Similarity of autoimmune diseases based on the profile of immune complex antigens.

Since immune complexes (IC) are a direct product of immune response through the binding between antigen and antibody, the profile of antigen-associated ICs may depend on each autoimmune disease. In this report, we examined the similarity of four neurological autoimmune diseases, Alzheimer's disease and healthy donors, and seven connective tissue diseases based on the profiling of IC-associated antigens which were previously or recently identified by immune complexome analysis of cerebrospinal fluid (CSF) or serum samples. The similarity between each pair of two diseases was assessed by correlation coefficients as distance matrix with the use of detection frequency (i.e., the percentage of patients who were positive for a certain antigen in each disease) of each IC-associated antigen in a certain disease. Among 15 pairs of five diseases and healthy control examined by the analysis of CSF samples, only 1 pair of neuropsychiatric systemic lupus erythematosus and multiple sclerosis corresponds to the higher correlation value (r = 0.73) than 0.7. On the other hand, among seven connective tissue diseases examined by the analysis of serum samples, 12 of 21 pairs show high correlation value (r > 0.70). Our finding suggested that the profile of IC-associated antigens identified by immune complexome analysis of CSF samples can be useful for evaluating the similarity of neurological autoimmune diseases; however, not by that of serum samples.

Proteomic approach to profiling immune complex antigens in cerebrospinal fluid samples from patients with central nervous system autoimmune diseases.

BACKGROUND: Immune complexes (ICs) may clearly reflect immunological abnormalities caused by disease, especially for autoimmune diseases. Although ICs have been detected in cerebrospinal fluid (CSF) from patients with CNS autoimmune diseases, identities of antigens in such ICs have not been comprehensively determined. METHODS: We used immune complexome analysis, in which nano-liquid chromatography-tandem mass spectrometry is employed to comprehensively identify antigens incorporated into ICs in biological fluids, to characterize ICs in CSF samples from patients with CNS autoimmune diseases, and to find disease-specific IC antigen to a certain CNS autoimmune disease. Also, we compared the IC antigens we identified with the reported CSF proteome or with the published plasma proteome to examine if the method is distinguished from the conventional CSF proteome analysis. RESULTS: We identified 176 antigens in 78 CSF samples. We then assessed the overlaps among these antigens, the CSF proteome, and the plasma proteome; 140 of the 176 antigens were found to be exclusively detected by our method. Notably, IC-associated suprabasin in CSF was 100% specific to neuropsychiatric systemic lupus erythematosus (NPSLE). CONCLUSIONS: This report is the first to comprehensively identify the antigens incorporated into ICs in CSF. There was limited overlap between the antigens we identified and the CSF proteome or the plasma proteome; therefore, our method can be distinguished from the conventional CSF proteome analysis. Although the sensitivity of disease-specific IC-antigens detected in immune complexome analysis screening, the sensitivity may be improved by developing an ELISA method specifically for detecting the ICs. Immune complexome analysis of CSF may be a new and promising path to biomarker discovery for diagnosis and study for CNS autoimmune diseases.

Immune complexome analysis of antigens in circulating immune complexes isolated from patients with IgG4-related dacryoadenitis and/or sialadenitis.

OBJECTIVES: IgG4-related disease (IgG4-RD) is characterized by various serological abnormalities. Some patients with IgG4-RD present with hypergammaglobulinemia, hypocomplementemia, and autoantibodies recognizing rheumatoid factors and nuclear factors. However, whether IgG4-RD is an autoimmune disease remains unclear. METHODS: Here, we used immune complexome analysis to comprehensively identify constituent antigens of circulating immune complexes isolated from 10 patients with IgG4-related dacryoadenitis and/or sialadenitis (IgG4-RDS) which is one condition associated with IgG4-RD. RESULTS: We detected each of 125 distinct antigens in independent samples from two or more patients with IgG4-RDS. Of them, 17 antigens were found to be specific to patients with IgG4-RDS by comparing 125 antigens with all the antigens found in other connective tissue diseases (antineutrophil cytoplasmic antibody-associated vasculitis, Takayasu's arteritis, mixed connective tissue disease, dermatomyositis, Sjögren's syndrome, systemic scleroderma, and systemic lupus erythematosus) or healthy donors. The number of disease-specific antigens associated with IgG4-RDS was comparable to those autoimmune diseases. CONCLUSIONS: Studies of the 17 IgG4-RDS-specific antigens might lead to a better understanding of the pathogenesis of IgG4-RD, but further study of the prevalence of these CIC-associated antigens that involves a selective and sensitive assay will be needed to determine their potential as diagnostic or pathogenic biomarkers.

