CCL8 deficiency in the host abrogates early mortality of acute graft-versus-host disease in mice with dysregulated IL-6 expression.

Although allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for diverse malignant and nonmalignant diseases, acute graft-versus-host disease (aGVHD) is strongly linked to mortality caused by HSCT. We previously reported that CC chemokine ligand 8 (CCL8) is closely correlated to aGVHD mortality in both humans and mice. To study the role of CCL8 in aGVHD, CCL8 knockout (CCL8-/-) mice were transplanted with fully allogeneic marrow grafts. These mice exhibited a significant reduction in mortality (90.0% vs. 23.4% survival for CCL8-/- vs. wild-type recipients at day 28, p < 0.0001). As a result, apparent prolonged median survival from 9 days in wild-type mice to 45 days in CCL8-/- mice was observed. Acute GVHD pathology and liver dysfunction in CCL8-/- mice were significantly attenuated compared with those in wild-type mice. In association with the reduced mortality, a surge of plasma interleukin (IL)-6 was observed in CCL8-/- recipients with allogeneic marrow, which was significantly increased compared with wild-type mice that received allografts. Donor T-cell expansion and plasma levels of interferon-γ and TNF-α during aGVHD were similar in both types of mice. Collectively, these findings indicate that CCL8 plays a major role in aGVHD pathogenesis with possible involvement of an IL-6 signaling cascade.

Proteomics analysis of site- and stage-specific protein expression after peripheral nerve injury.

BACKGROUND: The peripheral nervous system has greater regenerative potential than the CNS. This fact suggests the existence of molecules that act as key factors in nerve regeneration during molecular changes in the peripheral nervous system. METHODS: The right sciatic nerve of female Sprague-Dawley rats was exposed and transected at the mid-thigh level. Animals were sacrificed at 5, 10 or 35 days after nerve transection. Proximal and distal nerve segments (1-cm in length) were dissected. We then sought to observe overall molecular changes after peripheral nerve injury using a proteomic approach. For an overview of the identified proteins, each protein was classified according to its biological and molecular functions. We identified a number of proteins showing site- and stage-specific patterns of expression. RESULTS: Both proximal and distal molecular changes at 5, 10 and 35 days after nerve transection were investigated, and a total of 2353 proteins were identified. Among the various expression patterns observed, aFGF and GAP-43 were found to increase in the proximal stump at 10 days after transection, and PN-1, RPL9 and prosaposin increased in the distal stump at 5 days after transection. Among these proteins, aFGF, GAP-43, PN-1 and prosaposin were found to be associated with nerve regeneration. CONCLUSION: We demonstrated that aFGF, GAP-43, PN-1 and prosaposin expression increased at specific stages and in specific sites, such as the proximal or distal stump, after nerve transection by comprehensive measurement using proteomics analysis. We believe that these specific expression patterns might play important roles during regeneration after nerve injury.

Effects of dietary supplementation with milk fat globule membrane on the physical performance of community-dwelling Japanese adults: a randomised, double-blind, placebo-controlled trial.

We conducted a randomised, double-blind, placebo-controlled trial to elucidate the effects of dietary milk fat globule membrane (MFGM) on the physical performance of community-dwelling Japanese adults. For this 24-week study, 115 middle-aged subjects (range 50-70 years old) were invited, of whom 113 (seventy-two women, forty-one men) completed the trial. Participants were then divided into either the placebo control or MFGM group. Measurements of physical performance (without undertaking any mandatory exercise) examining muscle strength, agility and balance were tested every 6 weeks until 24 weeks. Analyses were performed using the intention-to-treat method for all participants. Although the effects of MFGM on muscle strength and agility were not significant, we noted that the parameter for balance (such as the ability to stand on one leg with eyes closed for longer durations) increased in the MFGM group (mean 10·1 (95 % CI 8·25, 12·4) s) compared with the placebo (mean 7·53 (95 % CI 6·11, 9·30) s) (P = 0·046). Similarly, application of the mixed-effect model for repeated measures under unstructured covariance also revealed that the effect of MFGM was significant when compared with the placebo (10·2 (95 % CI 8·33, 12·4) v. 7·61 (95 % CI 6·17, 9·30) s) (P = 0·045). In conclusion, we demonstrated that MFGM had an effect on the physical performance of community-dwelling Japanese adults despite mandatory exercise. However, studies using larger cohorts of individuals from different demographic backgrounds are required to further elucidate the mechanisms underlying these effects and to extend the application of MFGM.

