Complement profile in microscopic polyangiitis and granulomatosis with polyangiitis: analysis using sera from a nationwide prospective cohort study.

OBJECTIVE: The complement cascade, especially the alternative pathway of complement, has been shown in basic research to be associated with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV). We aimed to elucidate relationships between serum complement components and clinical characteristics in AAV. METHOD: In a nationwide prospective cohort study (RemIT-JAV-RPGN), we measured the serum levels of C1q, C2, C3, C3b/iC3b, C4, C4b, C5, C5a, C9, factor B, factor D, factor H, factor I, mannose-binding lectin, and properdin in 52 patients with microscopic polyangiitis (MPA) and 39 patients with granulomatosis with polyangiitis (GPA). RESULTS: The properdin level of MPA and GPA was significantly lower than that of healthy donors. The properdin level was negatively correlated with the Birmingham Vasculitis Activity Score (BVAS) (ρ = -0.2148, p = 0.0409). The factor D level at 6 months was significantly positively correlated with the Vasculitis Damage Index (VDI) at 6, 12, and 24 months (ρ = 0.4207, 0.4132, and 0.3115, respectively). Patients with a higher ratio of C5a to C5 had higher neutrophil percentage and serum immunoglobulin G levels, and significantly lower creatinine levels. Cluster analysis divided the MPA and GPA patients into three subgroups. A principal component (PC) analysis aggregated 15 types of complements into alternative pathway-related PC 1 and complement classical pathway and common pathway-related PC 2. CONCLUSIONS: The serum levels of properdin and factor D were correlated with the BVAS and the VDI in MPA and GPA, respectively. Our analyses suggested the pathological heterogeneity of MPA and GPA from the aspect of complement components.

Angiopoietin-like protein 2 promotes chondrogenic differentiation during bone growth as a cartilage matrix factor.

OBJECTIVE: Chondrocyte differentiation is crucial for long bone growth. Many cartilage extracellular matrix (ECM) proteins reportedly contribute to chondrocyte differentiation, indicating that mechanisms underlying chondrocyte differentiation are likely more complex than previously appreciated. Angiopoietin-like protein 2 (ANGPTL2) is a secreted factor normally abundantly produced in mesenchymal lineage cells such as adipocytes and fibroblasts, but its loss contributes to the pathogenesis of lifestyle- or aging-related diseases. However, the function of ANGPTL2 in chondrocytes, which are also differentiated from mesenchymal stem cells, remains unclear. Here, we investigate whether ANGPTL2 is expressed in or functions in chondrocytes. METHODS: First, we evaluated Angptl2 expression during chondrocyte differentiation using chondrogenic ATDC5 cells and wild-type epiphyseal cartilage of newborn mice. We next assessed ANGPTL2 function in chondrogenic differentiation and associated signaling using Angptl2 knockdown ATDC5 cells and Angptl2 knockout mice. RESULTS: ANGPTL2 is expressed in chondrocytes, particularly those located in resting and proliferative zones, and accumulates in ECM surrounding chondrocytes. Interestingly, long bone growth was retarded in Angptl2 knockout mice from neonatal to adult stages via attenuation of chondrocyte differentiation. Both in vivo and in vitro experiments show that changes in ANGPTL2 expression can also alter p38 mitogen-activated protein kinase (MAPK) activity mediated by integrin α5ß1. CONCLUSION: ANGPTL2 contributes to chondrocyte differentiation and subsequent endochondral ossification through α5ß1 integrin and p38 MAPK signaling during bone growth. Our findings provide insight into molecular mechanisms governing communication between chondrocytes and surrounding ECM components in bone growth activities.

A genome-wide search for human non-insulin-dependent (type 2) diabetes genes reveals a major susceptibility locus on chromosome 2.

Non-insulin-dependent (type 2) diabetes mellitus (NIDDM) is a common disorder of middle-aged individuals characterized by high blood glucose levels which, if untreated, can cause serious medical complications and lead to early death. Genetic factors play an important role in determining susceptibility to this disorder. However, the number of genes involved, their chromosomal location and the magnitude of their effect on NIDDM susceptibility are unknown. We have screened the human genome for susceptibility genes for NIDDM using non-and quasi-parametric linkage analysis methods in a group of Mexican American affected sib pairs. One marker, D2S125, showed significant evidence of linkage to NIDDM and appears to be a major factor affecting the development of diabetes mellitus in Mexican Americans. We propose that this locus be designated NIDDM1.

