Morphologie (2016) 100, 8594

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GENERAL REVIEW

Aluminum adjuvants of vaccines injected

into the muscle: Normal fate, pathology and
associated disease
Adjuvants aluminiques des vaccins injects dans le muscle :
destine, pathologie et maladie associe

R.K. Gherardi a,b,c, J. Aouizerate a,b,c, J. Cadusseau a,c,

S. Yara a,c, F.J. Authier a,b,c,

a
Garches-Necker-Mondor-Hendaye Reference Centre for Neuromuscular Diseases, 94000 Crteil, France
b
Expert Centre for Neuromuscular Pathology, Henri-Mondor Hospital, APHP, 51, avenue du
Marchal-de-Lattre-de-Tassigny, 94000 Crteil, France
c
Inserm U955-Team 10 Biology of Neuromuscular System Paris Est-Crteil University, Crteil, France

Available online 6 April 2016

KEYWORDS Summary Aluminum oxyhydroxide (Alhydrogel ) is a nano-crystalline compound forming

Aluminum; aggregates that has been introduced in vaccine for its immunologic adjuvant effect in 1926.
Vaccines; It is the most commonly used adjuvant in human and veterinary vaccines but mechanisms by
Myofasciitis; which it stimulates immune responses remain ill-dened. Although generally well tolerated
Granuloma; on the short term, it has been suspected to occasionally cause delayed neurologic problems
Myalgias; in susceptible individuals. In particular, the long-term persistence of aluminic granuloma also
Chronic fatigue termed macrophagic myofasciitis is associated with chronic arthromyalgias and fatigue and cog-
syndrome; nitive dysfunction. Safety concerns largely depend on the long biopersistence time inherent to
Cognitive this adjuvant, which may be related to its quick withdrawal from the interstitial uid by avid
dysfunction; cellular uptake; and the capacity of adjuvant particles to migrate and slowly accumulate in lym-
SPECT; phoid organs and the brain, a phenomenon documented in animal models and resulting from
PET-scan; MCP1/CCL2-dependant translocation of adjuvant-loaded monocyte-lineage cells (Trojan horse
CCL2 phenomenon). These novel insights strongly suggest that serious re-evaluation of long-term
aluminum adjuvant phamacokinetics and safety should be carried out.
2016 Elsevier Masson SAS. All rights reserved.

Corresponding author. Expert Centre for Neuromuscular Pathology, Henri-Mondor Hospital, APHP, 51, avenue du Marchal-de-Lattre-

de-Tassigny, 94000 Crteil, France.

E-mail address: authier@u-pec.fr (F.J. Authier).

http://dx.doi.org/10.1016/j.morpho.2016.01.002
1286-0115/ 2016 Elsevier Masson SAS. All rights reserved.
86 R.K. Gherardi et al.

Rsum Loxyhydroxyde daluminium (Alhydrogel ) introduit dans les vaccins en 1926 pour
MOTS CLS son effet adjuvant immunologique est un compos nanocristallin formant des agrgats. Il
Aluminum ; est ladjuvant le plus couramment utilis dans les vaccins humains et vtrinaires, mais les
Vaccins ; mcanismes par lesquels il stimule les rponses immunitaires restent mal dnis. Bien que
Myofaciite ; gnralement bien tolr sur le court terme, il a t soupconn de parfois provoquer des prob-
Granulome ; lmes neurologiques, sur le long terme, chez des individus sensibles. Les proccupations de
Myalgies ; scurit dpendante en grande partie de la longue bio-persistance inhrente cet adjuvant,
Syndrome de fatigue qui peut tre lie son retrait rapide du liquide interstitiel par une svre absorption cellulaire
chronique ; ; et la capacit des particules dadjuvant migrer et saccumuler lentement dans les organes
Dysfonction lymphodes et le cerveau, phnomne document dans des modles animaux et rsultant dune
cognitive ; translocation dpendante de MCP1/CCL2 des monocytes chargs en adjuvant (phnomne du
SPECT ; cheval de Troie). Ces lments indiquent que les modalits danalyse de la pharmacocintique
TEP scan ; et de la scurit des adjuvants aluminiques doivent tre compltement rvalues.
CCL2 2016 Elsevier Masson SAS. Tous droits rservs.