Reduction of HbA1c levels by fucoxanthin-enriched akamoku oil possibly involves the thrifty allele of uncoupling protein 1 (UCP1): a randomised controlled trial in normal-weight and obese Japanese adults.

Lifestyle-related problems are becoming a major health threat in East Asian countries. Therefore, finding an efficacious nutraceutical for this population is important. One candidate is fucoxanthin (Fx), a carotenoid abundantly found in edible brown seaweed that has been associated with a number of valuable health-promoting benefits. Unfortunately, clinical studies of Fx are limited. In the present study, we aimed to evaluate the effects of Fx on obesity-related parameters in Japanese subjects harbouring an SNP associated with lifestyle-related problems. In all, sixty normal-weight and obese Japanese adults with BMI over 22 kg/m2 were single-blinded and randomly assigned to three Fx-dose cohorts and administered Fx-enriched akamoku oil containing Fx at 0, 1 or 2 mg/d for 8 weeks (n 20 per group). Parameters relating to obesity and serum Fx metabolites were measured before and after intervention, but no significant differences were observed between and within the groups. Despite no changes in visceral fat areas and resting energy expenditures after intervention, we observed a significant decline in HbA1c levels in the 2 mg/d Fx group compared with that in the 0 mg/d group (P < 0·05), which was correlated with an increase in serum fucoxanthinol (Fx metabolite) levels. In addition, HbA1c levels declined more significantly in subjects with G/G alleles of the uncoupling protein 1 (UCP1) gene than in those with the A/A and A/G alleles (P < 0·05). We conclude that although Fx supplementation does not affect visceral fat areas, it may reduce HbA1c levels in those harbouring the thrifty allele of UCP1-3826A/G.

HLA-A24 ligandome analysis of colon and lung cancer cells identifies a novel cancer-testis antigen and a neoantigen that elicits specific and strong CTL responses.

This study focused on HLA-A24 and comprehensively analyzed the ligandome of colon and lung cancer cells without the use of MHC-binding in silico prediction algorithms. Affinity purification using the antibody specific to HLA-A24 followed by LC-MS/MS sequencing was used to detect peptides, which harbored the known characteristics of HLA-A24 peptides in terms of length and anchor motifs. Ligandome analysis demonstrated the natural presentation of two different types of novel tumor-associated antigens. The ligandome contained a peptide derived from SUV39H2, a gene found to be expressed in a variety of cancers but not in normal tissues (except for the testis). The SUV39H2 peptide is immunogenic and elicits cytotoxic CD8+ T-cell (CTL) responses against cancer cells and is thus a novel cancer-testis antigen. Moreover, we found that microsatellite instability (MSI)-colon cancer cells displayed a neoepitope with an amino-acid substitution, while microsatellite stable (MSS)-colon and lung cancer cells displayed its counterpart peptide without the substitution. Structure modeling of peptide-HLA-A24 complexes predicted that the mutated residue at P8 was accessible to T-cell receptors. The neoepitope readily elicited CTL responses, which discriminated it from its wild-type counterpart, and the CTLs exhibited considerably high cytotoxicity against MSS-colon cancer cells carrying the responsible gene mutation. The specific and strong CTL lysis observed in this study fosters our understanding of immune surveillance against neoantigens.

Evaluation of the Japanese Metabolic Syndrome Risk Score (JAMRISC): a newly developed questionnaire used as a screening tool for diagnosing metabolic syndrome and insulin resistance in Japan.

