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Serum HMGB1 as potential biomarker of peripheral demyelination

Colavita L., Cuppari C., Pizzino M.R., Sturiale M., Arrigo T., Salpietro C.D.
Department of Paediatrics, Unit of Genetics and Paediatric Immunology, University of Messina, Messina, Italy

The HMGB1 acts in the start and amplification of the inflammatory processes. More studies show increased serum and local HMGB1 concentration in inflammatory conditions, opening new futuristic possibilities for target therapies. We report a case of a 4 years old child with Guillan-Barrè syndrome (GBS), in which serum HMG1 concentration was evaluated in more disease phases. This is the first evaluation of serum HMGB1 in a pediatric patient affected from GBS. We found increased HMGB1 values (24.2 ng/ml) during the acute phase and a large reduction (6.7 ng/ml) after the completion of IVIG therapy and the resumption of ambulation. We observed a further reduction of HMGB1 values (3.4 ng/ml) after 3 weeks from the completion of IVIG therapy and the normalization of motor conduction. We may assume that HMGB1 can be considered an inflammatory marker also in GBS and that it correlates with disease activity and severity
The Guillan-Barrè syndrome (GBS) is an acute infllammatory disease of the peripheral nervous system that includes more variants (acute inflammatory demyelinating polyradiculoneuropathy, acute motor axonal neuropathy, acute motor-sensory axonal nauropathy, Miller-Fisher syndrome, acute autonomic neuropathy) . The commonest GBS variant is the acute inflammatory demyelinating polyradiculoneuropathy. It is clinically characterized by weakness, paresthesias and pain with diminished/absent reflexes in the limbs, tipically bilateral and relatively simmetric, with possible difficulty in walking. In the 90% of patients, the symptoms begin in the lower limbs and advance proximally. The severity and speed of disease progression are variable. The peak is reached within 2 weeks in 50% of cases and within 3 weeks in 80% of cases. Implication of respiratory muscles with respiratory failure occurs in about 25% of cases. Involvement of autonomic system are present in about 50% of cases and is clinically characterized by cardiac arrhythmias, dysregulation of blood pressure, urinary retention and intestinal dysmotility. The 20% of patients can present persistent neurological problems and long-term disability. The diagnosis is clinical. Some instrumental exams can be useful to confirm the diagnosis. A slowing of nerve conduction velocity is the typical finding. Nerve conduction evaluation can be negative if performed early. Cerebral Spinal Fluid (CSF) study shows normal white blood cells count and increased levels of proteins in the 90% of patients, when done at the clinical nadir. Spinal and brain magnetic resonance imaging (MRI) is often performed to exclude myelopathy or infiltrative or compressive causes of polyradicoloneuropathy. It can show an involvement of nerve roots or cranial nerves.
The therapy consists in intravenous immunoglobulins (IVIg) at a dose of 400 mg/Kg/day for 5 days and must be start within 14 days from symptoms onset. Corticosteroid therapy is not recommended. Another possible therapy is plasma exchange to remove the circulating immune-complexes, but its role in children’s therapy has not yet been established.
We describe a case of a 4 years old child with an acute inflammatory demyelinating polyradiculoneuropathy, the commonest variant of Guillan-Barrè syndrome (GS), and a good response to intravenous immunoglobulines (IVIG) therapy, in which serum HMG1 concentration was evaluated in more phases of the disease [1, 2, 3].
Case report

