In last centuries, vaccines reduced the incidence of several infectious diseases. In last decades, some vaccines aimed at preventing also some cancers, where viruses play a causative role. However, several adverse events have been described after vaccines, but a causal relationship has been established only in a minority of cases. Here, we describe a pseudo-neurological syndrome occurred shortly after the administration of the bivalent HPV vaccine. Some autoimmune disorders, including neurological demyelinating diseases, have been reported after HPV vaccines, but the patient showed no organic lesions. The patient was diagnosed as having a functional somatoform syndrome, which was supposed to be autoimmune/inflammatory syndrome induced by adjuvants (ASIA), seen the temporal link with vaccination and the presence of anti-phospholipid autoantibodies. Immunological mechanisms of vaccines-and of adjuvants-have not been completely elucidated yet, and although there is no evidence of statistical association with many post-vaccination events, a causal link with vaccine cannot be excluded in some individuals.

Aluminium hydroxide is used as an effective adjuvant in a wide range of vaccines for enhancing immune response to the antigen. The pathogenic role of aluminium hydroxide is now recognized by the presence of chronic fatigue syndrome, macrophagic myofasciitis and subcutaneous pseudolymphoma, linked to intramuscular injection of aluminium hydroxide-containing vaccines. The aim of this study is to verify if the subcutaneous pseudolymphoma observed in this patient in the site of vaccine injection is linked to an aluminium overload. Many years after vaccination, a subcutaneous nodule was discovered in a 45-year-old woman with subcutaneous pseudolymphoma. In skin biopsy at the injection site for vaccines, aluminium (Al) deposits are assessed by Morin stain and quantification of Al is performed by Zeeman Electrothermal Atomic Absorption Spectrophotometry. Morin stain shows Al deposits in the macrophages, and Al assays (inμg/g, dry weight) were 768.10±18 for the patient compared with the two control patients, 5.61±0.59 and 9.13±0.057. Given the pathology of this patient and the high Al concentration in skin biopsy, the authors wish to draw attention when using the Al salts known to be particularly effective as adjuvants in single or repeated vaccinations. The possible release of Al may induce other pathologies ascribed to the well-known toxicity of this metal.

The toxicity of aluminum in plant and animal cell biology is well established, although poorly understood. Several recent studies have identified aluminum as a potential, although highly controversial, contributory factor in the pathology of Alzheimer disease, amyotrophic lateral sclerosis, and dialysis dementia. For example, aluminum has been found in high concentrations in senile plaques and neurofibrillary tangles, which occur in the brains of subjects with Alzheimer disease. However, a mechanism for the entry of aluminum (Al3+) into the cells of the central nervous system (CNS) has yet to be found. Here we describe a possible route of entry for aluminum into the cells of the CNS via the same high-affinity receptor-ligand system that has been postulated for iron (Fe3+) delivery to neurons and glial cells. These results suggest that aluminum is able to gain access to the central nervous system under normal physiological conditions. Furthermore, these data suggest that the interaction between transferrin and its receptor may function as a general metal ion regulatory system in the CNS, extending beyond its postulated role in iron regulation.

Aluminum hydroxide (AlOOH) has been used for many years as a vaccine adjuvant, but little is known about its mechanism of action. We investigated in this study the in vitro effect of aluminum hydroxide adjuvant on isolated macrophages. We showed that AlOOH-stimulated macrophages contain large and persistent intracellular crystalline inclusions, a characteristic property of muscle infiltrated macrophages described in animal models of vaccine injection, as well as in the recently described macrophagic myofasciitis (MMF) histological reaction in humans. AlOOH-loaded macrophages exhibited phenotypical and functional modifications, as they expressed the classical markers of myeloid dendritic cells (HLA-DR(high)/CD86(high)/CD83(+)/CD1a(-)/CD14(-)) and displayed potent ability to induce MHC-II-restricted antigen specific memory responses, but kept a macrophage morphology. This suggests a key role of macrophages, in the reaction to AlOOH-adjuvanted vaccines and these mature antigen-presenting macrophages may therefore be of particular importance in the establishment of memory responses and in vaccination mechanisms leading to long-lasting protection.

This was a case study in which 3 patients with autoimmune/auto-inflammatory syndrome induced by adjuvants (ASIA) after quadrivalent human papillomavirus vaccination (HPV) were evaluated and described. All the patients were women. Diagnosis consisted of HLA-B27 enthesitis related arthritis, rheumatoid arthritis and systemic lupus erythematous, respectively. Our results highlight the risk of developing ASIA after HPV vaccination and may serve to increase the awareness of such a complication. Factors that are predictive of developing autoimmune diseases should be examined at the population level in order to establish preventive measures in at-risk individuals for whom healthcare should be personalized and participatory.

