High sensitivity of an ELISA kit for detection of the gamma-isoform of 14-3-3 proteins: usefulness in laboratory diagnosis of human prion disease.

BACKGROUND: The gamma-isoform of the 14-3-3 protein (14-3-3 gamma) is expressed in neurons, and could be a specific marker for neuronal damage. This protein has been reported as a detectable biomarker, especially in the cerebrospinal fluid (CSF) of Creutzfeldt-Jakob disease (CJD) patients by Western blotting (WB) or enzyme-linked immunosorbent assays (ELISAs). Western blotting for 14-3-3 gamma is not sensitive, and the reported data are conflicting among publications. An ELISA specific for 14-3-3 gamma is not available. METHODS: CJD patients (n=114 sporadic CJD patients, 7 genetic CJD, and 3 iatrogenic CJD) and 99 patients with other neurodegenerative diseases were examined in this study. The CSF samples obtained were analyzed by Western blotting for 14-3-3 gamma, and by ELISA for total tau protein. We evaluated the sensitivity and specificity of the newly developed sandwich ELISA for 14-3-3 gamma. RESULTS: The cut-off value of the 14-3-3 gamma ELISA was >1, 683 AU/ml; and sensitivity was 95.2%, with 72.7% specificity. This specificity was the same for the total tau protein ELISA. Seven CJD cases were negative by WB but positive using the 14-3-3 gamma ELISA, indicating that the ELISA is more sensitive. All 21 cases of early stage CJD could be diagnosed using a combination of the 14-3-3γ ELISA and diffusion weighted MR imaging (DWI-MRI). CONCLUSION: The 14-3-3 gamma ELISA was more sensitive than conventional WB, and was useful for laboratory diagnosis of CJD, similar to the ELISA for the tau protein. Using DWI-MRI and these ELISA tests on CSF, diagnosis of CJD will be possible even at early stages of the disease.

Establishment of a standard 14-3-3 protein assay of cerebrospinal fluid as a diagnostic tool for Creutzfeldt-Jakob disease.

Periodic sharp wave complexes observed on an electroencephalographic recording and the presence of a 14-3-3 protein in the cerebrospinal fluid (CSF) are both included in the diagnostic criteria for the Creutzfeldt-Jakob disease (CJD) supplied by the World Health Organization; however, the presence or absence of the 14-3-3 protein in the CSF is sometimes difficult to discern on a western blot because of equivocal bands. The goal of this study was to establish a standard 14-3-3 protein assay and to determine the threshold level of a 14-3-3 protein that can be assayed by western blot. We searched for the most suitable isoform of the 14-3-3 protein to test for in protein assays, and the most sensitive antibody among four antibodies with an affinity for 14-3-3. We measured the levels of all 14-3-3 isoforms in 112 patients with CJD and in 100 patients with other diseases. We compared the performances of four different antibodies. We carried out a semi-quantitative analysis of γ-isoform levels using the LAS 3000 system, which was capable of producing a digital image from the luminescence on a western blot. We determined that the most suitable isoform of the 14-3-3 protein for conducting a standardized assay was the γ-isoform. Among the four commercially available antibodies for this protein, the most sensitive and specific was 18647 (IBL, Japan). We report the high repeatability of the detection of the 14-3-3 protein by this antibody to the γ-isoform, showing that western blot can be used for semi-quantitative analysis.

Development of an ultra-rapid diagnostic method based on heart-type fatty acid binding protein levels in the CSF of CJD patients.

Creutzfeldt-Jakob disease (CJD) is a transmissible, fatal, neurodegenerative disease in humans. Recently, various drugs have been reported to be useful in the treatment of CJD; however, for such treatments to be useful it is essential to rapidly and accurately diagnose CJD. 124 CJD patients and 87 with other diseases causing rapid progressive dementia were examined. Cerebral spinal fluid (CSF) from CJD patients was analyzed by 2D-PAGE and the protein expression pattern was compared with that from healthy subjects. One of three CJD-specific spots was found to be fatty acid binding protein (FABP), and heart-type FABP (H-FABP) was analyzed as a new biochemical marker for CJD. H-FABP ELISA results were compared between CJD patients and patients with other diseases (n = 211). Visual readout accuracy of the Rapicheck(®) H-FABP test panel for CSF was analyzed using an independent measure of CSF H-FABP concentration. The distribution of H-FABP in the brains of CJD patients was examined by immunohistochemistry. ELISA sensitivity and specificity were 90.3% and 92.9%, respectively, and Rapicheck(®) H-FABP sensitivity and specificity were 87.9% and 96.0%, respectively. ELISA and Rapicheck(®) H-FABP assays provided comparable results for 14-3-3 protein and total tau protein. Elevated H-FABP levels were associated with an accumulation of abnormal prion protein, astrocytic gliosis, and neuronal loss in the cerebral cortices of CJD patients. In conclusion, Rapicheck(®) H-FABP of CSF specimens enabled quick and frequent diagnosis of CJD. H-FABP represents a new biomarker for CJD distinct from 14-3-3 protein and total tau protein.