Urinary podocalyxin is an early marker for podocyte injury in patients with diabetes: establishment of a highly sensitive ELISA to detect urinary podocalyxin.

AIMS/OBJECTIVE: Nephropathy, a major complication of diabetes, is the leading cause of end-stage renal disease. Recent studies have demonstrated that podocyte injury is involved in the onset of and progression to renal insufficiency. Here, we describe a novel, highly sensitive ELISA for detecting urinary podocalyxin, a glycoconjugate on the podocyte apical surface that indicates podocyte injury, particularly in the early phase of diabetic nephropathy. METHODS: Urine samples from patients with glomerular diseases (n = 142) and type 2 diabetes (n = 71) were used to quantify urinary podocalyxin by ELISA. Urine samples were obtained from 69 healthy controls for whom laboratory data were within normal values. Podocalyxin was detected in urine by immunofluorescence, immunoelectron microscopy and western blotting. RESULTS: Morphologically, urinary podocalyxin was present as a vesicular structure; western blotting showed it as a positive band at 165-170 kDa. Levels of urinary podocalyxin were elevated in patients with various glomerular diseases and patients with diabetes. In patients with diabetes, urinary podocalyxin was higher than the cut-off value in 53.8% patients at the normoalbuminuric stage, 64.7% at the microalbuminuric stage and 66.7% at the macroalbuminuric stage. Positive correlations were observed between urinary podocalyxin levels and HbA(1c), urinary ß(2) microglobulin, α(1) microglobulin and urinary N-acetyl-ß-D-glucosaminidase, although urinary podocalyxin levels were not correlated with other laboratory markers such as blood pressure, lipid level, serum creatinine, estimated GFR or proteinuria. CONCLUSIONS/INTERPRETATION: Urinary podocalyxin may be a useful biomarker for detecting early podocyte injury in patients with diabetes.

Is the addition of extranodal extension and lymph node yield of pN0 to the lymph node ratio useful as a prognostic parameter for patients with oral squamous cell carcinoma?

We investigated the value of the weighted lymph node ratio (WLNR), a new marker in pN0 patients that incorporates the number of metastatic lymph nodes with extranodal extension and the lymph node yield, for the prognosis and postsurgical management of oral squamous cell carcinoma (OSCC). We designed a retrospective study and enrolled patients with OSCC who were treated by neck dissection (ND). The predictor variable was WLNR, and the outcome variable was overall survival (OS). The Cox proportional-hazards model was used to identify independent prognostic factors. In 133 patients with OSCC, the WLNR cut-off value for predicting OS was 0.0363 (area under the curve 0.723, p<0.001). When stratified according to WLNR, there was a significant difference in OS (88.4% for low WLNR and 63.0% for high WLNR, p<0.001). Univariate analyses showed close associations between OS and age, dissection area, postoperative management, extranodal extension, number of positive lymph nodes, pN stage, WLNR, and nodal disease area. Cox multivariate analysis identified the WLNR as an independent predictive factor for OS (HR 3.273, 95% CI 1.227 to 8.731, p=0.018). As a predictive factor, a high WLNR (≥0.0363) in patients with pN0 disease, which included the addition of extranodal extension and lymph node yield to the LNR, was associated with diminished survival.

Laser microdissection-based analysis of cytokine balance in the kidneys of patients with lupus nephritis.