Introduction criteria, less-specic symptoms such as myalgia, arthral-

Vaccination is the most cost-effective method of infec-
tious disease control and prevents about 3 million global
deaths annually [1]. In general, vaccine safety has been
regarded as excellent at the level of the population [2],
but adverse effects have also been reported [3]. Aluminum-
based adjuvants are used in vaccines to enhance the
adaptive immune response, but many concerns about their
using have emerged [4]. Indeed, in 2001, aluminum adju-
vants were recognized to be involved in macrophagic
myofasciitis lesions [5,6] as well as suspected to migrate
in lymphoid organs, disseminate throughout the body within
monocyte-lineage cells and then accumulated in the brain
[7].
Macrophagic myofasciitis (MMF), rst reported in 1998
[5], is an emerging condition characterized by long-lasting
aluminic granulomas at deltoid muscle biopsy resulting
from previous intramuscular (i.m.) injection of aluminum-
adjuvanted vaccines [6,8,9]. Patients with long-lasting MMF
usually present with chronic fatigue, musculoskeletal pain,
and cognitive decits [10]. More than 600 cases were diag-
nosed in Henri-Mondor Hospital from 1993 to 2013. The rapid
emergence of MMF in France resulted from the replace-
ment of subcutaneous (s.c.) by the intramuscular (i.m) route
for vaccine injections in 1990s, the large-scale campaign
against the hepatitis B in the mid 1990s; and the preferential
choice of deltoid muscle for biopsy in France compared to
biceps brachialis and quadriceps muscles in other countries
[4]. After rst description and reports, a number of cases
have been reported in various countries including USA
[1113], UK [14,15], Ireland [16,17], Israel [18], Spain [19],
Portugal [20,21], Germany [2224], Italy [25], Saudi Arabia
[26], Brazil [27], South Korea [28] and Australia [29], so indi-
cating that MMF cannot be regarded as specically French.
MMF is classied as a rare disease in Orphanet database ((#
ORPHA 592; http://www.orpha.net) and is referenced under
M608 number in the International Statistical Classication of
Diseases and Related Health Problems (ICD).
Recently, it was proposed to incorporate MMF within the
group of autoimmune/inammatory syndromes induced
adjuvants (ASIA). ASIA is a term coined by Shoenfeld
and Agmon-Levin to identify conditions combining spe-
cic autoimmune diseases identied by well-established
gia, chronic fatigue, and cognitive impairment; and the
appearance of circulating anto-antibodies, which occurred
after exposure to chemical or natural products with
immunological adjuvant properties [30]. ASIA includes
siliconosis (complications observed in patients with leaky
breast silicone implants attributed to adjuvanticity of
silicone particles [31], Gulf War syndrome (a condition
strongly associated with the administration of multiple
aluminum-containing vaccinations to soldiers like anthrax
[13,32,33]), MMF, and post-vaccination phenomena.
Aluminum adjuvants of vaccines: old and
emerging concepts
The use of inactivated pathogens or selected antigens often
requires the addition of compounds known as adjuvants
to potentiate the immune response to vaccine. Aluminum
salts have been introduced in vaccines by Glenny in 1926
and two compounds, aluminum oxyhydroxide (Alhydrogel )
and aluminum hydroxyphosphate (Adjuphos ), remain the
most commonly used among licensed adjuvants for human
and veterinary vaccines. For example, the former is used
in vaccines against hepatitis B, hepatitis A, tetanus and
many others as well as in formulations used for sub-
cutaneous allergy immunotherapy, whereas the latter is
used in vaccine against herpes papilloma virus (HPV). Of
note, aluminum adjuvants strongly potentiate humoral (i.e.
antibody) responses (B lymphocyte priming and Th2 CD4+
lymphocyte activation) but not, or very little, cell-mediated
responses (Th1 CD8+ cytotoxic lymphocytes). Despite this
ancient and worldwide use the fate of aluminum adju-
vants upon injection and the mechanisms by which they
strongly stimulate Th2 immune responses still remain poorly
understood [34] as they were not subjected to intensive
investigation before the emergence of concerns about adju-
vant safety at the beginning of the XXIth century [6,35].
Several studies have demonstrated clear differences
between the two clinical aluminum adjuvants. Both are
particulate compounds, but their structure and physico-
chemical properties differ in the native state. For example,
aluminum oxyhydroxide has a crystalline morphology known