Familial Creutzfeldt-Jakob disease with a V180I mutation: comparative analysis with pathological findings and diffusion-weighted images.

BACKGROUND: Diffusion-weighted imaging (DWI) has been reported to be a useful technique for diagnosing Creutzfeldt-Jakob disease (CJD). The present study reported DWI results in cases of familial CJD with a V180I mutation (CJD180) in the prion protein gene as well as neurological findings. METHODS: A retrospective analysis of 3 patients with V180I was performed. Cerebrospinal fluid (CSF) analysis, brain MRI, single-photon emission computed tomography (SPECT), and magnetic resonance spectroscopy (MRS) were included. CSF was analyzed for biochemical markers, and each patient underwent brain MRI, SPECT, and MRS analysis. A brain biopsy from the frontal cortex, which corresponded to the area of increased DWI signals, was utilized for neuropathological analysis. RESULTS: CSF analysis results revealed elevated total tau protein and the absence of 14-3-3 protein, as well as decreased concentrations of neuron-specific enolase, S100 protein, and prostaglandin E(2). All patients presented with unique MRI features. Brain biopsy showed severe spongiform morphology, but comparatively preserved neurons and mild astrocytic gliosis. Accumulations of PrP(Sc) were not detected using the 3F4 antibody, and microglial activation was subtle. SPECT revealed hypoperfusion throughout both hemispheres. MRS revealed a reduced N-acetyl aspartate/creatine ratio. CONCLUSION: Results from this study suggested that increased DWI signals could reflect severe spongiform changes in CJD180 patients.

[A case of secondary central nervous system lymphoma presenting marked hypoglycorrhachia].

A 67-year-old male was transferred to our hospital with diplopia, decreased deep tendon reflex and ataxia. He had been suspected Fisher syndrome because of previous upper respiratory tract infection. A cerebrospinal fluid examination showed marked hypoglycorrhachia, pleocytosis and elevated protein, and cytological examination suggested malignant lymphoma. Abdominal computed tomography revealed a left adrenal mass. A biopsy of the left adrenal mass revealed diffuse large B-cell lymphoma. He was treated with a combination of R-CHOP (rituximab, cyclophosphamide, doxorubicin hydrochloride, oncovin and prednisolone) and intrathecal administration of methotrexate, cytarabine and prednisolone. Neurological symptoms were gradually improved. Malignancy should be considered in addition to bacterial, fungal or tuberculous meningitis in a case with marked hypoglycorrhachia.

Iatrogenic Removal of the Intima in the Middle Cerebral Artery by a Stent Retriever: A Report of Two Cases.

BACKGROUND: Mechanical thrombectomy improves functional outcomes in patients with acute ischemic stroke. However, stent retrievers have the risk of vascular damage. CASE DESCRIPTION: We present 2 cases of patients with acute internal carotid artery occlusion who experienced removal of the intima by a stent retriever. In both patients, a 6 × 30-mm Solitaire stent was fully deployed from the M2 portion and slowly withdrawn. White membranes were retrieved outside the strut in both patients. Histopathologic examination showed that one membrane consisted of thickened intima and internal elastic lamina and the other consisted of calcified intima and internal elastic lamina. One patient who suffered embolic stroke experienced recurrent infarction within 24 hours after operation, and the damaged vessel was occluded on magnetic resonance angiography 21 days after stroke. In another patient with carotid artery dissection, the damaged vessel showed asymptomatic stenosis on magnetic resonance angiography 90 days after stroke. Arteries with both atherosclerosis and vessel dissection may be vulnerable to high radial expansion force. CONCLUSIONS: Full deployment of a relatively large-sized stent into a vulnerable vessel may cause vessel dissection after removal of the intima. Appropriate material selection and treatment strategy while considering stroke etiology and the occlusion site are important to prevent vessel damage.

Acute motor sensory axonal neuropathy associated with Henoch-Schönlein purpura.