To determine the cytokine balance in patients with lupus nephritis (LN), we analysed kidney-infiltrating T cells. Renal biopsy samples from 15 systemic lupus erythematosus (SLE) patients were used. In accordance with the classification of International Society of Nephrology/Renal Pathology Society, they were categorized into Class III, Class III+V (Class III-predominant group, n = 4), Class IV, Class IV+V (Class IV-predominant group, n = 7) and Class V (n = 4) groups. The single-cell samples of both the glomelular and interstitial infiltrating cells were captured by laser-microdissection. The glomerular and interstitial infiltrating T cells produced interleukin (IL)-2, IL-4, IL-10, IL-13 and IL-17 cytokines in the Class III-predominant, Class IV-predominant and Class V groups. Interferon-gamma was detected only in the glomeruli of the Class III-predominant and Class V group samples. The expression level of IL-17 was correlated closely with clinical parameters such as haematuria, blood urea nitrogen level, SLE Disease Activity Index scores in both glomeruli and interstitium, urine protein level in glomeruli and serum creatinine and creatinine clearance levels in interstitium. This suggests that the glomerular infiltrating T cells might act as T helper type 1 (Th1), Th2 and Th17 cells while the interstitial infiltrating T cells, act as Th2 and Th17 cells in the Class III-predominant and Class V groups. In contrast, both the glomerular and interstitial infiltrating T cells might act as Th2 and Th17 cells in the Class IV-predominant group. The cytokine balances may be dependent upon the classification of renal pathology, and IL-17 might play a critical role in SLE development.

Postoperative delirium after reconstructive surgery for oral tumor: a retrospective clinical study.

The aim of this study was to perform a statistical evaluation of the risk factors for postoperative delirium after oral tumor resection and reconstructive surgery. The records of 69 consecutive patients who underwent major head and neck tumor resection and reconstructive surgery, and who received postoperative management in the high care unit (HCU) or intensive care unit (ICU) of Tsukuba University Hospital between January 2013 and December 2017, were analysed retrospectively. Delirium was diagnosed in 23 patients (33.3%) after surgery. There were significant differences in age, sex, history of diabetes mellitus and chronic obstructive pulmonary disease, recent hospitalization history, sedation period, duration of ventilator use, length of ICU/HCU stay, postoperative blood tests (haemoglobin and potassium), and postoperative medication with a major tranquilizer between those with and without delirium. Logistic regression analysis of selected independent variables revealed a hazard ratio (95% confidence interval) of 1.42 (1.09-1.86) for the sedation period. Delirium was hyperactive type in 15 cases, hypoactive type in five, and mixed type in three. There was no obvious difference in postoperative day of onset or delirium period according to subtype. In conclusion, a history of diabetes and the sedation period were found to be related to postoperative delirium. However, this study was small and retrospective, so further investigation is necessary.

Relationship Between Stress Coping Mechanisms and Depression in Kidney Transplant Recipients.

BACKGROUND: It has been reported that transplant recipients are exposed to physical and psychosocial stresses even after transplant surgery and exhibit psychological disorders such as depression. PURPOSE: In this study, we extracted trends concerning how recipients of kidney transplants cope with stress, and we also examined how they cope with depression and its countermeasures. METHOD: We administered questionnaire surveys to 109 kidney transplant recipients. These included items on personal attributes, medical information, depression, and stress-coping type scales. Statistical analysis was performed using factor analysis and multiple regression analysis. RESULTS: Fifteen out of 109 (13.8%) were found to be high-risk patients for depression based on responses to the questionnaire using the depression scale. We extracted 2 factors of stress-coping type, namely Factor 1, "Directly coping with the problem," of patients who try to directly resolve the problem in a positive manner and Factor 2, "Stress-release while avoiding the problem," for those who relieve their feelings in response to the stress without resolving the problem itself. When multiple regression analysis was conducted with the depression scale as the dependent variable and the stress-coping factor as the independent variable, Factor 1 tended to be associated with reduced depression and Factor 2 with increased depression. CONCLUSIONS: Results showed that to improve the mental health of those who receive kidney transplants, it is necessary to examine the depression and stress-coping types of such patients at an early stage and carry out education on stress-coping, focusing on resolving the actual problem.

Expression of a mitogen-inducible cyclooxygenase in brain neurons: regulation by synaptic activity and glucocorticoids.