as Boehmite, whereas aluminum hydroxyphosphate is
Aluminum adjuvants of vaccines injected into the muscle
amorphous. Aluminum oxyhydroxide is composed of elemen-
tary nano-needles of approximately 2.2 nm 4.5 nm 10 nm
[36] forming micronic aggregates through hydrogen bonds,
and displaying a nano-brous morphology at transmission
electron microscopy [37]. It is highly hydrated, forming a
stable gel which has a uniformly high adsorption capacity.
Electrostatic interactions and exchange of hydroxyl groups
by phosphate are major forces driving surface adsorption of
antigens. Alhydrogel has a signicantly higher adsorption
capacity than Adjuphos probably because the latter has
less hydroxyl groups available at its surface for phosphate
exchange. Bio-disposition kinetics of the two adjuvants
also signicantly differ, with lower solubilization rate
of Alhydrogel , pointing to differential reactions of the
two adjuvants upon interaction with phosphate, organic
acid and proteinaceous environments encountered in vivo
(Christopher Exley, personal communication).
Concerns linked to the use of aluminum adjuvants
have emerged following recognition of long-term biopersis-
tence of vaccine-derived aluminum oxyhydroxide particles
within phagocytic cells causing the so-called macrophagic
myofasciitis (MMF) lesion detected in patients with dif-
fuse myalgias, chronic fatigue, and cognitive alterations
[6,38]; observation that different types of traceable alu-
minum particles injected in mouse muscle migrate within
monocyte-lineage cells in lymphoid organs, disseminate
throughout the body, and may slowly accumulate in the brain
[7,39,40]; and demonstration that Alhydrogel can induce
neurotoxic effects in mice [4143].
It has long been believed that aluminum adjuvants ensure
a strong immune response through formation of an extra-
cellular depot progressively releasing the antigen under the
solubilizing effects of the interstitial uid which contains
Al chelators, namely citric, lactic and malic acids, three
organic acids containing alpha-hydroxy-carboxylic groups
[44,45]. This depot effect theory underpinned the classi-
cal view that aluminum adjuvants are completely solubilized
into Al3+ ions that could be then quickly eliminated from the
body by the urine. In support of this reassuring conception,
bio-disposition studies, using traceable isotopic 26 Al in place
of common 27 Al, showed that intravenous injections of sol-
uble (i.e. not particulate) Al citrate in human volunteers is
followed by elimination of 83% of the injected dose in the
urine and 1.8% in the feces at day 13 [46]. The remaining
15% retained in the body were very slowly released after-
wards (4% remaining un-excreted 1178 days after injection),
which may reect trapping of most residual Al in bone as
documented in rats [47].
The depot theory conception was challenged by the
observation that aluminum adjuvant particles are exclu-
sively detected within macrophages in muscle of humans [6],
and of mice, rats and monkeys injected with aluminum oxy-
hydroxide to understand the signicance of MMF [6,8,48].
This point is important since admittedly dissolving effects
of the interstitial uid could not operate in case of rapid
cellular uptake of aluminum particles [49]. The depot
theory has been abandoned in recent years as evidence
accumulated that immune-potentiation requires handling of
the aluminum adjuvant by dendritic cells [50], and neither
retention of the antigen by the adjuvant [51] nor persistence
of the adjuvanted vaccine longer than 2 hours [52] at site of
injection.
87
A core characteristic of aluminum oxyhydroxide that
emerged with the understanding of MMF is its unexpectedly
long-lasting biopersistence, exceeding 12 years after injec-
tion in some individuals [4]. In sharp contrast to the quick
elimination of soluble aluminum injected intravenously
[46], the reference study on particulate aluminum adju-
vant biodisposition conducted using isotopic 26 Al showed
that after intramuscular injection of aluminum hydroxide
in two rabbits no more than 6% of 26 Al was eliminated in
urine on day 28 endpoint [44]. At the same day 28 endpoint,
22% of 26 Al was excreted when aluminum phosphate was
injected. High retention rate of aluminum hydroxide appar-
ently raised no alarm since continuous solubilization of the
compound was the working hypothesis of authors who, at
that time, did not suspect cell incorporation and its inu-
ence on the fate of the compound. Tissue distribution of
26
Al was as follows for both aluminum hydroxide and phos-