OBJECTIVES: To prevent the onset of lifestyle-related diseases associated with metabolic syndrome (MetS) in Japan, research into the development of a useful screening method is strongly desired. We developed a new screening questionnaire (JAMRISC) utilizing a logistic regression model and evaluated its ability to predict the development of MetS, type 2 diabetes and other lifestyle-related diseases in Japanese populace. METHODS: JAMRISC questionnaire was sent to 1,850 individuals in Rumoi, a small city in Hokkaido. We received a total of 1,054 valid responses. To maximize the target individuals accurately diagnosed with MetS, logistic regression analysis was used to generate a unique metabolic syndrome score calculation formula as taking into consideration the clinical relevance of each question item as individual coefficients. RESULTS: The results of our comparative research utilizing both JAMRISC and Finnish Diabetes Risk Score (FINDRISC) questionnaires revealed the usefulness of JAMRISC for its ability to detect risks for MetS, pre-MetS, diabetes, and pre-diabetes. Study of disease risk detection via JAMRISC questionnaire targeting the 4283 residents of Rumoi indicated a high detection rate for pre-MetS (98.8 %), MetS (94.2 %), pre-diabetes (85.1 %) and type 2 diabetes (94.9 %). In addition, JAMRISC was useful not only as a MetS risk score test, but also as a screening tool for diagnosing insulin resistance. CONCLUSIONS: JAMRISC questionnaire is a useful instrument for the detection of early risk of not only MetS and type 2 diabetes but also insulin resistance.

Alteration of circulating type 2 follicular helper T cells and regulatory B cells underlies the comorbid association of allergic rhinitis with bronchial asthma.

Allergic rhinitis (AR), the most common allergic disorder of the airway, is often accompanied by bronchial asthma. However, little is known about the mechanism by which AR advances to AR comorbid with bronchial asthma (AR+Asthma). To determine the pathophysiologic features of AR and AR+Asthma, we examined subsets of follicular helper T (Tfh) cells and regulatory B (Breg) cells in peripheral blood from AR and AR+Asthma patients. The results showed polarization of Tfh2 cells within Tfh cell subsets in both AR and AR+Asthma cases. Interestingly, the %Breg cells in total B cells were decreased in AR cases and, more extensively, in AR+Asthma cases. Moreover, we found significant correlations of fractional exhaled nitric oxide and blood eosinophil levels with the index %Tfh2 cells per %Breg cells. Our findings indicate that relative decrease in Breg cells under the condition of Tfh2 cell skewing is a putative exaggerating factor of AR to bronchial asthma.

P4-006　　Natural peptidome presented by HLA-A24 of cancer and cancer stem cells.

  Circulating CD8+ T cells (CTL) survey the peptide/MHC class I complexes on cell surface to discriminate self and eliminate foreign/transformed cells. Thus, the MHC class I peptide repertoire directly influences cells fate, and provides information for immunotherapy strategy. Here we target HLA-A24 (A24), the most frequent serotype in Asia, and perform large-scale mass spectrometric profiling of natural peptides. Using the antibody specific to A24, we identified 264 peptides from colon (SW480, Colo320, HCT15/b2m) and 331 peptides from lung (LHK2, Sq-1) cancer cells. Although, known A24 binding anchors (Y/F at P2 and F/L/I at P9) are strongly conserved among the detected ligands, a subset of peptides with an unusual anchor (K/R at the C-terminal P9 or P10) is observed, suggesting diverse usage of anchors in certain types of cancer cells. Moreover, some peptides (and their genes) are exclusively expressed in cancer stem cells (cells able to exclude Hoechst dye). In summary, we identified approx. 500 non-overlapping natural peptides presented by A24 from colon and lung cancer cells. A combination of natural peptides specific to tumors could be an ideal way for a CTL-based immunotherapy.

Significant increase of plasma tetranectin in ovx mice as defined by proteomics analysis.

BACKGROUND: Proteomics is recognized as a useful tool in the dynamic screening of plasma protein expression. This study aimed to identify increased expressions of novel plasma proteins in ovariectomized mice (ovx) using selective reaction monitoring (SRM) validation in combination with electrospray ionized-quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) screening. MATERIALS AND METHODS: Twenty-week-old female C57BL/6 mice were ovariectomized or subjected to surgical exposure of the ovaries alone (sham). Blood plasma protein at 4 weeks after these operations was pooled for the ovx and sham group each and separated on SDS-PAGE, and then digested by peptides, which were first differentially displayed by ESI-Q-TOF-MS analysis. Mass spectra of peptides upregulated more than twofold in ovx compared to sham mice were selected for protein identification by ESI-Q-TOF-MS. The selected peptides were further validated in independent samples by SRM using electrospray ionized-triple quadrupole-linear ion trap mass spectrometry (ESI-QqLIT-MS). Optimum transitions for SRM were manually chosen for their high specificity in identifying peptides derived from the candidate proteins. RESULTS: Differential analysis of peptides revealed 1,108 upregulated peptides in ovx compared with sham control mice. Among the upregulated peptides, 231 nonredundant proteins were identified. Validation analysis for the potential use of these proteins as markers of bone turnover was performed using ESI-QqLIT-MS. The four proteins from the plasma samples, namely mannose-binding lectin-C, major urinary protein 2, type I collagen alpha 2 chain, and tetranectin, were evaluated in a blinded manner. A statistically significant elevation of all four proteins in the plasma of ovx mice was confirmed by SRM. Of the four upregulated plasma proteins, tetranectin increased by almost 50 times in the ovx mice compared with the sham mice. CONCLUSIONS: On the basis of proteomics analysis, this study demonstrated that four plasma proteins were significantly elevated in the ovx mice; of these, tetranectin was markedly upregulated by almost 50 times compared with the sham mice.