Prostaglandins play important and diverse roles in the CNS. The first step in prostaglandin synthesis involves enzymatic oxidation of arachidonic acid, which is catalyzed by prostaglandin H(PGH) synthase, also referred to as cyclooxygenase. We have cloned an inducible form of this enzyme from rat brain that is nearly identical to a murine, mitogen-inducible cyclooxygenase identified from fibroblasts. Our studies indicate that this gene, here termed COX-2, is expressed throughout the forebrain in discrete populations of neurons and is enriched in the cortex and hippocampus. Neuronal expression is rapidly and transiently induced by seizures or NMDA-dependent synaptic activity. No expression is detected in glia or vascular endothelial cells. Basal expression of COX-2 appears to be regulated by natural synaptic activity in the developing and adult brain. Both basal and induced expression of COX-2 are inhibited by glucocorticoids, consistent with COX-2 regulation in peripheral tissues. Our studies indicate that COX-2 expression may be important in regulating prostaglandin signaling in brain. The marked inducibility in neurons by synaptic stimuli suggests a role in activity-dependent plasticity.

Visinin: a novel calcium binding protein expressed in retinal cone cells.

Visinin is a retinal cone cell-specific protein (molecular weight 24,000, pI 5.1). To investigate its function, visinin cDNA was isolated from a chick retinal lambda gt11 cDNA library, using anti-visinin serum. The beta-galactosidase-visinin fusion protein was used for purifying epitope-selected antibody. The purified visinin antibody reacted only with a 24 kd protein in retinal cone cells. Visinin mRNA was expressed only in the retinal photoreceptor layer. The nucleotide sequence of the cDNA revealed that visinin has three E-F hand structures and is a Ca2+ binding protein. Visinin protein expressed in E. coli exhibited Ca2+ binding activity. These results suggest that visinin is a photoreceptor-specific Ca2+ binding protein and may be involved in phototransduction in the cone cells.

Arc, a growth factor and activity-regulated gene, encodes a novel cytoskeleton-associated protein that is enriched in neuronal dendrites.

Neuronal activity is an essential stimulus for induction of plasticity and normal development of the CNS. We have used differential cloning techniques to identify a novel immediate-early gene (IEG) cDNA that is rapidly induced in neurons by activity in models of adult and developmental plasticity. Both the mRNA and the encoded protein are enriched in neuronal dendrites. Analysis of the deduced amino acid sequence indicates a region of homology with alpha-spectrin, and the full-length protein, prepared by in vitro transcription/translation, coprecipitates with F-actin. Confocal microscopy of the native protein in hippocampal neurons demonstrates that the IEG-encoded protein is enriched in the subplasmalemmal cortex of the cell body and dendrites and thus colocalizes with the actin cytoskeletal matrix. Accordingly, we have termed the gene and encoded protein Arc (activity-regulated cytoskeleton-associated protein). Our observations suggest that Arc may play a role in activity-dependent plasticity of dendrites.

Dual inhibition of EZH1/2 breaks the quiescence of leukemia stem cells in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive and lethal blood cancer originating from rare populations of leukemia stem cells (LSCs). AML relapse after conventional chemotherapy is caused by a remaining population of drug-resistant LSCs. Selective targeting of the chemoresistant population is a promising strategy for preventing and treating AML relapse. Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27 to maintain the stemness of LSCs. Here, we show that quiescent LSCs expressed the highest levels of enhancer of zeste (EZH) 1 and EZH2, the PRC2 catalytic subunits, in the AML hierarchy, and that dual inactivation of EZH1/2 eradicated quiescent LSCs to cure AML. Genetic deletion of Ezh1/2 in a mouse AML model induced cell cycle progression of quiescent LSCs and differentiation to LSCs, eventually eradicating AML LSCs. Quiescent LSCs showed PRC2-mediated suppression of Cyclin D, and Cyclin D-overexpressing AML was more sensitive to chemotherapy. We have developed a novel EZH1/2 dual inhibitor with potent inhibitory activity against both EZH1/2. In AML mouse models and patient-derived xenograft models, the inhibitor reduced the number of LSCs, impaired leukemia progression, and prolonged survival. Taken together, these results show that dual inhibition of EZH1/2 is an effective strategy for eliminating AML LSCs.

Increased angiotensin-converting enzyme in peripheral blood monocytes from patients with sarcoidosis.