Proteomic profiling of antigens in circulating immune complexes associated with each of seven autoimmune diseases.

OBJECTIVE: Immune complexes (ICs) trigger humoral immune responses. Therefore, the identification of constituent antigens within ICs would have very different clinical significance than identification of free antigens. DESIGN AND METHODS: Here, we applied immune complexome analysis of serum to the study of seven major autoimmune diseases-anti-neutrophil cytoplasmic antibody-associated vasculitis, Takayasu's arteritis, mixed connective tissue disease, dermatomyositis, Sjögren's syndrome, systemic scleroderma, and systemic lupus erythematosus-and healthy donors to comprehensively identify antigens incorporated into circulating ICs and to find disease-specific antigens. RESULTS: We identified 468 distinct IC-associated antigens using this method. Importantly, 62 of those antigens were disease-specific antigens, and there were at least three disease-specific antigens for each of the seven autoimmune diseases. Of the disease-specific antigens identified, coiled-coil domain-containing protein 158 and spectrin were identified as potential autoantigens important to SSc and SS pathogenesis, respectively; notable titin and spectrin autoantibodies are reportedly found in SSc and SS patients, respectively. CONCLUSION: Immune complexome analysis may be generally applicable to the study of the relationship between ICs and autoimmune diseases in animals and humans.

Optimization of separation and digestion conditions in immune complexome analysis.

Immune complexome analysis is a method for identifying and profiling of antigens in circulating immune complexes (CICs); it involves separation of immune complexes from serum, direct tryptic digestion of these complexes, and protein analysis via nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS). To improve this method, we initially investigated the effects of two factors-the gradient elution program and nano-LC column type (C18-packed, C8-packed, or packed spray capillary column)-on the numbers of peptides and proteins identified. Longer gradient elution times resulted in higher identification capability throughout the range of 25-400 min. Moreover, the packed spray capillary column supported identification of more peptides and proteins than did any other column. In addition, microwave-assisted digestion was compared with conventional digestion, which involved incubation overnight at 37 °C. Microwave-assisted digestion produced more partially digested peptides than did conventional digestion. However, the percentages of miscleaved peptides in all of the identified peptides in microwave-assisted digestion of immune complexes (a protein mixture) were lower than those in the physical stimulation-assisted digestion of a model protein. Microwave-assisted digestion is slightly inferior to, or as effective as, conventional digestion, but it drastically reduces the digestion time.

Rocuronium and sugammadex used effectively for electroconvulsive therapy in a patient with Brugada syndrome.

We report the successful anesthetic management of a patient with Brugada syndrome who underwent electroconvulsive therapy to treat bipolar disorder. Suxamethonium and neostigmine were contraindicated to avoid the vagotonic effects that can precipitate ventricular fibrillation during anesthesia in patients with Brugada syndrome. The combination of 1.2 mg/kg rocuronium and 10 mg/kg sugammadex was effectively and safely used to induce and antagonize neuromuscular block for 8 consecutive electroconvulsive therapy sessions in this patient.

Protein kinase C stabilizes X-linked inhibitor of apoptosis protein (XIAP) through phosphorylation at Ser(87) to suppress apoptotic cell death.

