Morin Stain Research Articles

Morin Stain Detects Aluminum-Containing Macrophages in Macrophagic Myofasciitis and Vaccination Granuloma With High Sensitivity and Specificity.

Macrophagic myofasciitis (MMF) is an inflammatory condition associated with the intramuscular (i.m.) injection of aluminum adjuvant-containing vaccines. It is clinically characterized by myalgia, weakness, and chronic fatigue and histologically by aggregates of cohesive macrophages with abundant basophilic, periodic acid-Schiff (PAS)-positive, diastase-resistant granules that percolate through the peri- and endomysium without eliciting substantial myofiber damage. The definitive diagnosis of MMF requires demonstration of aluminum within these macrophages. We evaluated the Morin stain, a simple, 2-step histochemical stain for aluminum, as a confirmatory diagnostic tool for MMF. Among 2270 muscle biopsies processed at UTSW between 2010 and 2015, a total of 12 MMF cases and 1 subcutaneous vaccination granuloma case were identified (11 pediatric, 2 adults). With the Morin stain, all 13 cases showed strong granular reactivity within the cytoplasm of macrophages but not in myofibers or connective tissue. Three cases of inflammatory myopathy with abundant macrophages (IMAM), 8 cases of granulomatous inflammation and 23 other deltoid muscle biopsies used as controls were all negative. Morin stain could be used in both formalin-fixed paraffin-embedded and cryostat sections. Thus, Morin stain detects aluminum with high sensitivity and specificity in human muscle and soft tissue and may improve the diagnostic yield of MMF and vaccination granuloma.

Identification of blue staining vaccine-derived material in inflammatory lesions using cultured canine macrophages.

Vaccine reactions are described in cytology textbooks as having eosinophilic to magenta colored globules within and admixed with inflammatory cells. Recently, we have seen increased numbers of inflammatory lesions containing blue to blue-gray globular material, with historical information suggesting an association with rabies vaccination. The purpose of the study was to confirm the blue-gray and the eosinophilic material observed microscopically in some inflammatory lesions as being vaccine-derived. Three different vaccines were cytocentrifuged and Wright stained. Vaccine aliquots were also added to the culture media of canine-derived macrophages for 24 hours and the cells subsequently harvested, cytocentrifuged, and Wright stained. The globular material present in both preparations was compared to that observed in vaccine-induced inflammatory lesions. Morin staining was used to identify metal within vaccine material in both in vitro- and in vivo-derived cytology samples. Vaccine-derived material has a characteristic color and appearance. Appearance of the material was consistent in cytologic samples, in cells incubated with the vaccine, and in cytocentrifuged preparations of the vaccine vial contents. The blue-gray globules stained positively for Morin stain, while the eosinophilic material did not stain. Vaccine-induced inflammatory lesions may contain blue to blue-gray or magenta stained globular material. Blue-gray material was associated with administration of rabies vaccine Imrab 3 TF and the observed material may be metal-containing adjuvant. Magenta material was associated with other vaccines and negative for Morin stain, suggesting a metal-free adjuvant.

Slow CCL2-dependent translocation of biopersistent particles from muscle to brain

BackgroundLong-term biodistribution of nanomaterials used in medicine is largely unknown. This is the case for alum, the most widely used vaccine adjuvant, which is a nanocrystalline compound spontaneously forming micron/submicron-sized agglomerates. Although generally well tolerated, alum is occasionally detected within monocyte-lineage cells long after immunization in presumably susceptible individuals with systemic/neurologic manifestations or autoimmune (inflammatory) syndrome induced by adjuvants (ASIA).MethodsOn the grounds of preliminary investigations in 252 patients with alum-associated ASIA showing both a selective increase of circulating CCL2, the major monocyte chemoattractant, and a variation in the CCL2 gene, we designed mouse experiments to assess biodistribution of vaccine-derived aluminum and of alum-particle fluorescent surrogates injected in muscle. Aluminum was detected in tissues by Morin stain and particle induced X-ray emission) (PIXE) Both 500 nm fluorescent latex beads and vaccine alum agglomerates-sized nanohybrids (Al-Rho) were used.ResultsIntramuscular injection of alum-containing vaccine was associated with the appearance of aluminum deposits in distant organs, such as spleen and brain where they were still detected one year after injection. Both fluorescent materials injected into muscle translocated to draining lymph nodes (DLNs) and thereafter were detected associated with phagocytes in blood and spleen. Particles linearly accumulated in the brain up to the six-month endpoint; they were first found in perivascular CD11b+ cells and then in microglia and other neural cells. DLN ablation dramatically reduced the biodistribution. Cerebral translocation was not observed after direct intravenous injection, but significantly increased in mice with chronically altered blood-brain-barrier. Loss/gain-of-function experiments consistently implicated CCL2 in systemic diffusion of Al-Rho particles captured by monocyte-lineage cells and in their subsequent neurodelivery. Stereotactic particle injection pointed out brain retention as a factor of progressive particle accumulation.ConclusionNanomaterials can be transported by monocyte-lineage cells to DLNs, blood and spleen, and, similarly to HIV, may use CCL2-dependent mechanisms to penetrate the brain. This occurs at a very low rate in normal conditions explaining good overall tolerance of alum despite its strong neurotoxic potential. However, continuously escalating doses of this poorly biodegradable adjuvant in the population may become insidiously unsafe, especially in the case of overimmunization or immature/altered blood brain barrier or high constitutive CCL-2 production.