Angiotensin-converting enzyme (ACE) activity was measured in isolated peripheral blood monocytes and culture medium from 28 patients with sarcoidosis and compared with values obtained from monocytes of 25 normal control subjects. ACE activity was determined by radioimmunoassay of angiotensin II produced from angiotensin I. While there was no measurable ACE activity in monocytes or culture medium from normal controls under the conditions of our study, monocytes from patients with sarcoidosis all showed activity both in cells and culture medium. The mean ACE activity of monocytes from patients with sarcoidosis was 2.0 pg angiotensin II formed/min per 10(5) cells, and that released into medium over a 24-h interval was 30.4 pg angiotensin II/min per 10(5) cells. The monocyte ACE from patients with sarcoidosis was activated by chloride ions and inhibited by EDTA, captopril, and rabbit antiserum to purified human plasma ACE, indicating that enzymatic activity was effected specifically by ACE. Thus, our studies show a significant elevation and release of ACE by peripheral blood monocytes of patients with sarcoidosis under conditions where monocytes of normal control subjects do not demonstrate ACE activity.

Mononuclear cell infiltration and its relation to the expression of major histocompatibility complex antigens and adhesion molecules in pancreas biopsy specimens from newly diagnosed insulin-dependent diabetes mellitus patients.

We examined pancreas biopsy specimens from 18 newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients to elucidate the mechanism underlying beta cell destruction. Pancreas islets were seen in all patients and insulitis in eight patients. Infiltrating mononuclear cells consisted of CD4+T, CD8+T, B lymphocytes, and macrophages. Among them, CD8+T lymphocytes were predominant and macrophages followed. The expression of MHC class I antigens was increased in islet and endothelial cells in nine patients. MHC class II expression was increased in endothelial cells of the same patients. The expression of intercellular adhesion molecule-1 was increased in endothelial cells in two of the nine patients with MHC hyperexpression; in one of them, lymphocyte function-associated antigen-3 expression was also increased. Out of the eight patients with insulitis, seven showed MHC class I hyper-expression, whereas 2 of the 10 patients without insulitis showed the phenomenon (P < 0.05). The relation between insulitis and the hyperexpression of adhesion molecules was not evident. In conclusion, we revealed the close relation between CD8+T lymphocyte-predominant insulitis and MHC class I hyperexpression in islet cells. This suggests that infiltrating CD8+T lymphocytes recognize islet autoantigens in association with increased MHC class I molecules and act as major effector cells in autoimmune response against islet cells in IDDM pancreases. The role of adhesion molecules in the pathogenesis of IDDM still remains to be elucidated.

Loss of cytokeratin 13 expression in squamous cell carcinoma of the tongue is a possible sign for local recurrence.

Cytokeratin (CK) 13 is an intermediate filament protein that is expressed in a cell-type-specific manner, in the tongue and occasionally in tongue squamous cell carcinoma (SCC). Correlations between the clinical features of patients with SCC and CK13 expression in the tumor are here investigated along with CK13's utility as a marker for tongue cancer status. Samples from 121 patients with SCC of the tongue were examined by immunohistochemistry with antibodies against CK13. Correlations between the expression level of CK13 in the tumor and the patients' clinical features were statistically analyzed by univariate and multivariate methods. Univariate analysis showed a more relevant number of local recurrence (P = 0.04) in CK13-negative staining patients. In addition, CK13-negative cases were associated with local recurrence by multiple logistic regression analysis (OR: 3.36; 95% CI: 1.044-10.78; P = 0.04). Our results suggest that the loss of CK13 expression indicates tumors with a high potential for recurrence, and thus CK13 could be useful for determining the best course of treatment.

Zonal necrosis prevented by transduction of the artificial anti-death FNK protein.