phate: kidney > spleen > liver > heart > lymph nodes > brain.
Of note, only 2 rabbits were analyzed per adjuvant, a single
injection was done, delayed time points were not inves-
tigated, bones were not examined, the injected muscles
were not examined, and only mesenteric lymph nodes (not
the ones draining the injected site) were analyzed in this
unique reference study on aluminum adjuvants pharmacoki-
netics. Since the understanding that adjuvant particles are
avidly taken up by monocyte-lineage cells, biopersistence
of adjuvant-loaded cells was documented in a variety of
laboratory animal models up to 612 months post-IM injec-
tion, in both the injected muscle [6,8,40,48] and, quite
abundantly, in the draining lymph nodes and spleen [39].
These results may suggest that progressive accumulation
of adjuvant particle-loaded cells can occur in lymphoid
organs following repeated immunizations. For example, the
exact safety level of the US pediatric vaccination schedule
which includes 16 aluminum adjuvant shots from birth to 18
months probably merits precise re-evaluation [53] despite
reassuring predictive models based on the historical, but
respectively non-relevant and fragmentary, studies of Priest
and Flarend [54].
There has long been a missing conceptual link between
long-term persistence of adjuvant particles within
macrophages at the site of previous immunization and
the occurrence of adverse neurological events in patients
with MMF. It is well established that aluminum is a neuro-
toxic metal, affecting cognition, memory, and psychomotor
control, damaging the blood-brain barrier, altering neu-
rotransmission and synaptic activity, exerting pro-oxidant
effects, activating microglia, depressing the cerebral glu-
cose metabolism and mitochondrial functions, interfering
with gene transcription, and promoting beta-amyloid and
neurolament aggregation (review in Tomljenovic, 2011)
[55]. However, concerns about bio-persistent adjuvants
largely depend on ability of Al3+ to reach remote organs
and the brain and exert its toxicity in critical sites. Several
studies from the literature have suggested such an occur-
rence [44,5659], but whether adjuvant-derived aluminum
reaches the brain in its native particulate or in a soluble
form remained undetermined. To assess the fate of partic-
ulate material in C57B6 mice, we performed i.m. injections
of alum-containing vaccine [7], uorescent latex beads
[7], uorescent nanohybrids coated with precipitated alum
[7], and Alhydrogel tagged with uorescent naodiamonds
88
[40]. All these materials were quickly captured by CD11b+
monocyte-lineage cells which rapidly formed a persistent
local granuloma but also served as a vehicle for both rapid
(within days) or delayed (within months) transportation of
particle from the injected muscle to the draining lymph
node and spleen [7,39,40]. Particle-laden cells left the
lymphatic system and reached the blood circulation (pre-
sumably via the thoracic duct), allowing them to eventually
reach the brain in extremely low amount where they were
mainly distributed to microglial cells. By using relevant
recombinant chemokine injections and KO mice, we showed
that systemic biodistribution of particles crucially depends
on MCP-1/CCL2 the major monocyte chemoattractant [7].
Extremely low brain incorporation of aluminum particles in
normal conditions was in accordance with the good overall
tolerance of aluminum adjuvants in the general population.
However, brain translocation was signicantly increased
in case of altered blood-brain barrier (BBB) or in case of
abundant MCP-1/CCL2 signaling in muscle and brain [7].
The imbalance between the huge number of vaccinated
individuals and the relatively low number of MMF cases
suggests that individual susceptibility factors, related to
ageing or other environmental factors or genetic traits, may
play a crucial role in intolerance to aluminum adjuvants.
For example, BBB is immature in the rst year of age in
humans, and becomes weaker in the elderly or in case of
hidden neuropathological processes. Among the genetic sus-
ceptibility factors, one should investigate genes impacting
either the immunologic responses to adjuvanted vaccines,
e.g. HLA genotypes [60], intracellular biodisposition of
mineral particles, i.e. xeno/autophagy genes (Gherardi
et al., in preparation), or inammatory molecules driving
neuromigration of particle-loaded cells, e.g. MCP1/CCL2
[7]. For example, we have identied selective increase
of circulating MCP-1/CCL2 as a circulating biomarker in