Deficiency of NOX1/nicotinamide adenine dinucleotide phosphate, reduced form oxidase leads to pulmonary vascular remodeling.

OBJECTIVE: Involvement of reactive oxygen species derived from nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase has been documented in the development of hypoxia-induced model of pulmonary arterial hypertension (PAH). Because the PAH-like phenotype was demonstrated in mice deficient in Nox1 gene (Nox1(-/Y)) raised under normoxia, the aim of this study was to clarify how the lack of NOX1/NADPH oxidase could lead to pulmonary pathology. APPROACH AND RESULTS: Spontaneous enlargement and hypertrophy of the right ventricle, accompanied by hypertrophy of pulmonary vessels, were demonstrated in Nox1(-/Y) 9 to 18 weeks old. Because an increased number of α-smooth muscle actin-positive vessels were observed in Nox1(-/Y), pulmonary arterial smooth muscle cells (PASMCs) were isolated and characterized by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. In Nox1(-/Y) PASMCs, the number of apoptotic cells was significantly reduced without any change in the expression of endothelin-1, and hypoxia-inducible factors HIF-1α and HIF-2α, factors implicated in the pathogenesis of PAH. A significant decrease in a voltage-dependent K(+) channel, Kv1.5 protein, and an increase in intracellular potassium levels were demonstrated in Nox1(-/Y) PASMCs. When a rescue study was performed in Nox1(-/Y) crossed with transgenic mice overexpressing rat Nox1 gene, impaired apoptosis and the level of Kv1.5 protein in PASMCs were almost completely recovered in Nox1(-/Y) harboring the Nox1 transgene. CONCLUSIONS: These findings suggest a critical role for NOX1 in cellular apoptosis by regulating Kv1.5 and intracellular potassium levels. Because dysfunction of Kv1.5 is among the features demonstrated in PAH, inactivation of NOX1/NADPH oxidase may be a causative factor for pulmonary vascular remodeling associated with PAH.

Innervation pattern at the undersurface of the extensor carpi radialis brevis tendon in recalcitrant tennis elbow.

PURPOSE: We investigated the general, sensory, and sympathetic innervation patterns at the undersurface of the extensor carpi radialis brevis (ECRB) origin in patients with recalcitrant tennis elbow. METHODS: Eight elbows in eight consecutive patients (6 females and 2 males) with tennis elbow who underwent arthroscopic surgery were included in this study. The mean age was 45 years (38-66 years), and the mean duration of symptoms before surgery was 23 months (13-52 months). Operative treatment consisted of an arthroscopic inspection and debridement of the ECRB origin. Control tissues were obtained from biopsy of the ECRB capsule in two patients with osteochondritis dissecance of the capitellum who underwent arthroscopic resection of loose bodies. The tissue specimens were investigated immunohistochemically with antibodies delineating general (PGP9.5), sensory (SP/CGRP), and sympathetic (NPY) nerve patterns. RESULTS: In the non-tendinosis control tissue, SP/CGRP and NPY immunoreactions were heterogeneously distributed in association with blood vessels. Pathologic evaluation of the biopsy tissue showed atypical fibrous granulation containing numerous vessels and nerve structures in all eight patients. Marked reactions to PGP 9.5 took the form of nerve fibers associated with arteries and arterioles in the atypical granulation. Most of the perivascular innervation was found to express NPY. The immunoreactions for SP and CGRP were invariably weak. CONCLUSION: Increased perivascular sympathetic innervation accompanied with loss of sensory innervation at the undersurface of the ECRB tendon may play a role in chronic pain generation in recalcitrant tennis elbow. LEVEL OF EVIDENCE: Diagnostic, Level IV.