In the recent literature an inverse relationship between iron status and serum aluminum levels has repeatedly been reported in dialysis patients. To check whether this observation is, at least in part, due to an interference of iron with the protein binding of aluminum, we studied the effect of the latter element on both the number of free binding sites on transferrin (Tf) and on the affinity of the protein for aluminum. For the purpose of this, a recently developed HPLC-ETAAS hybrid method was used, allowing protein-binding studies at clinical relevant metal concentrations and under contamination-free conditions. After we incubated apo-Tf with iron and aluminum which were added in amounts equivalent to the calculated number of metal-binding sites on the protein (i.e., 2 mol metal/mol Tf), we found that Tf can be saturated for 100% with iron. However, for aluminum only a 23% aluminum-Tf saturation was observed. In Tf solutions with iron saturations ranging between 0 and 60% as well as in the serum of 15 subjects with iron-Tf saturations varying between 12 and 48%, a significant (p<0.001) negative correlation between the degree of iron-Tf saturation and the percentage of aluminum (added in amounts equivalent to the number of the remaining binding sites on Tf) bound to Tf was noted (y = -0.26x + 24.5, r = -0.87 in serum). It is concluded that the iron-Tf saturation influences the Tf binding of aluminum not only by occupying binding sites otherwise available for aluminum, but also by lowering the affinity of Tf for aluminum. The effects of iron on serum aluminum levels and bone aluminum deposition are discussed.

In order to assess the risk of parenteral aluminium (Al) exposure, we evaluated the effects of intraperitoneal administration of aluminium hydroxide, a compound widely used in medicine. Mice (strain Pzh:SFIS) received intraperitoneally, every two weeks 1 mg Al or 0.1 mg Al for five days a week. Controls received injections of saline. Al concentrations in liver, bone and brain were evaluated by electrothermal atomic absorption spectrometry after exposure to 2 mg, 4 mg, and 6 mg Al. The concentration was the highest in liver and occurred after exposure to only 2 mg Al (265.1 +/- 27.7 mg/kg, 233.5 +/- 28.0 mg/kg). Generally further accumulation was not dose- and treatment-dependent. The only exception was a significant Al increase in the liver after exposure to 6 mg Al, injected 0.1 mg Al five days/week. Development of resorption granulomas was observed in the liver, Al being revealed by Morin fluorescence in constituent macrophages and giant cells. By electron probe X-ray microanalysis, Al was identified predominantly in lysosomes of macrophages and Kupffer cells. In tibia of mice, a dose-dependent Al accumulation was observed. The highest level of Al concentration after the 6 mg treatment was 23.5 +/- 3.82 mg/kg and 25.06 +/- 2.3 mg/kg. The Al concentration in the brain of mice had not changed significantly during Al treatment.

Parenteral feedings containing more than 4 to 5 μg/kg/d of aluminum have been shown to result in neurodevelopmental delay in preterm infants.1 However, an infant at the 2-month checkup receives multiple aluminum-containing vaccines that in combination may have as high as 1225 μg of intramuscular aluminum; this is a much higher intramuscular aluminum dose than the safely recommended intravenous aluminum dose.2 Our first objective was to measure prevaccine and postvaccine levels of aluminum in preterm infants, a population at higher risk of aluminum neurotoxic effects. Our second objective was to measure prevaccine and postvaccine levels of essential elements (EE). Inflammation from trauma can cause declines in serum levels of specific EE such as zinc and selenium3-5; there may be similar EE perturbations secondary to vaccination-induced inflammation.

The use of vaccines including aluminum (Al)-based adjuvants is widespread among small ruminants and other animals. They are associated with the appearance of transient injection site nodules corresponding to granulomas. This study aims to characterize the morphology of these granulomas, to understand the role of the Al adjuvant in their genesis, and to establish the presence of the metal in regional lymph nodes. A total of 84 male neutered lambs were selected and divided into 3 treatment groups of 28 animals each: (1) vaccine (containing Al-based adjuvant), (2) adjuvant-only, and (3) control. A total of 19 subcutaneous injections were performed in a time frame of 15 months. Granulomas and regional lymph nodes were evaluated by clinicopathological means. All of the vaccine and 92.3% of the adjuvant-only lambs presented injection-site granulomas; the granulomas were more numerous in the group administered the vaccine. Bacterial culture in granulomas was always negative. Histologically, granulomas in the vaccine group presented a higher degree of severity. Al was specifically identified by lumogallion staining in granulomas and lymph nodes. Al median content was significantly higher ( P<.001) in the lymph nodes of the vaccine group (82.65μg/g) compared with both adjuvant-only (2.53 μg/g) and control groups (0.96 μg/g). Scanning transmission electron microscopy demonstrated aggregates of Al within macrophages in vaccine and adjuvant-only groups. In these two groups, Al-based adjuvants induce persistent, sterile, subcutaneous granulomas with macrophage-driven translocation of Al to regional lymph nodes. Local translocation of Al may induce further accumulation in distant tissues and be related to the appearance of systemic signs.