patients with MMF [61]. Such a MCP-1/CCL2 overproduction
may point to a constitutive genetically-determined charac-
ter [7] and/or assess the longstanding effect of aluminum
adjuvant overload within immune cells [62].
Histopathological characteristics of aluminic
granuloma
MMF lesion is very stereotyped and easy to recognize. It
corresponds to aluminic granuloma at site of previous i.m.
injection of aluminum-adjuvanted vaccine. For this reason,
MMF lesion is focal and mainly found in deltoid muscle, less
frequently in quadriceps muscle. It is localized at the periph-
ery of muscle, within fascicles or in peri- or epimysium, and
in perimuscular fascia or deep fat tissue. The lesion is made
of cohesive inltration of large basophilic macrophages with
PAS-positive granular intracytoplasmic content, without for-
mation of multinucleated giant cells. T-cells are constantly
present, intermingled with macrophages [6] (Fig. 1). A
B-cell component can be observed as well as plasmo-
cytes, which sometimes may form lymphoid follicles within
macrophagic inltrates [8]. Perivascular lymphocytis cuffs
or vasculitis are frequent, remote from macrophagic inl-
trates. Myobers have normal appearance, excepted those
engulfed by macrophages that appear roundedly atrophic.
Myobers in close vicinity to macrophagic inltrates may
R.K. Gherardi et al.
re-express major histocompatibility complex (MHC)-type 1
antigens but this feature remains strictly focal (Fig. 2). Ubiq-
uituous myober MHC-I re-expression should lead to consider
the possibility of an associated diffuse immune myopathy.
Fibrotic reaction may be seldom observed with collagen
lamellae interposed between macrophages. Morin stain-
ing shows strong green uorescence of aluminum-loaded
macrophages. Electron microscopy evidence the presence
of accumulations of spiculated osmiophilic crystals (Fig. 3)
[63]. MMF lesions display up-regulation of gene expres-
sion of CD206/MRC1, SOCS1 and TGFB genes witnessing
Th2-induced alternative activation of macrophages as in
muscular sarcoidosis granulomas. TYROBP and TREM2, genes
regulating giant cell formation, are only weakly expressed in
MMF lesions compared to sarcoidosis granulomas, a feature
in keeping with the lack of multinucleated cells [64].
Experimental modeling of MMF lesions in rats con-
rmed that aluminic granuloma constantly forms within
three weeks following the i.m. injection of a single dose
of aluminum hydroxyde-containing vaccine [8]. These
experiments showed that MMF lesions persist longer than
commonly alleged, but disappear within one year. Similar
ndings have been reported in other animal models including
monkeys [48] and it can be reasonably assumed the situation
is similar in human. Therefore, the presence of MMF lesions
at muscle biopsy can be considered as pathological only if
delay is superior to 2 years. In patients, the median delay
elapsed between last vaccination and biopsy was found
at 53 months with a peak between 4 and 5 years after
vaccination, indicating abnormal long-lasting persistence
of MMF lesions [10]. These data are in complete opposition
with the view that MMF lesion corresponds to a trivial
post-vaccinal scar fortuitously detected on the occasion of
a muscle biopsy. In contrast, they support the hypothesis on
an individual predisposition manifesting by impaired ability
to clear aluminum from the deltoid muscle [65].
Clinical manifestations associated with
long-lasting MMF lesion
Adult patients with persistent MMF lesions at muscle biopsy
suffer from chronic musculoskeletal pain (arthromyalgias),
chronic fatigue, and cognitive disorders. These clinical man-
ifestations were described in a series of clinical studies
[46,38,6672], as well as in two reports from French
governmental agencies, InVS [73] and ANSM [74] which
have allowed dening precisely the MMF-associated clinical
symptoms. From clinical studies, it appears that patients
are mainly women (7080%) with a mean of age of 45
years at diagnosis (i.e. muscle time of biopsy), who com-
plained from myalgias, with or without arthralgias, and
chronic fatigue, these symptoms usually developing within
the year after aluminum-loaded vaccine administration with
a median delay of 7 months [38,75].
Musculoskeletal pain
A general survey in different French neuromuscular centers
has detected a strong statistical association between myal-
gias and MMF (myalgias in 90% of patients with MMF vs 44%
without MMF) [6]. Myalgias are most frequent presenting
Aluminum adjuvants of vaccines injected into the muscle 89