Evaluation of annexin A5 as a biomarker for Alzheimer's disease and dementia with lewy bodies.

BACKGROUND: Alzheimer's disease (AD) differs from other forms of dementia in its relation to amyloid beta peptide (Aß42). Using a cell culture model we previously identified annexin A5, a Ca(2+), and phospholipid binding protein, as an AD biomarker. Plasma level of annexin A5 was significantly higher in AD patients compared to that in a control group. On the other hand, AD has been identified to share a number of clinical and pathological features with Dementia with Lewy bodies (DLB). The present study was done to examine whether or not plasma annexin A5 is a specific marker for AD, when being compared with the levels of DLB patients. As Apolipoprotein E (ApoE) gene subtype ε4 (ApoE-ε4) has been noticed as the probable genetic factor for AD, we also examined and compared ApoE genotype in both AD and DLB. METHODS: Blood samples were obtained from 150 patients with AD (aged 77.6 ± 6.5 years), 50 patients of DLB (79.4 ± 5.0) and 279 community-dwelling healthy elderly individuals of comparable age and sex (75.6 ± 8.1). All AD patients met NINCDS-ADRDA criteria and all DLB patients were diagnosed as probable DLB according to the latest consensus diagnostic criteria. Quantification was done using the Chemiluminescent Enzyme Immunoassay (CLEIA) Technique (SphereLight assay) using the monoclonal antibodies against annexin A5. DNA genotyping of ApoE was performed by distinguishing unique combinations of Hha1 fragments of PCR-amplified genomic DNA products. RESULTS: The plasma level of annexin A5 was significantly higher in AD patients than in the healthy individuals (control) (P < 0.0001). The plasma annexin A5 level was also significantly higher in DLB patients than in the control group (P < 0.0001). From the ROC curves with plasma annexin A5 concentrations, the mean areas under the curve were 0.863 and 0.838 for the AD/control and DLB/control, respectively. The rate of ApoE4 carrier status and the frequency of the ε4 allele were significantly higher in AD or DLB than in control and there was no significant difference between AD and DLB. CONCLUSIONS: These results suggest that both annexin A5 and ApoE4 are common markers for AD and DLB.

Oral administration of monogalactosyl diacylglycerol from spinach inhibits colon tumor growth in mice.

Previously, we observed that purified monogalactosyl diacylglycerol (MGDG), a major glycoglycerolipid from spinach, selectively inhibits the activities of mammalian replicative DNA polymerases (α, δ and ε). However, the function of MGDG following ingestion is not well-known. In the present study, spinach MGDG suppressed the proliferation of Colon26 mouse colon cancer cells with an LD(50) of 24 µg/ml in vitro. γ-cyclodextrin (CD)-MGDG complex was prepared and administered orally following Colon26 mouse tumor adhesion for 26 days. It was observed that 20 mg/kg equivalent (eq.) of the CD-MGDG complex reduced tumor volume by ∼60% compared with that of the vehicle-treated controls. In immunohistochemical analysis, the CD-MGDG complex group showed a decreased number of proliferating cell nuclear antigen (PCNA)-positive cells and reduction of mitosis in the tumor cells compared with the control group. In addition, the CD-MGDG complex increased the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive apoptotic cells and inhibited CD31-positive tumor blood vessel growth significantly. These results suggest that MGDG has the potential for cancer prevention and health promotion.

Metallothionein deficiency in the injured peripheral nerves of complex regional pain syndrome as revealed by proteomics.

