Identification of the novel autoantigen candidate Rab GDP dissociation inhibitor alpha in isolated adrenocorticotropin deficiency.

Isolated adrenocorticotropin deficiency (IAD) is characterized by low or absent adrenocorticotropic hormone (ACTH) production. IAD is presumed to be caused in part by an autoimmune mechanism, and several lines of evidence have suggested the presence of anti-pituitary antibodies in IAD. However, the exact autoantigens remain unknown. The present study was designed to identify the autoantigen(s) in IAD using chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Rat anterior pituitary lysate was subjected to SDS-PAGE, and immunoblotting was performed using the sera from two patients with IAD and from a healthy subject. The bands detected by the patient serum samples, but not by the healthy subject sample, were excised, in-gel digested using trypsin, and subjected to LC-MS/MS analysis. On immunoblots, a 51-kDa band in the insoluble pellet was detected by the sera from the IAD patients but not from the healthy subject. Mass spectrometric analysis revealed the 51-kDa band contained Rab guanine nucleotide dissociation inhibitor (GDI) alpha. Consistent with the mass spectrometric analysis, a recombinant full-length human Rab GDI alpha was recognized by the two IAD patient samples but not by the healthy subject sample using immunoblotting. In total, anti-Rab GDI alpha antibodies were detected in serum samples from three of five patients with IAD (60%) but were absent in 5 healthy subjects. In addition, Rab GDI alpha was expressed in the anterior pituitary. In conclusion, it appears that Rab GDI alpha is a candidate autoantigen involved in IAD, and that anti-Rab GDI alpha antibodies are present predominantly in patients with IAD.

Minocycline prevents osmotic demyelination associated with aquaresis.

Overly rapid correction of chronic hyponatremia can cause osmotic demyelination syndrome (ODS). Minocycline protects ODS associated with overly rapid correction of chronic hyponatremia with hypertonic saline infusion in rats. In clinical practice, inadvertent rapid correction frequently occurs due to water diuresis, when vasopressin action suddenly ceases. In addition, vasopressin receptor antagonists have been applied to treat hyponatremia. Here the susceptibility to and pathology of ODS were evaluated using rat models developed to represent rapid correction of chronic hyponatremia in the clinical setting. The protective effect of minocycline against ODS was assessed. Chronic hyponatremia was rapidly corrected by 1 (T1) or 10 mg/kg (T10) of tolvaptan, removal of desmopressin infusion pumps (RP), or administration of hypertonic saline. The severity of neurological impairment in the T1 group was significantly milder than in other groups and brain hemorrhage was found only in the T10 and desmopressin infusion removal groups. Minocycline inhibited demyelination in the T1 group. Further, immunohistochemistry showed loss of aquaporin-4 (AQP4) in astrocytes before demyelination developed. Interestingly, serum AQP4 levels were associated with neurological impairments. Thus, minocycline can prevent ODS caused by overly rapid correction of hyponatremia due to water diuresis associated with vasopressin action suppression. Increased serum AQP4 levels may be a predictive marker for ODS.

Time-dependent changes in proinflammatory and neurotrophic responses of microglia and astrocytes in a rat model of osmotic demyelination syndrome.

Osmotic demyelination syndrome (ODS) is a serious demyelinating disease in the central nervous system usually caused by rapid correction of hyponatremia. In an animal model of ODS, we previously reported microglial accumulation expressing proinflammatory cytokines. Microglia and astrocytes secreting proinflammatory cytokines and neurotrophic factors are reported to be involved in the pathogenesis of demyelinative diseases. Therefore, to clarify the role of microglial and astrocytic function in ODS, we examined the time-dependent changes in distribution, morphology, proliferation, and mRNA/protein expression of proinflammatory cytokines, neurotrophic factors, and matrix metalloproteinase (MMP) in microglia and astrocytes 2 days (early phase) and 5 days (late phase) after the rapid correction of hyponatremia in ODS rats. The number of microglia time dependently increased at demyelinative lesion sites, proliferated, and expressed tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, inducible nitric oxide synthase, and MMP2, 9, and 12 at the early phase. Microglia also expressed leukemia inhibitory factor (a neurotrophic factor) and phagocytosed myelin debris at the late phase. The number of astrocytes time dependently increased around demyelinative lesions, extended processes to lesions, proliferated, and expressed nerve growth factor and glial cell line-derived neurotrophic factor at the late phase. Moreover, treatment with infliximab, a monoclonal antibody against TNF-α, significantly attenuated neurological impairments. Our results suggest that the role of microglia in ODS is time dependently shifted from detrimental to protective and that astrocytes play a protective role at the late phase. Modulation of excessive proinflammatory responses in microglia during the early phase after rapid correction may represent a therapeutic target for ODS.