BACKGROUND: Multiple protein kinases have been shown to be involved in the apoptotic neuronal loss of Alzheimer's disease (AD). Although some studies support the role of protein kinase C (PKC) in amyloid precursor protein processing as well as in tau phosphorylation, a direct role for PKC in apoptotic neuronal death remains to be clarified. In the present study, we report on the possible role of PKC in cell survival during conditions of stress through phosphorylation of the X-linked inhibitor of apoptosis protein (XIAP). METHODS: Phosphorylation of XIAP at Ser87 was confirmed by western blot analysis employing phosphorylation dependent anti-XIAP antibody after incubation of recombinant XIAP with active PKC in vitro. And increased phosphorylation of XIAP at the site was also confirmed in SH-SY5Y cells treated with PKC activator, phorbol 12-myristate 13-acetate (PMA). A mutant XIAP construct in which Ser87 was substituted by Ala, was prepared, and transfected to cells. After the transfection of wild or mutant XIAP, cells viability was evaluated by counting living and dead cells treated with PMA during etoposide-induced apoptosis. RESULTS: Recombinant XIAP was phosphorylated at Ser(87) by PKC in vitro and treatment of XIAP-transfected SH-SY5Y cells with a PKC activator, phorbol 12-myristate 13-acetate (PMA) induced phosphorylation of XIAP at Ser(87) . Pulse chase experiments revealed that, when phosphorylated at Ser(87) , wild-type XIAP is more stable than XIAP with a Ser87Ala substitution, which is degraded faster. Importantly, the phosphorylation of XIAP at the site by PKC significantly increased cell survival up to approximately 2.5 times under the condition of apoptosis induced by 25 µg/ml etoposide. CONCLUSION: The findings of the present study indicate a role for PKC, through phosphorylation of XIAP at Ser(87) and its stabilization, in cell survival under conditions of stress and lend strength to the idea that PKC is crucial in regulating neuronal homeostasis, which may be impaired in AD.

Clinical course of pain in a patient with neuropathic pain induced by ligation of an intercostal nerve.

A patient with severe right chest pain and mechanical allodynia induced by an intercostal drainage tube to his chest is presented. It was not relieved by treatment with diclofenac sodium and was worsened by movement and touch to the right chest wall. Mechanical allodynia was also present. The patient's wrenching pain disappeared immediately after stitch removal, but the dull pain and mechanical allodynia persisted, gradually decreasing to zero in 7 days.

Adiponectin deficiency enhances colorectal carcinogenesis and liver tumor formation induced by azoxymethane in mice.

AIM: To investigate the causal relationship between hypoadiponectinemia and colorectal carcinogenesis in in vivo experimental model, and to determine the contribution of adiponectin deficiency to colorectal cancer development and proliferation. METHODS: We examined the influence of adiponectin deficiency on colorectal carcinogenesis induced by the administration of azoxymethane (AOM) (7.5 mg/kg, intraperitoneal injection once a week for 8 wk), by using adiponectin-knockout (KO) mice. RESULTS: At 53 wk after the first AOM treatment, KO mice developed larger and histologically more progressive colorectal tumors with greater frequency compared with wild-type (WT) mice, although the tumor incidence was not different between WT and KO mice. KO mice showed increased cell proliferation of colorectal tumor cells, which correlated with the expression levels of cyclooxygenase-2 (COX-2) in the colorectal tumors. In addition, KO mice showed higher incidence and frequency of liver tumors after AOM treatment. Thirteen percent of WT mice developed liver tumors, and these WT mice had only a single tumor. In contrast, 50% of KO mice developed liver tumors, and 58% of these KO mice had multiple tumors. CONCLUSION: Adiponectin deficiency enhances colorectal carcinogenesis and liver tumor formation induced by AOM in mice. This study strongly suggests that hypoadiponectinemia could be involved in the pathogenesis for colorectal cancer and liver tumor in human subjects.

Blocking CD147 induces cell death in cancer cells through impairment of glycolytic energy metabolism.