Aluminium overload after 5 years in skin biopsy following post-vaccination with subcutaneous pseudolymphoma

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 (in μg/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.

Aluminum hydroxide injections lead to motor deficits and motor neuron degeneration

Gulf War Syndrome is a multi-system disorder afflicting many veterans of Western armies in the 1990-1991 Gulf War. A number of those afflicted may show neurological deficits including various cognitive dysfunctions and motor neuron disease, the latter expression virtually indistinguishable from classical amyotrophic lateral sclerosis (ALS) except for the age of onset. This ALS "cluster" represents the second such ALS cluster described in the literature to date. Possible causes of GWS include several of the adjuvants in the anthrax vaccine and others. The most likely culprit appears to be aluminum hydroxide. In an initial series of experiments, we examined the potential toxicity of aluminum hydroxide in male, outbred CD-1 mice injected subcutaneously in two equivalent-to-human doses. After sacrifice, spinal cord and motor cortex samples were examined by immunohistochemistry. Aluminum-treated mice showed significantly increased apoptosis of motor neurons and increases in reactive astrocytes and microglial proliferation within the spinal cord and cortex. Morin stain detected the presence of aluminum in the cytoplasm of motor neurons with some neurons also testing positive for the presence of hyper-phosphorylated tau protein, a pathological hallmark of various neurological diseases, including Alzheimer's disease and frontotemporal dementia. A second series of experiments was conducted on mice injected with six doses of aluminum hydroxide. Behavioural analyses in these mice revealed significant impairments in a number of motor functions as well as diminished spatial memory capacity. The demonstrated neurotoxicity of aluminum hydroxide and its relative ubiquity as an adjuvant suggest that greater scrutiny by the scientific community is warranted.

Makrophagische Myofasziitis vs. atypischer M. Whipple

A 39-year-old man presented pronounced asthenia and diffuse myalgias 8 months after a vaccination against the tetanus toxoid. Muscle biopsy disclosed infiltration of the perimysium and endomysium by densely packed, PAS-positive macrophages. Tropheryma whippelii detection in blood and faeces allowed the initial assumption of atypical Whipple's disease with isolated muscle involvement. However, the histological detection of aluminium-containing macrophages by means of Morin stain immunofluorescence established the diagnosis of macrophagic myofascitis in an obviously asymptomatic T. whippelii carrier.

Granulome vaccinal avec hyperplasie lymphocytaire : 10 cas: Présence d’aluminium dans les biopsies

Few cases of cutaneous lymphocytic hyperplasia secondary to vaccination have been published, although such lesions are not rare.We report a series of 10 cases registered between 1993 and 2003.Mean age was 25. The clinical aspect was solitary or multiple subcutaneous nodules, located on the arm, developing after a delay of 1 to 18 months after vaccination. Histologic examination showed a lymphocytic infiltration of the subcutaneous fat, with diffuse and/or follicular pattern, without nuclear atypia, the morphological and immunohistochemical analysis of which revealed the benign nature. In all cases, there was fibrosis and granuloma composed of lymphocytes, plasma cells, eosinophils and macrophages with basophilic cytoplasm. Morin stain showed intralesional aluminium in the 6 investigated cases. Evolution was always benign, with no relapse following exeresis.Cutaneous lymphocytic hyperplasia secondary to vaccination has to be suspected in a young patient with subcutaneous nodules appearing at a vaccination site. Evidence of aluminium in the lesions supports the diagnosis and the hypothesis that aluminium in the vaccine excipient might have a role in the onset of such lesions.

Do aluminium and/or glutamate induce Alz-50 reactivity? A light microscopic immunohistochemical study.