Complex regional pain syndrome (CRPS) is characterized by persistent and severe pain after trauma or surgery; however, its molecular mechanisms in the peripheral nervous system are poorly understood. Using proteomics, we investigated whether injured peripheral nerves of CRPS patients have altered protein profiles compared with control nerves. We obtained nerve samples from 3 patients with CRPS-2 who underwent resection of part of an injured peripheral nerve. Sural nerves from fresh cadavers with no history of trauma or neuropathic pain served as controls. Proteomic analysis showed that the number and functional distribution of proteins expressed in CRPS and control nerves was similar. Interestingly, metallothionein was absent in the injured nerves of CRPS-2, although it was readily detected in control nerves. Western blotting further confirmed the absence of metallothionein in CRPS-2 nerves, and immunohistochemistry corroborated the deficiency of metallothionein expression in injured nerves from 5 of 5 CRPS patients and 2 of 2 patients with painful neuromas. In contrast, all control nerves, including 5 sural nerves from fresh cadavers and 41 nerves obtained from surgically resected tumors, expressed MT. Furthermore, expression of S100 as a marker for Schwann cells, and neurofilament M as a marker of axons was comparable in both CRPS-2 and controls. Metallothioneins are zinc-binding proteins that are probably involved in protection against injury and subsequent regeneration after CNS damage. Their absence from the injured peripheral nerves of patients with CRPS-2 suggests a potential pathogenic role in generating pain in the damaged peripheral nerves.

Early expression of plasma CCL8 closely correlates with survival rate of acute graft-vs.-host disease in mice.

OBJECTIVE: To elucidate the significance of early expression of CC-chemokine ligand motif 8 (CCL8) in mice with graft-vs.-host disease (GVHD), we investigated its induction mechanisms and correlation with overall survival rate in GVHD mice. Plasma CCL8 increases on day 5 of allogeneic transplantation, when signs of GVHD are barely detectable. Increase of allogeneic splenocytes in grafts exacerbates GVHD and leads to upregulation of plasma CCL8 on day 5. Overall survival is the gold standard in determining the severity of acute GVHD in mice, but the absence of clinical and/or pathological manifestations in the early phase make it difficult to estimate vital outcomes at this stage of allogeneic marrow transplantation. MATERIALS AND METHODS: After lethal irradiation, BALB/c mice received bone marrow transplantation from C57BL/6 mice. Survival rate was monitored and clinical and pathological scores of GVHD were examined. Coculture of BALB/c-derived dendritic cells and C57BL/6-derived splenocytes was performed. CCL8 was measured by immunoassay. RESULTS: The plasma CCL8 level at day 5 of transplantation was closely correlated with survival rate and clinical/pathological scores on day 14. In vitro study revealed that the BALB/c-derived dendritic cells expressed CCL8 upon stimulation of C57BL/6 CD4(+) T cells by cell interactions through major histocompatibility complex class II molecules. CONCLUSIONS: These investigations indicate that early and preclinical expression of CCL8 in plasma predicts overall survival of GVHD mice. Together with an involvement of allo-recognition in CCL8 expression, it suggests that CCL8 plays an important role in GVHD pathology.

Activation of glial cells in the spinal cord of a model of lumbar radiculopathy.

PURPOSE: Glial cells in the spinal cord of a lumbar radiculopathy model were investigated using immunohistochemical methods. Neuropathic pain is a consequence of neural plasticity. In models of neuropathic pain models, roles for glial cells in the development of pain behaviors have been reported. Accumulating evidence suggests that activation of p38 mitogen-activated protein kinase (p38) in glial cells contributes to the pathogenesis of neuropathic pain. We examined whether activation of glial cells is involved in the development of neuropathic pain-like behavior observed in a model of lumbar radicular pain that we developed. However, the pathogenesis of lumbar radiculopathy and in particular the effect of spinal glial activation on pain transmission in the dorsal horn of the spinal cord are still not fully known. METHODS: The left L5 spinal root of Sprague-Dawley rats was ligated proximal to the DRG to produce models of lumbar radiculopathy. Protein levels of phosphorylated-p38 (p-p38) in the spinal cord were quantified by Western blot analysis. Double-immunofluorescense studies of p-p38 and specific markers of glia and neurons were performed to determine when and which types of cells were activated in the spinal cord. RESULTS: We observed p38 activation in hyperactive microglia in the dorsal horn ipsilateral to surgery at 1 and 7 days after root constriction, but not in astrocytes or neurons. CONCLUSIONS: Constriction of the lumbar root activated microglia in the spinal cord at 1 and 7 days after surgery, and then returned to normal state at 28 days after surgery, while pain behavior continued. These findings suggest that development of lumbar radicular pain may be initiated by activation of microglia.

Apolipoprotein E4 frequencies in a Japanese population with Alzheimer's disease and dementia with Lewy bodies.