Figure 1 Macrophagic myofasciitis (MMF) lesions: histopathological ndings. A and B. Focal inammatory inltrate made of cohe-
sive large mononucleated macrophages intermingled with lymphocytes; macrophages displayed granular intracellular inclusions (B:
arrows). C and D. Perimysial MMF lesion (stars): macrophages strongly positive with PAS technique (D). E and F. Higher magnication
showing collagen strips interposed between macrophages (E: arrows) and macrophages lled by PAS-positive granular inclusions
(F: arrows). Deltoid muscle biopsy, parafn sections; hematoxylin-eosin-saffron (AC and E) and periodic acid-Schiff (PAS; D, F);
light microscopy. Histological analyses were carried out on Axioskop 2 light microscope (Zeiss) and digital image capture using
Coolscope (Nikon) device.
Lsions histologiques de myofasciite macrophagique (MFM). A et B. Inltrat inammatoire focal form de grands macrophages
mononucls cohsifs, avec prsence de lymphocytes ; les macrophages prsentent des inclusions intracellulaires granuleuses (B
: ches). C et D. Lsion de MFM primysiale (toile) : macrophages fortement positifs avec la technique du PAS (D). E et F.
Fort grossissement montrant des bres de collagne interposes entre les macrophages (E : ches) et des macrophages avec des
inclusions granuleuses PAS-positives (F : ches). Biopsie de muscle deltode, coupes en parafne ; hmatoxyline-osine-safran
(AC et E) et periodic acid-Schiff (PAS ; D, F) ; microscopie optique. Lanalyse histologique a t ralise avec un microscope
Axioskop 2 (Zeiss) et la numrisation dimage avec lappareil Coolscope (Nikon).