Alzheimer's disease is thought to be characterized by conformational and phosphorylation changes in tau protein, leading to the formation of aggregations of paired helical filaments within neurons. Potential agents for inducing conformational changes in tau, namely aluminium and glutamate, were investigated in this study. Explant cultures of cortical neurons were established from embryonic day 17 rat fetuses. Cultures were exposed to aluminium, glutamate, aluminium/glutamate, aluminium/citric acid and citric acid (since aluminium is thought to enter cells via the transferrin receptor by complexing with citric acid) from 7-12 days in vitro. Control explants were exposed to basal medium only. On day 12, explants were paraffin-embedded. Four-six explants were serially sectioned per condition. For each explant, every 10th and adjacent 4 microm section were randomly selected and processed, with controls, for: (1) alcoholic morin histochemistry (to detect intracellular aluminium), (2) Perls' iron histochemistry (to control for the morin stain which also detects iron), (3) neurofilament immunohistochemistry (to estimate total neuronal number per explant) and (4) Alz-50 immunohistochemistry (to detect possible conformational changes in tau). The absolute number of stained/immunoreactive neurons was determined per explant. The percentage incidence was then determined per explant and averaged per condition. Explants in the aluminium conditions contained significant increases in the incidence of morin-positive aluminium containing neurons. There was also a significant increase in the incidence of Alz-50 positive neurons for the aluminium compared with control explants. These results suggest: (1) aluminium enters neurons and (2) aluminium alone induces possible conformational changes in tau as detected by the Alz-50 antibody, while aluminium combined with glutamate, or glutamate alone, do not.

Enhanced neurite growth in cultured neuroblastoma cells exposed to aluminum

Patients with aluminum-induced encephalopathy syndromes have been shown to have a high level of aluminum concentration in the brain. In the present study, the effects of aluminum were studied in mouse neuroblastoma cells (N-2A) grown in medium supplemented with aluminum (100 μM). It was found that aluminum enhanced neurite growth within 2 days of exposure. The mean total length of neurites in the control after 14 days in culture was 29.8 ± 4.7 μm, whereas the neurite length of cells pre-exposed to aluminum for 2 days and then maintained in normal media for an additional 12 days was 56.4 ± 8.9 μm. Further, the duration of exposure did not significantly promote a greater neurite response. The neurite length of cells exposed to aluminum for 14 days (60.7 ± 9.6 μm) was not statistically different from that of cells exposed to aluminum for 2 days. Using morin stain, intracellular aluminum was detected within 24 h of exposure in the majority of aluminum-exposed cells. Intracellular aluminum did not disappear from those cells even after they were grown for 12 days in control medium. Our finding suggests that a brief exposure (2 days) to low level aluminum (100 μM) is sufficient to cause long-lasting effects on the morphology of neuroblastoma cells in culture. Such neurite behavior associated with aluminum exposure may suggest a morphological basis for the dementia seen in aluminum encephalopathy.

Aluminum(III) influences the permeability of the blood-brain barrier to [ 14C]sucrose in rats

To determine the influence of the metal coordination sphere on the permeability of the blood-brain barrier (BBB), rats were injected intraperitoneally with aluminum lactate (Al(lact) 3), aluminum acetylacetonate (Al(acac) 3), aluminum maltolate (Al(malt) 3) at pH 7.5, or with physiological saline. Two h after each treatment, [ 14C]sucrose physiological saline solution was injected in animals, and the radioactivity was measured in 5 brain regions (cerebral cortex, mesencephalon, diencephalon, medulla-pons, cerebellum). Radioactivity was significantly elevated in brains from animals treated with Al(malt) 3 (hydrolytically stable and hydrophilic), and with Al(acac) 3 (hydrolytically stable and lipophilic) but not with Al(lact) 3. Time-course study carried out at 2, 4 and 24 h with different aluminum compounds showed a persistent radioactivity 24 h after treatment only in the brain from animals treated with Al(acac) 3. Morin stain localized Al III only in neurons from animals treated with Al(acac) 3. These findings indicate that Al III alters the BBB function in the rat either permanently or transiently depending on the physicochemical properties of the metal coordination sphere. Implications of these results, in terms of Al III as a potential toxic factor in humans, are considered and discussed.

Histochemical localization of aluminum in the rabbit CNS

Aluminum was observed in the nucleolus, interchromatin granules, rough endoplasmic reticulum, free ribosomes, euchromatin, and the heterochromatin of the neuron. The association of aluminum with the first four r-RNA-containing cellular components and with the last two DNA-containing chromatins suggests the association of aluminum with the nucleic acids. The aluminum may interfere with the normal mechanism of the protein synthesis of r-RNA and of the transcription or gene modulation of DNA. Aluminum was also observed in the astrocytic process and in the nuclei of endothelial cells, pericytes, and the muscle cells of the blood vessels. The detection of aluminum in the pyrimidal cells of the cerebral cortex and hippocampus and in the spinal cord neurons, was observed 1 h after i.v. injection, indicating a rapid entry of aluminum from the injection site through the blood-brain barrier (BBB) to the neurons. Using Morin stain, pyramidal neurons of the cerebral cortex and hippocampus, motoneurons of spinal cord, ganglion cells, and bipolar cells of retina and Purkinje cells of cerebellum, exhibited yellow fluorescence, with peak intensity at 560 nm. Tangles were observed in these six types of neurons. The granule cells of hippocampus and cerebellum and the photoreceptors of the retina exhibited green fluorescence with the peak intensity at 490-500 nm. Tangles were not observed in these three types of neurons.