The success of vaccination programs in reducing and eliminating infectious diseases has contributed to an ever-increasing number of vaccines given at earlier ages (newborns and infants). Exposure to low levels of environmental toxic substances (including metals) at an early age raises plausible concerns over increasingly lower neuro-cognitive rates. Current immunization schedules with vaccines containing aluminum (as adjuvant) are given to infants, but thimerosal (as preservative) is found mostly in vaccines used in non-industrialized countries. Exclusively, breastfed infants (in Brazil) receiving a full recommended schedule of immunizations showed an exceedingly high exposure of Al (225 to 1750μg per dose) when compared with estimated levels absorbed from breast milk (2.0 μg). This study does not dispute the safety of vaccines but reinforces the need to study long-term effects of early exposure to neuro-toxic substances on the developing brain. Pragmatic vaccine safety needs to embraceconventional toxicology, addressing especial characteristics of unborn fetuses, neonates and infants exposed to low levels of aluminum, and ethylmercury traditionally considered innocuous to the central nervous system.

Iron, an essential element for all living organisms, is central importance in a number of crucial metabolic pathways, including the regulation of immune function. Iron delivery to cells is accomplished by the complexing of iron to transferrin (Tf), a monomeric iron-binding protein in the plasma, followed by specific binding of Tf to cell-surface receptors, endocytosis of the receptor-ligand complexes and ultimately, release of iron from endosomal vesicles to the cytoplasm. The purpose of this study was to evaluate the effect of cytokines, alone and in combination, on the factors that can affect the iron delivery in thioglycollate-elicited macrophages. In this study, IFN gamma induced a marked increase in Tf synthesis by macrophages, while IL-1, IL-6 and TNF alpha produced a more modest increase. Combinations of these cytokines were shown to be less effective in promoting macrophage Tf synthesis than the cytokines by themselves. IFN gamma alone and in combination with other cytokines was effective in inducing nitrite (NO) production and inducible nitric oxide synthetase (iNOS) expression in macrophages, while IL-1, TNF alpha and IL-6 individually, as well as in various combinations, were not. While all tested cytokines individually and in combination inhibited the expression of the transferrin receptor (TfR) on macrophages, IFN gamma alone and in combination with other cytokines most strongly repressed the TfR expression. TfR localization in macrophages after IFN gamma stimulation showed that TfR fluorescence was most intense in the perinuclear region after 6 hours and scattered diffusely throughout the cytoplasm after 24 hours. This data suggests that IFN gamma may enhance iron uptake during the early phase of macrophage activation, and in later phases, down-regulate TfR expression by inducing NO, thus contributing to intracellular oxidative stress reduction.

We report the case of a 14-year-old girl who developed postural orthostatic tachycardia syndrome (POTS) with chronic fatigue 2 months following Gardasil vaccination. The patient suffered from persistent headaches, dizziness, recurrent syncope, poor motor coordination, weakness, fatigue, myalgias, numbness, tachycardia, dyspnea, visual disturbances, phonophobia, cognitive impairment, insomnia, gastrointestinal disturbances, and a weight loss of 20 pounds. The psychiatric evaluation ruled out the possibility that her symptoms were psychogenic or related to anxiety disorders. Furthermore, the patient tested positive for ANA (1:1280), lupus anticoagulant, and antiphospholipid. On clinical examination she presented livedo reticularis and was diagnosed with Raynaud's syndrome. This case fulfills the criteria for the autoimmune/auto-inflammatory syndrome induced by adjuvants (ASIA). Because human papillomavirus vaccination is universally recommended to teenagers and because POTS frequently results in long-term disabilities (as was the case in our patient), a thorough follow-up of patients who present with relevant complaints after vaccination is strongly recommended.

The term"ASIA-Autoimmune/inflammatory Syndrome Induced by Adjuvants"describes an umbrella of clinical conditions sharing similar signs or symptoms, including post-vaccination phenomena. No information is available on the epidemiology of the ASIA syndrome, especially following HPV vaccination. We carried out an analysis of the VAERS database to retrieve all cases of suspected ASIA syndrome according to the Shoenfeld and Agmon-Levin's guideline for the diagnosis. After causality assessment and case validation, 2,207 cases were considered probably or possibly related to vaccination. These represent the largest ASIA cohort ever reported and allowed us to estimate epidemiological and clinical characteristic of this syndrome. The commonest clinical manifestation observed were pyrexia (58%), myalgia (27%) and arthralgia or arthritis (19%), and the estimated reporting rate was of 3.6 cases per 100,000 doses of HPV vaccine distributed (95% CI 3.4-3.7). This study presents the first systematic estimation of ASIA incidence and expands the knowledge on this pathology. Further analyses are needed to identify genetic and non-genetic risk factors for ASIA syndrome.