Minocycline prevents osmotic demyelination syndrome by inhibiting the activation of microglia.

Rapid correction of chronic hyponatremia can lead to osmotic demyelination syndrome (ODS), a severe demyelination disease. The microglia that accumulate in the demyelinative lesions may play a detrimental role in the pathogenesis of ODS by producing proinflammatory cytokines, suggesting that they may be a target for therapeutic intervention. Here, we investigated whether minocycline, a selective and potent inhibitor of microglial activation, could protect against ODS in rats. We induced hyponatremia by liquid diet feeding and dDAVP infusion. Rapid correction of the hyponatremia 7 days later resulted in neurologic impairment with severe demyelinative lesions. Activated microglia accumulated at the site of demyelination. Treatment with minocycline within 24 hours of rapid correction, however, was protective: rats exhibited minimal neurologic impairment, and survival improved. Histologic analysis showed that minocycline inhibited demyelination and suppressed the accumulation of microglia at the site of demyelination. Real-time RT-PCR and immunohistochemical analyses showed that minocycline inhibited the activity of microglia and the expression of inflammatory cytokines (e.g. IL-1ß, inducible nitric-oxide synthase, and TNF-α), monocyte chemoattractant protein-1, and matrix metalloproteinase-12 in microglia. These results demonstrate that minocycline can protect against ODS by inhibiting the activation and accumulation of microglia at the site of demyelinative lesions, suggesting its possible use in clinical practice.

Rabphilin-3A as a Targeted Autoantigen in Lymphocytic Infundibulo-neurohypophysitis.

CONTEXT: Central diabetes insipidus (CDI) can be caused by several diseases, but in about half of the patients the etiological diagnosis remains unknown. Lymphocytic infundibulo-neurohypophysitis (LINH) is an increasingly recognized entity among cases of idiopathic CDI; however, the differential diagnosis from other pituitary diseases including tumors can be difficult because of similar clinical and radiological manifestations. The definite diagnosis of LINH requires invasive pituitary biopsy. OBJECTIVE: The study was designed to identify the autoantigen(s) in LINH and thus develop a diagnostic test based on serum autoantibodies. DESIGN: Rat posterior pituitary lysate was immunoprecipitated with IgGs purified from the sera of patients with LINH or control subjects. The immunoprecipitates were subjected to liquid chromatography-tandem mass spectrometry to screen for pituitary autoantigens of LINH. Subsequently, we made recombinant proteins of candidate autoantigens and analyzed autoantibodies in serum by Western blotting. RESULTS: Rabphilin-3A proved to be the most diagnostically useful autoantigen. Anti-rabphilin-3A antibodies were detected in 22 of the 29 (76%) patients (including 4 of the 4 biopsy-proven samples) with LINH and 2 of 18 (11.1%) patients with biopsy-proven lymphocytic adeno-hypophysitis. In contrast, these antibodies were absent in patients with biopsy-proven sellar/suprasellar masses without lymphocytic hypophysitis (n = 34), including 18 patients with CDI. Rabphilin-3A was expressed in posterior pituitary and hypothalamic vasopressin neurons but not anterior pituitary. CONCLUSIONS: These results suggest that rabphilin-3A is a major autoantigen in LINH. Autoantibodies to rabphilin-3A may serve as a biomarker for the diagnosis of LINH and be useful for the differential diagnosis in patients with CDI.