manifestation and often associate with fatigue. The pro-
gression of pain is usually slow, over several months, and is
usually exacerbated by exercise [4,710]. Myalgias typically
begin in the lower limbs, and not at the previous immuniza-
tion site [10,73] and appear within the rst year following
immunization (median delay 7 months [38]). Myalgias gradu-
ally extend toward the top of the body, affect the paraspinal
muscles, and become diffuse [38]. Diffuse myalgias were
observed with a prevalence ranging from 55 to 96% [10].
At physical examination, MMF patients exhibit few tender
90 R.K. Gherardi et al.

Figure 2 Macrophagic myofasciitis (MMF): immunoperox-

idase. A. MHC-I/HLA-ABC: macrophages appeared strongly
positive (arrowheads). In the close vicinity to MMF lesions,
a small subset of myobers abnormally re-expressed HLA-
ABC (arrows) while the others remained negative (stars). Figure 3 Macrophagic myofasciitis (MMF): ultrastuctural
B. CD56/NCAM: NCAM-positive myobers (arrows) close to a appearance. A. Endomysial macrophage showing numerous
macrophagic inltrate (arrowheads). Deltoid muscle biopsy, speculated aggregates within cytoplasm. B. At higher magni-
frozen 7 m sections; immunoperoxidase technique for MHC- cation, inclusions resemble bundles of sticks and correspond
I/HLA-ABC (W6/32, 1/4000, Dako, Glostrup, Denmark) and to aluminum hydroxide particular aggregates. Courtesy of Dr M.
CD56/neural cell adhesion molecules (NCL-CD56-1B6, 1/100, Coquet (Bordeaux).
Novocastra, Antony, France), using Bond-III automaton (Leica, Myofasciite macrophagique (MFM) : aspect en microscopie
Nanterre, France). lectronique. A. Macrophages endomysiaux prsentant de nom-
Myofasciite macrophagique (MFM) : immunoproxydase. A. breux agrgats spiculaires intracytoplasmiques. B. plus
MHC-I/HLA-ABC : les macrophages apparaissent fortement fort grossissement, les inclusions se prsentent comme des
positifs (pointes de che). proximit des lsions de paquets daiguilles et correspondent aux agrgats articulaires
MFM, on observe une r-expression anormale des antignes dhydroxyde daluminum. Document aimablement fourni par le
HLA-ABC par les myocytes les plus proches de linltrat Dr M. Coquet (Bordeaux).
(ches), les autres myosites plus distance restant ngat-
ifs (toiles). B. CD56/NCAM : myocytes NCAM-positifs (ches) point sites or not, so they rarely fulll 1990 ACR criteria for
proches de linltrat macrophagique (pointe de ches). Biop- bromyalgia [71]. Arthralgias are less often reported (from
sie de muscle deltode, coupes en conglation de 7 m ; 14 to 84%) but spinal pain, especially dorsal, is frequently
technique immunoproxidase pour MHC-I/HLA-ABC (W6/32, expressed [71]. Finally, MMF patients typically present with
1/4000, Dako, Glostrup, Denmark) et CD56/neural cell adhe- diffuse arthromyalgias affecting both lower and upper limbs
sion molecules (NCL-CD56-1B6, 1/100, Novocastra, Antony, at proximal and distal parts. Pain is present at the wak-
France), ralise avec un automate Bond-III (Leica, Nanterre, ing up and increases with effort and daily activities [10].
France). Muscle strength is frequently normal at physical examina-
tion, but the presence of a decit should encourage the
search of a diffuse muscular inammatory/dysimmune pro-
cess such as inclusion body myositis that may be associated
Aluminum adjuvants of vaccines injected into the muscle
with MMF [68]. It must also be noted that most patients had
neuropathic pain in addition to arthromyalgias, laser evoked
potentials showing full-blown small sensory ber neuropathy
in 28% [76].
Chronic fatigue
Chronic fatigue is the second important symptom [75,77]
that may be, in some patients, the only symptom at onset
and may precede pain by several months [10]. It is more
frequent and more severe in patients with MMF compared
to those without MMF [74]. Depending on the study, its
prevalence is ranging from 36 to 100% (91% in InVS study)
[10]. In a study with 30 MMF patients, chronic fatigue (> 6
months) was present in 28/30 patients (93%) [67]. Fatigue
was described as severe and weakening in 26/30 patients
(87%), causing reduction in daily activities in 24/30 patients
(80%), present more than half of the time in 19/30 patients
(63%), and affecting both physical and mental functioning in
16/30 patients (53%) [67]. Finally, 53% of these patients met
the 1991 Oxford or the 1994 CDC international criteria for
encephalomyelitis/chronic fatigue syndrome [67].
Cognitive dysfunction
The prevalence of cognitive dysfunction in MMF varies from
20 to 68% according to used criteria [38,60,67,78]. It forms
with arthromyalgias and fatigue the characteristic clinical
triad associated with long-lasting persistence of MMF lesions
[69,70]. MMF patients typically complain of memory loss,
mood changes, and foggy brain [69]. At neuropsychological
testing, cognitive dysfunction typically combines cortico-
subcortical impairment impacting visual memory, working
memory, and inter-hemispherical disconnection [69], most
patients fullling criteria for non-amnestic/dysexecutive
mild cognitive impairment (MCI) [70]. Importantly, MMF-
associated cognitive dysfunction does not correlate with
disease duration or other factors that can impair atten-
tion and concentration such as fatigue, pain, depression,
or drug intake [69,70]. This cognitive dysfunction suggests
cortico-subcortical brain lesions, probably of inammatory
or toxic origin. Aluminum has long been identied as a
neurotoxic metal able to affect memory, cognition and psy-
chomotor control, neurotransmission and synaptic activity,
and the blood-brain barrier (BBB) [55]. It exerts a pro-
oxidant effects, activates neuroinammation and microglia,
depresses the cerebral glucose metabolism and mitochon-
drial functions, interfers with transcriptional activity, and
promotes beta-amyloid and neurolament aggregation [55].
