Soluble amyloid precursor protein α in the cerebrospinal fluid as a diagnostic and prognostic biomarker for idiopathic normal pressure hydrocephalus.

BACKGROUND: Cognitive impairment is difficult to improve after shunt operation in patients with idiopathic normal pressure hydrocephalus (iNPH). This study aims to identify cerebrospinal fluid (CSF) biomarkers predictive of improvement in cognitive function. METHODS: This study was conducted between January 2008 and December 2010 on consecutive, unselected admissions to our program for the treatment of patients with clinically suspected iNPH. Lumbar CSF concentrations of total tau (Tau), tau phosphorylated at threonine 181 (p-tau), soluble amyloid precursor protein (sAPP), sAPPα, sAPPß, and ß-amyloid(1-42) (Aß42) were analyzed by ELISA. RESULTS: Concentrations of p-tau, sAPP, sAPPα, and sAPPß were strong diagnostic biomarkers for distinguishing between iNPH and Alzheimer's disease (AD). sAPPα exhibited the highest accuracy in differentiating iNPH from patients with AD and normal controls, with an area under the curve value of 0.994. We examined the prognostic value of p-tau and sAPPα for cognition function after surgery. With a cutoff value of 198 ng/ml or less for sAPPα, sensitivity and specificity are 66.7% and 82.9%, respectively, whilst the Mini-Mental State Examination score at 6 months after surgery is expected to be 25 or more. CONCLUSION: Our results show that sAPPα is a suitable biomarker for the diagnosis and prognosis of iNPH.

Selective down-regulation of Th2 cell-mediated airway inflammation in mice by pharmacological intervention of CCR4.

BACKGROUND: The chemokine receptor CCR4 has been implicated in Th2 cell-mediated immune responses. However, other T cell subsets are also known to participate in allergic inflammation. OBJECTIVE: The role of CCR4 in Th1, Th2, and Th17 cell-mediated allergic airway inflammation was investigated. METHOD: We generated an allergic airway inflammation model by adoptive transfer of in vitro-polarized ovalbumin (OVA)-specific Th1, Th2, and Th17 cells. The effect of a low-molecular weight CCR4 antagonist, Compound 22, on this model was examined. RESULTS: Upon in vitro polarization of DO11.10 naïve T cells, Th1- and Th2-polarized cells dominantly expressed CXCR3 and CCR4, respectively, while Th17-polarized cells expressed CCR6 and CCR4. Intranasal OVA-challenge of mice transferred with each T cell subset induced accumulation of T cells in the lungs. Eosinophils were also massively accumulated in Th2-transferred mice, whereas neutrophils were preferentially recruited in Th1- and Th17-transferred mice. Compound 22, as well as anti-CCL17 or anti-CCL22 antibody selectively suppressed accumulation of Th2 cells and eosinophils in the lungs of Th2-transferred and OVA-challenged mice. Compound 22 also inhibited bronchial hyperresponsiveness but had little effect on goblet cell hyperplasia in Th2-transferred and OVA-challenged mice. CONCLUSIONS AND CLINICAL RELEVANCE: There were notable differences in allergic lung inflammation mediated by different T cell subsets. CCR4 blockage was selectively effective for suppression of Th2-mediated allergic inflammation by blocking infiltration of Th2 cells.

Lipopolysaccharide-lipophorin complex formation in insect hemolymph: a common pathway of lipopolysaccharide detoxification both in insects and in mammals.

The formation of the lipophorin-lipopolysaccharide (LPS) complex in Bombyx mori hemolymph and its role in LPS detoxification were explored. LPS, an antibacterial protein inducer in insects, was injected into B. mori larvae. Analytical density gradient ultracentrifugation revealed that after injection the LPS peak shifts to a zone of lower density with time. The shifted peak was identified as the lipophorin-LPS complex. This complex formation was also achieved in an in vitro mixture of cell-free hemolymph and LPS at 25 degrees C but not at 1 degree C. The lipophorin-LPS complex had a significantly lower capacity to elicit the mRNA of cecropin B, an antibacterial protein. The biological activity of reextracted LPS from the complex was slightly reduced in the Limulus test and no structural modification was observed in sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These results suggested that the formation of lipophorin-LPS strikingly reduces the cecropin inducibility of LPS without any structural change in LPS. Similar serum lipoprotein-LPS complex formation and reduction of biological activities of LPS were also observed in mammals. We, therefore, suggest that the formation of the serum lipoprotein-LPS complex is a common pathway to inactivate LPS both in insects and in mammals.