Protection of cells from necrosis would be important for many medical applications. Here, we show protein transduction domain (PTD)-FNK therapeutics based on protein transduction to prevent necrosis and acute hepatic injury with zonal death induced by carbon tetrachloride (CCl4). PTD-FNK is a fusion protein comprising the HIV/Tat PTD and FNK, a gain-of-function mutant of anti-apoptotic Bcl-x(L). PTD-FNK protected hepatoma HepG2 from necrotic death induced by CCl4, and additionally, increased the apoptotic population among cells treated with CCl4. A concomitant treatment with a pan-caspase inhibitor Z-VAD-FMK (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone), which alone could not prevent the necrosis, protected these cells from the apoptosis. When pre-injected intraperitoneally, PTD-FNK markedly reduced zonal liver necrosis caused by CCl4. Moreover, injection of PTD-FNK accompanied by Z-VAD-FMK suppressed necrotic injury even after CCl4 administration. These results suggest that PTD-FNK has great potential for clinical applications to prevent cell death, whether from apoptosis or necrosis, and organ failure.

Glutamate reduces secretion of l-serine in astrocytes isolated from stroke-prone spontaneously hypertensive rats.

In the CNS, l-serine (l-Ser) plays an essential role in neuronal survival by evoking a variety of biological responses in glial cells. Initially, we examined whether glutamate, hydrogen peroxide (H(2)O(2)), interleukin-1 (IL-1) beta, and sodium nitroprusside (SNP) induce the secretion of l-Ser in astrocytes isolated from Wistar Kyoto rats (WKY). The secretion of l-Ser was significantly induced with glutamate and SNP in cultured astrocytes. Next, to gain insight into the involvement of l-Ser in glutamate-induced neuroprotection, we compared the secretion of l-Ser in astrocytes isolated from stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive rats, WKY. We also examined the mRNA expression of the enzyme that produces l-Ser, 3-phosphoglycerate dehydrogenase (PHGDH), and a neural amino acid transporter, ASCT1, in the cultured astrocytes. A dose-dependent study of glutamate in astrocytes of SHRSP indicated differences in the secretion of l-Ser, and gene expression of PHGDH and ASCT1, compared with levels in the WKY astrocytes. We demonstrated that both the secretion and the gene expression were significantly attenuated in glutamate-treated astrocytes from SHRSP. Cerebral ischemia in SHRSP induced a massive efflux of glutamate, causing delayed neuronal death in region CA1 of the hippocampus. The results suggest that the attenuated secretion of l-Ser in astrocytes is involved in neuronal vulnerability and survival in SHRSP during the production of glutamate, as the secretion of l-Ser, which is stimulated by glutamate, is closely related to the protective effect against glutamate-mediated neurotoxicity. We conclude that glutamate and SNP up-regulate the secretion of l-Ser in primary astrocytes. Secretion of l-Ser is regulated in astrocytes in response to glutamate and nitric oxide and may correspond to the level of l-Ser needed for neuronal survival during brain insults such as ischemic stroke in SHRSP.

A prospective study to evaluate a new dental management protocol before hematopoietic stem cell transplantation.

Pre-hematopoietic stem cell transplantation (HSCT) dental treatment is essential to prevent serious infections from oral sources during immunosuppression, in patients who undergo HSCT therapy. This study was planned to establish a dental management protocol for such patients. Forty-one patients scheduled for HSCT to treat hematological malignancies were consecutively enrolled in the prospective trial. The dental status of all patients was evaluated by clinical and radiographic examination at a median of 47 days before the commencement of HSCT therapy. Thirty-six patients had one or more dental diseases; the remaining five had none. Caries was found in 26 patients, apical periodontitis in 19, marginal periodontitis in 24 and a partially erupted third molar in 11. Our policy is to preserve patients' teeth whenever possible, and therefore minimal dental intervention was planned. Treatment was completed for all 36 patients with dental pathologies, before the conditioning regimen began. All patients received the scheduled HSCT therapy without alteration, interruption or delay, and did not show any signs or symptoms associated with odontogenic infection while they were immunosuppressed. This protocol, therefore, appears to be appropriate for the pre-HSCT dental treatment of patients with hematological diseases.

Submandibular abscess caused by Salmonella.

Salmonella species represent uncommon causative agents in focal infections of the head and neck. This article presents a rare case of Salmonella submandibular abscess in a 59-year-old patient with diabetes mellitus, chronic hepatitis and liver cirrhosis. The patient was successfully treated with drainage of the abscess and oral administration of quinolone. An immunocompromised condition can be a critical predisposing factor for Salmonella submandibular abscess.