BACKGROUND: The apolipoprotein E (APOE) ε4 allele has been reported to be a risk factor for Alzheimer's disease (AD) and dementia with Lewy bodies (DLB). Previous neuropathological studies have demonstrated similar frequencies of the APOE ε4 allele in AD and DLB. However, the few ante-mortem studies on APOE allele frequencies in DLB have shown lower frequencies than post-mortem studies. One reason for this may be inaccuracy of diagnosis. We examined APOE genotypes in subjects with AD, DLB, and a control group using the latest diagnostic criteria and MRI, SPECT, and MIBG myocardial scintigraphy. METHODS: The subjects of this study consisted of 145 patients with probable AD, 50 subjects with probable DLB, and a control group. AD subjects were divided into two groups based on age of onset: early onset AD (EOAD) and late onset AD (LOAD). All subjects had characteristic features on MRI, SPECT, and/or myocardial scintigraphy. RESULTS: The rate of APOE4 carrier status was 18.3% and the frequency of the ε4 allele was 9.7% in controls. The rate of APOE4 carrier status and the frequency of the ε4 allele were 47% and 27% for LOAD, 50% and 31% for EOAD, and 42% and 31% for DLB, respectively. CONCLUSION: The APOE4 genotypes in this study are consistent with previous neuropathological studies suggesting accurate diagnosis of AD and DLB. APOE4 genotypes were similar in AD and DLB, giving further evidence that the ε4 allele is a risk factor for both disorders.

Rab7 is a critical mediator in vesicular transport of tyrosinase-related protein 1 in melanocytes.

How melanosomal proteins such as enzymic proteins (tyrosinase and tyrosinase-related proteins, Tyrps) and structural protein (gp100) are transported from Golgi to melanosomal compartments is not yet fully understood. A number of small GTPases have been found to be associated with melanosomes and we have identified one of them, Rab7, a regulator of vesicular transport, organelle motility, phospholipid signaling and cytosolic degradative machinery, as being involved in the transport of Tyrp1 from Golgi to stage I melanosomes. This study further characterizes the role of Rab7 as a regulator of differential sorting of melanosomal proteins in this process. Murine melanocytes were transiently transfected with a plasmid encoding either wild-type (Rab7WT), constitutively active (Rab7Q67L) or dominant-negative (Rab7N125I and Rab7T22N) Rab7. Through immunocytostaining and confocal laser scanning microscopy, we quantitatively compared the bio-distribution of melanosomal proteins between Rab7WT-expressing cells and mutant Rab7-expressing cells. We also characterized their differential elimination from melanosomal compartments by Rab7 by utilizing a proteasome inhibitor, MG132. Our findings indicate that Rab7 plays an important role in differential sorting of tyrosinase, Tyrp1 and gp100 in early melanogenesis cascade, and that it is more specifically involved with Tyrp1 than tyrosinase and gp100 in the trafficking from Golgi to melanosomes and the specific exit from the degradative process.

Investigation of annexin A5 as a biomarker for Alzheimer's disease using neuronal cell culture and mouse model.

Alzheimer's disease (AD) differs from other forms of dementia in its relation to amyloid beta peptide (Abeta). Abeta, a proteolytic product of amyloid precursor proteins (APP), has a toxic effect on neuronal cells, which involves perturbation of their Ca(2+) homeostasis. This effect implies that changes of protein expression in neuronal cells with calcium stress should provide a molecular marker for this disease. In the present study, we used the supernatant from a neuronal cell culture after incubation with or without Abeta and isolated a Ca(2+)-dependent acidic phospholipid binding fraction to perform a proteomic study. Several unique proteins were identified after incubation with Abeta. We focused on annexin A5, among these proteins, because it binds both Ca(2+) and lipids likely to be involved in calcium homeostasis. Tg2576 transgenic mice (AD model) overexpressing mutant human APP showed a significant increase of annexin A5 in the brain cortex but not in other organs, including liver, kidney, lung, and intestine. In human plasma samples, the level of annexin A5 was significantly increased in a proportion of AD patients compared with a control group (P < 0.0001 in the logistic regression analysis). From the receiver operating characteristic (ROC) curve with plasma annexin A5 concentrations, the mean area under the curve (AUC 0.898) suggests that annexin A5 is a favorable marker for AD.