Fibroblast growth factor (FGF)-9 immunoreactivity in senile plaques.

We examined fibroblast growth factor (FGF)-9 immunoreactivity in human hippocampal sections of Alzheimer's disease (AD). FGF-9 immunoreactivity was observed in dystrophic neurites of senile plaques in AD and control cases, in addition to the hippocampal and cortical neurons. The amyloid core and neurofibrillary tangles lacked immunoreactivity. FGF-9 immunoreactive astrocytes were conspicuous in AD brains. FGF-9 may be involved in the neuropathology of AD.

Detection of Borna disease virus genome in normal human brain tissue.

Borna disease virus (BDV), a neurotropic virus naturally infecting horses and sheep, has been suggested to be associated with human psychiatric disorders. Thus far no extensive studies have been done, providing the evidence of BDV genome in normal human brain tissue. We therefore examined four brain regions of 30 normal autopsy brains for BDV p24 genome. By highly sensitive nested reverse transcriptase (RT)-mediated PCR analysis, we found positive PCR products in two brains: one in frontal and temporal cortices and hippocampus and another in frontal cortex and olfactory bulb. Our results suggest that BDV can infect human brain tissue latently, without causing an apparent neuropsychiatric disorder.

Neuronal localization of a novel mosaic apolipoprotein E receptor, LR11, in rat and human brain.

A new type of mosaic protein was recently discovered as a new member of the low density lipoprotein receptor (LDLR) family, designated as LR11. The predominant expression of LR11 transcripts in brain tissue and the presence of elements found in neural adhesion molecules suggested a function(s) in the central nervous system (CNS). In order to gain insight about this complex receptor in the CNS, we raised a rabbit polyclonal antibody and examined immunohistochemically rat and human brain tissue. A strong LR11 immunoreactivity was found to be localized mainly in neurons throughout the brain in both species. A detailed mapping in the rat brain showed a distribution of LR11 immunoreactivity in a widespread population of neurons, though the intensity varied between different locations. The most prominent immunoreactivity was observed in neurons of the hippocampus, some nuclei of brain stem and Purkinje cells, whereas neurons of the thalamus and the hypothalamus showed weak staining. Uniquely, the single LR11 immunoreactive cytoplasmic puncta were observed in the proximity of apical dendrites, most conspicuously in the pyramidal neurons of hippocampus. In the human brain, one to four immunoreactive puncta were seen within individual neurons. The neuronal localization of LR11 and its unique association of cytoplasmic structure, presumably botrysome, may suggest the roles of LR11 in both the lipoprotein metabolism and intracellular trafficking in certain neuronal population of the CNS.

Tau accumulation in a patient with pallidonigroluysian atrophy.

We studied the brain of a patient with pallidonigroluysian atrophy (PNLA) in whom argyrophilic and abnormally phosphorylated tau positive neurons and glia were identified in the brain on Gallyas-Braak silver staining and immunohistochemical analysis although neurofibrillary tangles were not seen by Bodian silver stain. Immunohistochemical studies using six anti-tau antibodies that recognize the different phosphorylated epitopes of tau protein revealed that these epitopes in neurons and glial cells share common characteristics with neurofibrillary tangles in Alzheimer's disease. Immunoblot analysis of phosphorylated tau protein showed major bands of 64 and 68 kDa and after dephosphorylation, tau consisted mainly of 4 repeat tau. No mutations were detected in the coding exons and their flanking intronic regions of the tau gene. This study suggests that PNLA is one of tauopathy and the biochemical characteristics of phosphorylated tau are similar to those found in progressive supranuclear palsy and corticobasal degeneration.

Motor neurons in human and rat spinal cord synthesize fibroblast growth factor-9.

Fibroblast growth factor (FGF)-9, initially referred to as a glia-activating factor, is a recently identified member of the FGF family. In the present study we demonstrated that spinal cord motor neurons and dorsal root ganglion neurons were strongly immunostained with specific antibodies to FGF-9 in human and rat tissues. By in situ hybridization using digoxigenin-labeled antisense probe to FGF-9 mRNA, we found specific signals in these neurons in rat. By immunoblotting analysis, we detected a 30/29 kDa doublet band in human spinal cord proteins, which corresponded to the doublet band of originally isolated FGF-9 from culture media. Our results indicate that these neurons synthesize FGF-9.

High serum adenosine deaminase activity and its correlation with lymphocyte subsets in myasthenia gravis.

