, IL-17 and TNF-α should also be
considered as candidates for AAT therapy
(107). To date, unlike in the case of AAT,
antiinflammatory approaches that aim at
ameliorating acute and chronic conditions
that depend on the combination of in-
flammation, hypoxia and tissue damage
(157,158) often succeed in blocking the
unwanted arms of the inflammatory re-
sponse, yet are at risk of failing to sustain
the imperative benefits of inflammation.
The author thanks Charles A Dinarello
and Sabina Janciauskiene for indispensi-
ble insights and advice regarding the bi-
ology of AAT. The author also thanks
Galit Shahaf, Eyal Ozeri, Mark Mizrahi,
Hadas Moser, Keren Bellacen, Noa Kalay,
Efrat Ashkenazi, Avishag Abecassis and
David Ochayon for exceptional assistance
in writing this manuscript.
The author declares that he has no
competing interests as defined by Molec-
ular Medicine, or other interests that
might be perceived to influence the re-
sults and discussion reported in this
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