A. was admitted at our pediatric emergency word at the age of 4 years and 7 months for onset, 4-5 hours before, of pain in the soles of feet and in the retromalleolar area, with refusal to walk. Twelve days before symptoms onset, the child had a febrile acute gastroenteritis, which resolved in 5 days. In our emergency department the child appeared in general clinical good condictions. On examination, vital signs (oximetry, heart and breath frequency) were normal and he had not febrile temperature. The physical examination of chest and abdomen was normal; no lymphadenopathy nor skin alterations; no signs of trauma in the head or in other parts of the body. Pupills were equal and showed a normal reactivity to light. The deep tendon reflexes were absent without clonus of arms and legs. The child showed difficulty to walk and to sustain his weight in order to stand upright. Besides he complained pain in the soles of the feet and in the retromalleolar area. Absent cranial nerve deficits. The blood tests showed a mild increase of C Reactive Protein (CRP) (0.60 mg/dl) with normal white blood cells count, hemoglobin, indices of liver and kidney function and serum electrolytes concentrations. The child was hospitalizaed in our department of Genetics and Pediatric Immunology of the University Hospital of Messina. During hospitalization motor conduction velocity was performed and showed slight decrease in the conduction velocity of the internal popliteal nerve and superficial peroneal nerve of the right leg. A lumbar puncture documented: glucose 56 mg/dl, protein 29 mg/dl, chlorine 126 mmol/L and white blood cells count of 3 cells/mmc in the liquor. A nuclear magnetic resonance of brain and lumbosacral spinal showed slight leptomeningeal impregnation contrast enhancement on the front and rear surface of the conus. For these clinical and instrumental signs, indicative of acute inflammatory demyelinating polyradiculoneuropathy, a Guillan-Barrè syndrome was suspected and, from the second day of hospedalization, IVIG therapy was started: 400 mg per Kg per day for 5 days. In the second day of therapy, onset of fever and headache during IVIG infusion, treated with slowdown of the infusion rate and acetaminophen, with benefit. No additional adverse reactions to subsequent IVIG infusions. During the subsequent 5 days the child showed a rapid improvement in overall clinical condition and neurological picture with recovery of walking and deep tendon reflexes. A second motor conduction velocity was perfomed after completion of IVIG therapy, with normal result. A written informed consent was obtained by child's parents and after more serum HMGB1 (High-Mobility Group Box 1 protein) dosages were perfomed. We have found increased HMGB1 values (24.2 ng/ml) during the acute phase (blood sample collected in the 2nd day of hospetalization, before IVIg therapy initiation) and a large reduction (6.7 ng/ml) in the second sample, collected after the completion of IVIG therapy, when the clinical symptoms were significantly improved. A third sample was collected after 3 weeks from the completion of IVIG therapy and the normalization of motor conduction velocity, with further reduction of HMGB1 values (3.4 ng/ml) . The dosage was done using ELISA (Enzyme-Linked ImmunoSorbent Assay) methodic.
Our patient was affected from an acute inflammatory demyelinating polyradiculoneuropathy, the commonest variant of Guillan-Barrè syndrome (GBS) . The phatophysiological machanism of GBS is the cross-reaction between antigens of specific infectious agents (for example, Campylobacter jejuni express lipooligosaccharides similar to our gangliosides; other common pathogens are Epstein-Barr and influenza virus, citomegalovirus, parvovrus B19, Mycoplasma pneumoniae) and neural antigens. The cross-reactive antibodies cause an inflammatory process that involves specific components of the nervous system: in our case the myelin (acute inflammatory demyelinating polyradiculoneuropathy) . The consequent inflammatory neuropathy is characterized by cellular infiltrate both in the perivascular that endoneural areas. Autoreactive T cells start the inflammatory demyelinating process; the effector mechanisms are, however, complement-fixing antibodies and macrophages’ activity. Macrophages get to basement membrane of Schwan cells and cause the shallow myelin sheath destruction. The autoreactive T cells and/or complement activation, through inflammatory cytokines and chemokines release, are due to increased vascular permeability and consequent further transmigration of macrophages and autoreactive T cells. Recently, a role of Th17, Th22 and their cytokines (IL17 and IL22) in the pathogenesis of GBS has been speculated for the detection of their increased serum levels in the acute phase and their reduction after treatment. IgG and IgM deposits and membranolytic attack complex (MAC), expression of the presence of complement-fixing antibodies against myeline, are found through immunohystochemical studies on GBS patients’ peripheral nerves.
Various clinical and laboratory findings support the hypothesis that serum factors are important in the pathogenesis of GBS (for example: the beneficial effect of plasmapheresis; the detection of various autoantibodies; the ability of the GBS patients’ serum, taken during the acute phase, to determine demyelination of rodent dorsal root ganglionic extracts or demyelination and conduction block, if injected into rat sciatic nerves) [4, 5, 6, 7].
High mobility group box 1 (HMGB1) is a high-conserved nuclear protein (215 residues, 30 kD) binding DNA, encoded by a gene located on chromosome 13 (13q12) . Inside the cells, the HMGB1 is important to maintain nucleosome structure, regulate gene transcription and modulate the steroid hormone receptors activity. When HMGB1 is present in the extra-cellular fluid, as result of passive release (cellular necrosis) or active secretion (from monocyte-macrophages after stimulation by LPS, TNF-α or IL-1.), it is an inflammatory mediator. In fact, it is the ligand for membrane receptors RAGE or toll-like receptors such as TLR-2 and TLR-4, with consequent activation of a cascade of intracellular signaling through TRAF6, NEMO, and IKK IKBα and the final induction of NF-kB transcriptional activity. This causes the production and secretion of various pro-inflammatory cytokines (IL-12, IL-6, IL-1, IL-8, TNF- α) and the amplification of the inflammatory process. So HMGB1 has been classified as damage-associated molecular pattern (DAMP) molecule, that acts in the onset and perpetuation of inflammatory processes [8, 9, 10, 11].
More studies have shown that there is a systemic and local increase of HMGB1 levels in inflammatory conditions, evaluating its role as new biomarkers for various diseases and opening new futuristic possibilities for target therapies. It was demonstrated that the HMGB1 plays an important role in various pediatric diseases: sepsis, obesity, tumor progression, inflammatory and autoimmune diseases, like rheumatoid arthritis, systemic lupus erythematosus, vasculitis and inflammatory bowel diseases (IBD) . In IBD, arthritis, allergic rhinitis, asthma, cystic fibrosis and chronic obstructive pulmonary diseases have been demonstrated increased HMGB1 levels in specific samples (respectively in stool samples, synovial fluid, nasal lavage fluid and sputum), expression of a local HMGB1 production in the site of inflammation [10, 11, 12, 13, 14, 15, 16]. Regarding neurological diseases, HMGB1 was studied in patients affected from autistic disorders; anorexia nervosa; traumatic brain injury, in which HMGB1 levels are increased in CSF as expression of cell necrosis; bacterial and aseptic meningitis; epilepsy and febrile seizures, where HMGB1 and pro-infalmmatory cytokines play an important pathogenetic role, with consequent possible creation of a novel antiepileptic strategy based on pharmacological modulation of HMGB1-TLR/RAGE axis; neuromyelitis optica and multiple sclerosis, with increased values in both serum and CSF [10, 17, 18].
This is the first evaluation of serum HMGB1 in a pediatric patient affected from Guillan-Barrè syndrome (GBS) .
We have demostrated that in our patient affected from acute inflammatory demyelinating polyradiculoneuropathy, the commonest variant of GBS, serum HMGB1 levels are elevated in the acute phase (24.2 ng/ml) . Reduced HMGB1 levels (6.7 ng/ml) were observed after completion of IVIg therapy, when the clinical symptoms were significantly improved. Further reduction of serum HMGB1 concentration (3.4 ng/ml) was found after 3 weeks from completion of IVIg therapy and the normalization of motor conduction velocity.
As previously described, the HMGB1 is released from immune cells and acts in the start and amplification of the inflammatory process. So, we can assume that it play an important role in the GBS pathogenesis. Figure 1.