Serum adenosine deaminase (ADA) activity and peripheral lymphocyte subsets of patients with myasthenia gravis (MG) were simultaneously measured. The ADA activity in MG (n = 30) was significantly higher as compared with normal control (n = 150) and multiple sclerosis (n = 12) (P less than 0.05). The ADA activity of generalized MG was higher than that of ocular MG, while a significant elevation of ADA activity was observed in grade IIB as compared with grade I of Osserman's classification (P less than 0.05). A trend of high ADA activity was demonstrated in those whose disease had advanced to a severe degree associated with unstable clinical features (P less than 0.05). In addition, there was a significant elevation of ADA activity in patients who disclosed positive anti-Ach-receptor-antibody as compared with negative one (P less than 0.05). There was no specific trend among the proportions of the subsets of peripheral lymphocytes which could reflect the severity of MG, however, the proportion of OK Ia1+ tended to be higher with advancing the grade of MG. Interestingly enough, a close correlation was found between the ADA activity and the proportion of OK Ia1+ cells (P less than 0.05). From the above results, it was concluded that high ADA may be responsible for the pathophysiology of MG through the alteration of peripheral lymphocyte function.

A correlation study between blink reflex habituation and clinical state in patients with Parkinson's disease.

The neurologic states and activities of daily life of patients with Parkinson's disease were evaluated using a rating scale with subitems, and subsequently the neurologic disturbance scores and the daily activity impairment scores were obtained. Subjects consisted of 19 normal controls, and 55 ambulatory patients without marked dyskinesia who were on various anti-parkinsonian drugs. Blink reflex was elicited by paired electrical stimulation over the supraorbital nerve. The interval time between the conditioning stimulation and the test stimulation was set at 200 ms, and 5 serial ipsilateral maximal R2 amplitudes on the stimulated side were measured. The mean of the paired maximal R2 amplitude ratio (test/conditioning), expressed as a percentage, was defined as the habituation index. The habituation indices in normal controls and those with Parkinson's disease were 17.1 +/- 7.6 and 51.9 +/- 29.3, respectively (P less than 0.01). The degree of akinesia, rigidity, balance/gait and dysarthria was positively correlated with the habituation index (P less than 0.01), while tremor was not. On the whole the habituation index was found to have a significant correlation not only with the neurologic disturbance score but also with the daily activity impairment score (P less than 0.01).

Primary culture and expression of cytokine mRNAs by lipopolysaccharide in bovine Kupffer cells.

Kupffer cells are sessile tissue macrophages that have a role in liver defense against endogenous endotoxins. Because little information is available on the role of bovine Kupffer cells, we developed a primary culture method to investigate the function of bovine Kupffer cells. Kupffer cells were isolated from the caudate lobe of calf liver by perfusion with collagenase and pronase. Then, the cells were purified by gradient centrifugation followed by counterflow centrifugal elutriation. With the methods, a mean number of 1.5 x 10(6) Kupffer cells with a final viability of over 98% was obtained from 1 g of the liver. Over 95% of the isolated cells were positive for non-specific esterase activity and had surface molecule of CD68. The cultured Kupffer cells expressed mRNAs of tumor necrosis factor-alpha, interleukin (IL)-1 alpha, IL-1 beta and IL-6 by stimulation for 3 h with lipopolysaccharide. The primary culture of bovine Kupffer cells could be useful to investigate the systemic inflammatory response in bovine liver.

Association of interleukin-6 in the cerebrospinal fluid during crisis of calf with ammoniated feed syndrome.

Ammoniated feed syndrome (AFS) in cattle is a neurotoxic syndrome caused by feeding specific ammoniated forage. To clarify the pathophysiology of AFS, we examined the association of interleukin-6 (IL-6) in the brain. By feeding milk either from cows fed such ammoniated forage or milk added with 4-methyl-imidazole, newborn calves showed a neurotoxic crisis of hyperexcitability, ataxia, muscle tremor, circling, roaring, epileptoid seizure, sweating and marked fever response. Although these calves had no pathological lesions in the brain, we detected a rise in IL-6 in the cerebrospinal fluid (CSF). Tumor necrosis factor-alpha (TNF-alpha) was not detected in the CSF. In the sera, IL-6 and TNF-alpha hardly changed during the experiment. Administration of recombinant human IL-6 into the lateral ventricle resulted in fever. Thus, we believe IL-6 in the CSF is related to the fever response in newborn calves with AFS.

Prevalence of spotted fever rickettsial antibodies in dogs and rodents in the Philippines.