Laboratory investigations in MMF patients
Biological tests
Standard biology testing is usually unremarkable. In partic-
ular, serum creatine kinase (CK) levels are usually within
normal range. However, in one third of MMF patients, CK
levels may be found initially increased and then get back to
normal. In addition, few patients have sustained inexplica-
bly increased CK levels. In these patients, it is recommended
91
to carefully check the possibility of MMF-associated inam-
matory or immune myopathy [10]. Baseline aluminum serum
levels remain within reference value [6]; increased body
burden of aluminum was reported in one patient, suggest-
ing that coincident aluminum overload may contribute to
the severity of these conditions in a given individual at a
given time [14].
Aluminum hydroxide is a potent activator of the immune
system, a feature in keeping with the increased prevalence
(19%) of autoimmune diseases reported in MMF patients,
including multiple sclerosis and also autoimmune thyroidi-
tis, inclusion body myositis, dermatomyositis, rheumatoid
arthritis, and Sjogrens syndrome [38,66,68,79,80]. More-
over, MMF patients have frequently an increase in circulating
lymphocytes B and monocyte chemoattractant protein 1
(CCL2/MCP-1) as well as the presence of anti-nuclear and
anti-phospholipid antibodies [10]. These ndings witness
protracted immunological activation, a phenomenon known
to cause chronic fatigue and arthralgias [81].
Electrophysiological investigations
Electromyographic study is usually unremarkable. However
in some patients, it may disclose diffuse myopathic fea-
tures [38] that have not been related to specic muscular
change except the few patients with full-blown inamma-
tory/dysimmune myopathy in addition to MMF. In patients
with neuropathic pain, laser evoked potentials may show
small sensory ber neuropathy (SFNp) whose origin remains
unclear to date. Not exceptionally, auditory, visual and sen-
sory evoked potentials may show abnormalities suggestive
of central nervous system involvement, including in the lack
of multiple sclerosis.
Musculoskeletal and cerebral imaging
Magnetic resonance imaging (MRI) of skeletal muscle is not
helpful in the diagnostic workup of MMF patients apart
from the case of suspected associated diffuse myositis.
Gallium-67 (Ga67 ) is a radioisotope which binds to the trans-
ferrin receptor expressed on the surface of macrophages
and different types of activated lymphocytes. In MMF
patients, Ga67 scintigraphy disclosed a characteristic pat-
tern of hyperxations in fascias and periarticular areas in
lower limbs [82]. Nowadays, Ga67 scintigraphy is replaced
in by 18-uorodeoxygluocose positron emission tomogra-
phy/computed tomography (18 FDG-PET/CT) in its classic
indications and therefore becomes usually unavailable.
The neuropsychological prole of MMF-associated cog-
nitive dysfunction suggests the presence of an underlying
organic brain involvement, of inammatory or toxic ori-
gin. Routine brain MRI does not remain informative, except
in patients in whom MMF associates with multiple sclero-
sis [66,69,70]. In contrast, brain functional imaging through
single photon emission computerized tomography (SPECT)
was abnormal in most MMF patients. SPECT discloses hypop-
erfusions mainly affecting hippocampus, amygdala, and
caudatus nucleus [69]. These changes are not random but
strikingly correlate with cognitive decits and therefore
provide a neurobiological substrate for brain dysfunction
in MMF patients. At present, we used 18 FDG-PET/CT to
92
investigate cerebral functioning in MMF patients. Although
still under evaluation, preliminary results indicate that
18
FDG-PET/CT brain imaging may display an atypical pattern
of hypometabolism, involving symmetrically the occipi-
tal cortex, temporal lobes, and limbic system (including
amygdalo-hippocampal complexes), and the cerebellum in
MMF [83]. If conrmed, this PET/CT imaging pattern could
constitute reliable marker for MMF-associated cognitive dys-
function.
Individual genetic susceptibility
Aluminum-containing vaccines are generally well tolerated
as indicated by the relative rarity of adverse events consid-
ering the large number of vaccinees worldwide. A genetic
predisposition causing the prolonged persistence of the
post-vaccination lesion and the development of systemic
dysimmune manifestations has been long suspected [65],
a hypothesis supported by familial cases and experimen-
tal studies [60,78,84,85]. Current approaches relate to the
understanding of pathophysiological mechanisms leading to
long-term persistence of aluminum hydroxide within body
and subsequent biological events. CCL2/MCP-1, a chemokine
crucially implicated in the penetration of nanomaterials
within brain, was found increased in MMF patients [7,61]. Its
expression is subjected to signicant inter-individual varia-
tions depending in part to genetic factors and the analysis
of CCL2 gene in 252 MMF patients and 516 controls found an
association between some gain-of-function haplotypes and
the disease [7,61]. Although overrepresented in MMF, these
haplotypes concern only a minority of patients suggesting
that other factors are probably needed to explain the occur-
rence of disease. Further studies are currently in progress,
more specically targeting the mechanisms involved in the
clearance of aluminum hydroxide particles.
Disclosure of interest
The authors declare that they have no competing interest.
Acknowledgements
This work was supported by grants from Region le-de-
France (PICRI Program 2014), Association francaise contre
les myopathies (AFM, Translamuscle Program 2015), Entraide
des Malades de la Myofasciite Macrophages (E3M), Agence
nationale de scurit du mdicaments et des produits
de sant (ANSM) and Childrens Medical Safety Research
Institute (CMSRI).
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