Figure 1.

GBS pathogenesis and hypothesized role of HMGB1 in the demyelinating inflammatory process. Virus and bacteria activate macrophages, dendritic cells and T cells, with consequent production of inflammatory cytokines and antibodies, that cross react with neural antigens (myelin). The consequence is an inflammatory demyelinating process through the action of auto-antibodies and complement (MAC), the local infiltration of autoreactive T cells and macrophages that release inflammatory cytokines. HMGB1 is released by macrophages, dendritic cells and T cells. HMGB1 stimulates macrophages, dendritic cells, T and B cells activity and favors its own production and that of inflammatory cytokines.

Legend: Pa = pathogens; M = macrophages; TC = T cell; DC = dendritic cell; BC = B cell; P = plasma cell; Ab = antibody; C = complement
The IVIg mechanism of action has been largely studied and depends on the interaction with more cellular and soluble components of the innate and adaptive immune system. IVIg bind FcγRs on immune cells surface (dendritic cells, macrophages and granulocytes) and so block the interaction with immune complexes, induce the expression of the inhibitory FcγRIIb and suppress the expression of IFNγR2, hereby inhibiting macrophages activation. IVIg therapy reduces T cells activation through the promotion of antibody-dependent cellular cytotoxicity of dendritic cells by NK cells and inhibit B cells proliferation and antibody production, while promote B cells apoptosis. The IVIg also interact with Treg cells and cause their expansion and stimulation (Treg epitope Peptides) . Relating to IVIg action on soluble components, they reduce complement activation; neutralize super-antigens, pathogenic autoantibodies and cytokines; modulates cytokine secretion; hide self antigens; reduce the expression of adhesion molecules on endothelial cells [19, 20].
We may assume that among the mechanisms of action of IVIg there is also the binding and neutralization of HMGB1, with the consequent further blocking of the inflammatory cascade and demyelination (Figure 2) . Further studies are obviously needed to confirm this hypothesis.


Figure 2.

Mechanism of action of intravenous immunoglobulins (IVIg).

Legend: M = macrophages; TC = T cell; DC = dendritic cell; BC = B cell; P = plasma cell; T reg = T regulatory cell; Th = T helper

We can conclude that HMGB1 can be considered an inflammatory marker also in Guillan–Barrè syndrome and that its serum concentration correlates with disease activity and severity.
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