CD147 is a multifunctional transmembrane protein and promotes cancer progression. We found that the anti-human CD147 mouse monoclonal antibody MEM-M6/1 strongly induces necrosis-like cell death in LoVo, HT-29, WiDr, and SW620 colon cancer cells and A2058 melanoma cells, but not in WI-38 and TIG-113 normal fibroblasts. Silencing or overexpression of CD147 in LoVo cells enhanced or decreased the MEM-M6/1 induced cell death, respectively. CD147 is known to form complex with proton-linked monocarboxylate transporters (MCTs), which is critical for lactate transport and intracellular pH (pHi) homeostasis. In LoVo cells, CD147 and MCT-1 co-localized on the cell surface, and MEM-M6/1 inhibited the association of these molecules. MEM-M6/1 inhibited lactate uptake, lactate release, and reduced pHi. Further, the induction of acidification was parallel to the decrease of the glycolytic flux and intracellular ATP levels. These effects were not found in the normal fibroblasts. As cancer cells depend on glycolysis for their energy production, CD147 inhibition might induce cell death specific to cancer cells.

Induction by lysophosphatidic acid of peritoneal and pleural metastases of intestinal cancers induced by azoxymethane in Wistar rats.

The effects of 1-oleoyl lysophosphatidic acid on the induction of metastasis from intestinal adenocarcinomas induced in rats by azoxymethane and on RhoA activity in the tumors were investigated in male Wistar rats. Rats were given a weekly s.c. injection of azoxymethane (7.4 mg/kg body weight) for 10 weeks and, from week 16, s.c. injection of lysophosphatidic acid (5 or 15 microg/kg body weight) every other day until the end of the experiment in week 45. Lysophosphatidic acid at both dosages significantly increased the incidence of peritoneal metastasis. Its administration at higher dosage also significantly enhanced the development of pleural metastasis. Although lysophosphatidic acid at both dosages had little or no effect on the location, histologic type, depth of involvement or infiltrating growth patterns of the tumors, its administration at both dosages significantly increased the incidence of vessel invasion of adenocarcinomas. Lysophosphatidic acid also increased the activity of RhoA in the tumors, but not the cellular proliferation and vascularity of the colon tumors. Our findings indicate that lysophosphatidic acid significantly increased the incidence of peritoneal and/or pleural metastases from intestinal adenocarcinomas induced in rats by azoxymethane through RhoA activation.

Geranylgeranylacetone attenuates suppression by Helicobacter pylori extract of human umbilical vein epithelial cell growth.

BACKGROUND/AIMS: Helicobacter pylori infection delays gastric ulcer healing. Angiogenesis is important for the healing of gastric ulcers. Therefore, the effects of H. pylori water extract and a novel antiulcer drug, geranylgeranylacetone, on the viability of human umbilical vein epithelial cells (HUVECs) were investigated. METHODOLOGY: H. pylori (ATCC43504) was prepared by sonication. The HUVEC viability after treatment with H. pylori water extract alone or in combination with geranylgeranylacetone was estimated by an MTT assay. RESULTS: H. pylori water extract significantly decreased cell viability in a concentration-dependent manner after 48 h. However, combined use of H. pylori water extract and geranylgeranylacetone significantly increased the HUVEC viability over that of H. pylori extract alone. CONCLUSIONS: A novel antiulcer drug, geranylgeranylacetone, attenuates the H. pylori-induced inhibition of angiogenesis.

Glycine-extended gastrin induces matrix metalloproteinase-1- and -3-mediated invasion of human colon cancer cells through type I collagen gel and Matrigel.