Antibodies against spotted fever group rickettsiae have been detected in blood samples of dogs and rodents obtained from selected areas in the Philippines. In this serosurvey, the positive percentage rates are 8.3% (11/132) in dogs and 12.2% (6/49) in rats. Positive results were read from samples tested with Rickettsia japonica antigen. No positive result was obtained in blood samples of rats and house mice using R. akari antigen. The findings of this study are the first to confirm the detection of spotted fever group rickettsial antibodies in the Philippines.

Tissue distribution of monomeric glutathione peroxidase in broiler chicks.

It is known that there are two kinds of enzymes which show glutathione peroxidase activity, 'classical' glutathione peroxidase and glutathione S-transferase. Recently, a third enzyme was found, monomeric glutathione peroxidase, in broiler chick liver cytosolic fraction. To gain an insight into the possible physiological role of the monomeric glutathione peroxidase, the distribution of this enzyme in other broiler tissues was studied. The monomeric glutathione peroxidase was found in all tissues examined and in erythrocytes. The percentage of the total glutathione peroxidase activity accounted for by the monomeric enzyme ranged from 4 per cent in erythrocytes to 28 per cent in liver. Livers from three avian and two mammalian species contained the monomeric glutathione peroxidase. The contribution of the monomeric enzyme to total glutathione peroxidase activity with cumene hydroperoxide was high in poultry livers, while only trace monomeric glutathione peroxidase activity was found in mammalian livers. Chick monomeric glutathione peroxidase showed high activity toward phospholipid hydroperoxide. Thus, monomeric glutathione peroxidase might be an important enzyme in reducing membrane lipid hydroperoxides in birds.

Elevated levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) activities in the sera and milk of cows with naturally occurring coliform mastitis.

The presence of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) activities was determined in milk and serum of cows with naturally occurring coliform mastitis (CFM). TNF-alpha was detected in the sera from 26 of 32 cows with CFM. TNF-alpha levels were higher in the sera than in the milk. IL-6 was high in the sera of surviving CFM animals, but was low in animals that died and in healthy controls. Furthermore, the mean level of IL-6 was 20-fold higher in the milk than in the sera of mastitic cows. The level of IL-6 in the serum was correlated to that in the milk in individual animals. The presence of IL-6 and TNF-alpha in the sera appears to relate to severe clinical condition of CFM, in the milk whereas they may play a role in generating inflammation of the mammary gland.

The influence of age and health status on the serum alpha 1-acid glycoprotein level of conventional and specific pathogen-free pigs.

Serum alpha-1-acid glycoprotein (alpha 1AG) was measured in 212 Landrace White pigs between birth and finishing age. The alpha 1AG level of healthy pigs five to ten months of age was 338 +/- 79 micrograms/mL, and the upper normal limit in mature swine has been established as 500 micrograms/mL. In both specific pathogen-free (SPF) and conventional pigs, the alpha 1AG level within one day of birth was 14,263 +/- 2,393 micrograms/mL, 40 times the normal adult value, but rapidly decreased to 699 +/- 186 micrograms/mL by four weeks of age. In conventional pigs, alpha 1AG began to increase after four weeks, averaged 1,428 micrograms/mL by eight weeks, but gradually decreased to adult levels by 20 weeks of age. In comparison, alpha 1AG of SPF pigs was only 800 micrograms/mL at eight weeks and decreased more rapidly to normal by 16 weeks of age. The conventional pigs had a high incidence of clinical pneumonia and specific antibodies to Actinobacillus pleuropneumoniae and Mycoplasma hyopneumoniae at the age of eight weeks. As the clinical pneumonia disappeared, serum alpha 1AG level also gradually declined. In contrast, SPF pigs had little clinical illness, low alpha 1AG, and little serological evidence of microbial infection. Conventional pigs with nonrespiratory infections, encephalitis, or with hernias had increased alpha 1AG. While the very high alpha 1AG level of the neonatal pig may be due to genetic influences, increases later in life are likely in response to stimuli from its external environment. Monitoring of serum alpha 1AG in several herds aided in the recognition of disease processes and may have potential use in swine herd health management.

Serum apolipoprotein B-100 concentrations in healthy and diseased cattle.