A 41-year-old man was admitted with progressive tetraparesis with hypoesthesia. He also presented with purpura in both legs. After admission, joint pain, gastrointestinal tract bleeding, and renal dysfunction developed. A nerve conduction study revealed reduced amplitude of the motor and sensory nerve action potential, with normal conduction velocity. A skin biopsy showed leukocytoclastic vasculitis, indicating Henoch-Schönlein purpura (HSP). After administration of corticosteroids, the symptom completely disappeared. The present case is the first report in Japan of HSP associated clinically and electrophysiologically with confirmed acute motor sensory axonal neuropathy. Common pathogenesis might have a role for development for two distinct disorders.

[Garcin syndrome in a patient with rhinocerebral mucormycosis].

Garcin syndrome is characterized by an unilateral cranial nerves involvement without sensory or motor long-tract disturbances. It is usually caused by tumor infiltrating in the skull base with osteolytic changes on radiological study. We report a case of 64-year-old man with history of alcohol overintake, who admitted local hospital, because of right periorbital edema and facial swelling. He noted right ptosis 2 weeks prior to admission. Neurological examination revealed right multiple cranial nerves involvement including II, III, IV, V, and VI cranial nerves. MR imaging of the brain showed marked paranasal sinusitis and abnormal infiltration of right orbital fat. Orbital apex syndrome related to paranasal sinusitis was diagnosed, and antibiotics was administered. But a few days after admission, he developed a right VII, IX, X cranial nerve palsy. He was transferred to our hospital because of acute development of left hemiparesis and deteriorated consciousness. MR imaging of the brain showed right internal carotid artery (ICA) occlusion, and infarction in right middle cerebral artery (MCA)'s territory. The diagnostic biopsy of the paranasal sinus showed mucorales hyphae, indicating that the pathological diagnosis was mucormycosis. Despite of antibiotic therapy included of amphotericin-B administration and strict control of diabetic mellitus, his sinusitis was gradually spread. His condition progressively deteriorated, and finally died of sepsis. Post-mortem examination revealed a widespread mucor infiltration in the dura mater without skull bone invasion. This case presented with unilateral multiple cranial nerve involvements (Garcin syndrome) followed by left hemiparesis associated with rhinocerebral mucormycosis. It is suggested that mucormycosis should be considered in case of Garcin syndrome without osteolysis in the skull base.

[Cheiromegaly and podomegaly associated with primary progressive multiple sclerosis: a case report].

Cheiromegaly is an extremely rare syndrome characterized by unilateral hand hypertrophy with hyperhydrosis. Unilateral foot hypertrophy has been described as podomegaly. Cheiromegaly/podomegaly is usually seen in patients with syringomyelia. Although the underlying pathophysiology remains unknown, it is speculated that chronic cervical cord injury including central descending sympathetic tract is essential for development of this syndrome. We report a cheiromegaly/podomegaly in a case with primary progressive multiple sclerosis. This 29-year-old woman developed cheiromegaly and podomegaly in her right hand and foot associated with chronic progressive cervical myelopathy. At age two, she developed transient paraparesis with full recovery, suggesting her symptomatic onset of her longstanding illness. At age 10, she had left leg weakness, and gradually worsened. On admission at age 29, she had muscular weakness in her left upper and both lower extremities. Sensory impairment was seen below her 5th cervical and 6th thoracic dermatome level. Her deep tendon reflexes were exaggerated in all extremities with bilateral Babinski's signs. MR imaging showed multiple plaques in the white matter of the brain associated with cervical spinal cord lesions. Taken together with the presence of oligoclonal band IgG in the cerebrospinal fluid, this patient met the criteria of multiple sclerosis. This is the first report presenting cheiromegaly/podomegaly not associated with syringomyelia, but with multiple sclerosis. It is suggested that cheiromegaly/podomegaly in present case may be caused by chronic cervical cord injury affecting descending central sympathetic tract associated with primary progressive multiple sclerosis.

Successful treatment of a patient with primary Sjögren's syndrome complicated with pericarditis during pregnancy.

A 35-year-old woman with primary Sjögren's syndrome (pSS) developed fever and chest pain during pregnancy. When the dose of prednisolone was reduced, she experienced chest pain with elevated CRP and D-dimer, resulting in admission to our hospital with marked cardiomegaly and pleural effusion. Because there was no evidence of other autoimmune disorders or infection, oral prednisolone was increased to 30 mg daily with heparin, and hypercoagulopathy was carefully monitored. The patient's condition improved rapidly, and she delivered a healthy baby. This is the first case to support the beneficial effect of prednisolone in pericarditis with pSS, and illustrates its safety during pregnancy.