The effects of glycine-extended gastrin (G-Gly) on the invasion by colon cancer cells through stromal extracellular matrix and the role of metalloproteinases (MMPs) in this invasion were investigated. We found that 10(-9)-10(-6) M G-Gly significantly increased the invasiveness of 2 human colon cancer cell lines, LoVo and HT-29, both expressing the G-Gly-specific binding site but little gastrin/CCK-B receptor (gastrin receptor). LoVo cells expressed MMP-1, -2, -3 and -9. An amount of 10(-7) M G-Gly enhanced collagenase MMP-1 expression. Overexpression of enhanced green fluorescent protein (EGFP)-fused MMP-1 in LoVo cells, by cDNA transfection, enhanced invasiveness through type I collagen gel. Immunofluorescence study revealed that G-Gly increased the number of cytoplasmic vesicles containing MMP-1, some vesicles being released from the cells. The MMP-1 vesicles contained one of the ubiquitous coat proteins, Golgi-localized, gamma-adaptin ear-containing, ARF-binding proteins-2 (GGA-2). MMP-1 also colocalized with CD147 (EMMPRIN, an extracellular matrix metalloproteinase inducer in adjacent stromal cells). It was suggested that G-Gly increased the number of vesicles containing MMP-1 and that MMP-1 interacted with CD147 to increase invasion. G-Gly significantly enhanced the production of MMP-3, an activator of MMP-1 and -9, as well as gelatinase MMP-9 activity. The G-Gly-mediated MMP-9 increase was inhibited by treatment with anti-MMP-3 IgG and MMP-3 siRNA. Furthermore, G-Gly increased the proMMP-2 level, although no activated MMP-2 was found in conditioned medium in either the presence or the absence of G-Gly. By contrast, gastrin (10(-7) M) had no effect on the levels of these MMPs or the invasiveness of colon cancer cells in type I collagen gel and Matrigel. These effects of G-Gly on the activity and expression of MMPs and the invasiveness of colon cancer cells were inhibited by treating the cells with a broad-spectrum metalloproteinase inhibitor (CGS27023A) and nonselective gastrin/CCK receptor antagonists (proglumide and benzotript). But a gastrin/CCK-B receptor antagonist (YM022) did not inhibit the increased invasion by G-Gly. Together, these results demonstrate that G-Gly renders colon cancer cells more invasive by increasing MMP-1 and MMP-3 expressions via the putative G-Gly receptor and would thus be a good molecular target in a clinical setting.

Attenuation by cyclic phosphatidic acid of peritoneal metastasis of azoxymethane-induced intestinal cancers in Wistar rats.

The effect of cyclic phosphatidic acid, a unique analogue of lysophosphatidic acid, on the induction of bombesin-enhanced peritoneal metastases from intestinal adenocarcinomas induced by azoxymethane was investigated in male Wistar rats. Rats were given 10 weekly injections of azoxymethane (7.4 mg/kg body weight, s.c.) and of bombesin (40 microg/kg body weight, s.c.) every other day from the start of the experiment, and from week 16, they received injections of cyclic phosphatidic acid (3 or 6 mg/kg body weight, s.c.) every other day until the end of the experiment in week 45. Cyclic phosphatidic acid at both dosages significantly decreased the incidence of bombesin-enhanced cancer metastases to the peritoneum but had little or no effect on the location, histologic type, depth of involvement or infiltrating growth patterns of the tumors. Cyclic phosphatidic acid at either dose decreased significantly the incidence of lymphatic vessel invasion of adenocarcinomas and the activity of RhoA protein in the tumors, both of which were enhanced by bombesin. Our findings indicate that cyclic phosphatidic acid inhibits cancer metastasis through inhibition of RhoA protein activation.

Suppression by nimesulide of bombesin-enhanced peritoneal metastasis of intestinal adenocarcinomas induced by azoxymethane in Wistar rats.