The purpose of this study was to establish the normal range of serum apolipoprotein B-100 (APO B-100) concentration in clinically normal cattle, and to assess its abnormalities with clinical diseases. We measured the serum concentration of APO B-100 in cattle of varying ages, breeds and sex, maintained under normal field conditions. Blood samples were obtained from 735 apparently healthy cattle and 146 cows with various diseases. The concentration of serum APO B-100 in cattle was assayed by the single radial immunodiffusion method. The concentration of serum APO B-100 in healthy adult breeding bulls (mean +/- SD: Holstein; 101 +/- 46 microg/ml, Japanese Black; 106 +/- 46 microg/ml) was significantly (P<0.001) lower than that in cows (Holstein; 259 +/- 63, Japanese Black; 210 +/- 46 microg/ml), while that of APO B-100 in steers (Holstein; 290 +/- 86 microg/ml, Japanese Black; 302 +/- 90 microg/ml) was similar to the level in cows. The concentration of serum APO B-100 in cattle varied with sex and breed. APO B-100 concentration in cattle was decreased in association with metabolic disorders such as ketosis, displaced abomasum and fatty liver. From these results, it is assumed that the level of serum APO B-100 will be applied to diagnosis of metabolic diseases in cattle.

Turbidimetric-kinetic assay of endotoxin in rumen fluid or serum of cattle fed rations containing various levels of rolled barley.

A new, automated turbidimetric-kinetic (ATK) assay was used to quantitate bacterial endotoxin in rumen fluid or in serum of Holstein steers. The ATK method used Limulus amebocyte lysate (LAL) reagent with added beta-glucan (LAL-ES) which improved specific sensitivity to endotoxin. Design of the feeding trial permitted comparison of endotoxin levels found during consumption of a basal ration with those higher levels detected at various times following the introduction of increasing percentages of rolled barley to that basal ration. Both serum and ruminal endotoxin levels were significantly higher in steers on the higher concentrate rations. Peak endotoxin levels were detected 20 days following the change to the highest concentrate ration which contained 60% barley. Endotoxin levels from both sources subsequently decreased. Ruminal endotoxin stabilized at about 10 times the level, and serum endotoxin stabilized at 2 to 4 times the level, of that previously found during feeding of the basal ration. Test protocol included sample dilution and heating in order to avoid the effects of endotoxin inhibitors. Recovery rates for added endotoxin to either serum or rumen fluid supernates ranged from 120 to 136%. Coefficient of variation for endotoxin concentration in serum was lower than 10%, and in rumen fluid only slightly higher. There was significant correlation between ruminal concentration as measured by the ATK method and an alternative chromogenic substrate assay procedure. Changes in endotoxin level in experimental steers were those predictable from experience with naturally occurring incidents of grain engorgement. The ATK assay appears to be an accurate and rather simple technique which will prove useful for experimental and clinical studies in the future.

Serum growth hormone and insulin-like growth factor-1 concentrations in Japanese black cattle with growth retardation.

Serum concentrations of growth hormone (GH) and insulin-like growth factor-1 (IGF-1) were determined in 5 calves in the same lineage with growth retardation. They had normal appetites, activities, body proportion, and laboratory test results. Calves with growth retardation had higher serum GH concentrations and lower serum IGF-I concentrations. These findings suggested defects in the GH-IGF-1 axis, such as in the GH-receptor.

Characterization of serum alpha 1-acid glycoprotein in fetal and newborn calves during development.

Serum alpha 1-acid glycoprotein (alpha 1-AG) in bovine fetuses and newborn calves was characterized. Serum alpha 1-AG concentration increased during fetal development and neonatal stages. Mean +/- SD serum alpha 1-AG concentration reached a peak of 1,368 +/- 207 micrograms/ml immediately after birth, but thereafter gradually decreased to 249 +/- 100 micrograms/ml, similar to the normal adult bovine range. By use of isoelectric focusing of thin-layer gels, we detected 7 microheterogeneity bands ranging from pI 3.2 to 3.8 in adult bovine serum. Twelve bands ranging from pI 2.6 to 3.8 were found in 9-month fetuses and in neonates. The 5 most-acidic bands, which are absent in adult serum, ranged between pI 2.6 and 3.1 and decreased with maturation as band patterns assumed adult characteristics. By crossed affinity electrophoresis, alpha 1-AG of adult bovine serum was separated into 4 peaks according to its differential affinity to concanavalin A (conA). Seventy-five percent of the alpha 1-AG concentration was represented by peak 3 (P-3) and peak 4 (P-4), which had moderate or strong binding to conA. In contrast, fetal sera contained only peak 1 (P-1), which did not have conA-binding affinity. In neonatal sera, 4 peaks were recognized, of which P-1 comprised 70% of the total alpha 1-AG. Thereafter, with aging, percentage of P-3 and P-4 increased as band composition approached the normally expected adult pattern.