The effects of the cyclooxygenase (COX)-2 inhibitor nimesulide on bombesin-enhanced peritoneal metastasis of azoxymethane (AOM)-induced intestinal adenocarcinomas were investigated in male Wistar rats. From the beginning of the study, the rats were given 10 weekly s.c. injections of AOM (7.4 mg/kg body weight) and s.c. injections of bombesin (40 microg/kg body weight) every other day. From week 16, the rats were given chow pellets containing 200 ppm or 400 ppm nimesulide ad libitum until termination of the study at week 45. Nimesulide at the higher dose significantly decreased the incidence of bombesin-enhanced metastasis to the peritoneum at week 45, although its administration had little or no effect on the location, histologic type, depth of involvement or infiltrating growth patterns of the tumors. Nimesulide also significantly decreased the incidence of bombesin-enhanced lymphatic vessel invasion by adenocarcinomas. Finally, it also inhibited bombesin-induced matrix metalloproteinase (MMP)-9 and pro-MMP-9 inductions. Our findings indicate that nimesulide may inhibit cancer metastasis through inhibition of pro-MMP-9 and MMP-9 inductions.

Enhancement by interleukin-1 beta of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats: a possible mechanism for Helicobacter pylori-associated gastric carcinogenesis.

The effect of prolonged administration of the cytokine interleukin (IL)-1beta on gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was examined in Wistar rats. In addition, we examined the effects on the proliferating cell nuclear antigen (PCNA) labeling index and the hepatocyte growth factor (HGF) immunoreactivity of the gastric mucosa. The rats received intraperitoneal injections of 0.1 or 0.3 microg/kg body weight of IL-1beta every other day after oral treatment with MNNG for 25 weeks. Long-term administration of IL-1beta at high dose, but not at low dose, significantly increased the incidence of gastric cancer in week 52. Administration of IL-1beta at high dose also significantly increased the labeling index and the HGF immunoreactivity of the gastric antral mucosa, and induced inflammatory cell infiltration and glandular atrophy of the gastric mucosa. Because IL-1beta production in the gastric mucosa is increased in patients with Helicobacter pylori-associated gastritis and eradiation of the organism significantly decreases the IL-1beta production, these findings suggest that Helicobacter pylori-associated gastric carcinogenesis may be in part mediated through IL-1beta.

Suppression by iron chelator phenanthroline of sodium chloride-enhanced gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats.

The effect of prolonged administration of iron chelator phenanthroline on sodium chloride-enhanced gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine, and the labeling and apoptotic indices in the gastric cancers was investigated in Wistar rats. After 25 weeks of carcinogen treatment, the rats were given chow pellets containing 10% sodium chloride and intraperitoneal injections of phenanthroline at doses of 15 or 30 mg/kg body weight every other day. At week 52, feeding of sodium chloride significantly increased the incidence of gastric cancers, as compared with the control group. Prolonged injections of phenanthroline at both doses significantly reduced the incidence of gastric cancers, which was enhanced by oral supplementation with sodium chloride. Phenanthroline at both doses significantly decreased the labeling index of gastric cancers, which was enhanced by sodium chloride, and significantly increased the apoptotic index of gastric cancers, which was lowered by sodium chloride. In vitro examination using electron spin resonance revealed that sodium chloride promotes the production of hydroxyl radical during Fe(2+) oxidation by Fenton's reaction. These findings suggest that enhancement by sodium chloride of gastric carcinogenesis may be mediated by hydroxyl radicals.

Acute promyelocytic leukemia developing in untreated essential thrombocythemia.

We describe a patient with untreated essential thrombocythemia (ET) who developed microgranular variant of acute promyelocytic leukemia, 9 years after the initial diagnosis of ET. He achieved complete remission (CR) but relapsed 11 months later. After achieving the second CR, he received peripheral stem cell transplantation from his HLA complete-matched sibling. Five months after the transplantation, he relapsed again with meningeal infiltration of leukemic cells. In this paper, we review cases of promyelocytic transformation of myeloproliferative diseases (MPD) other than chronic myeloid leukemia (CML). To our knowledge, this is the first case of promyelocytic transformation of Philadelphia chromosome negative untreated ET, in whom both t(15;17) and PML-RAR alpha fusion were proven.