Targeted IgA Fc receptor I (FcαRI) therapy in the early intervention and treatment of pristane-induced lupus nephritis in mice.

The Fc receptor I for IgA (FcαRI) down-regulates humoral immune responses and modulates the risk of autoimmunity. This study aimed to investigate whether FcαRI targeting can affect progression of pristine-induced lupus nephritis. In the first experiment (early intervention), four groups of animals were evaluated: untreated FcαRI/FcRγ transgenic (Tg) mice and Tg mice administered control antibody (Ctr Fab), saline and anti-FcαRI Fab [macrophage inflammatory protein (MIP)-8a], respectively, three times a week for 29 weeks, after being injected once intraperitoneally with 0·5 ml pristane. In the second experiment, antibody injection started after the onset of nephritis and was carried out for 2 months, with similar groups as described above. MIP-8a improved proteinuria, decreased the amounts of glomerular injury markers, serum interleukin (IL)-6, IL-1 and monocyte chemoattractant protein (MCP)-1, and F4/80 macrophages in the interstitium and glomeruli, in both experiments. When MIP-8a was used as early intervention, a decrease in mouse serum anti-nuclear antibody (ANA) titres and reduced deposition of immunoglobulins in glomeruli were observed. This effect was associated with reduced serum titres of immunoglobulin (Ig)G2a but not IgG1, IgG2b and IgG3. Furthermore, pathological analysis showed lower glomerular activity index and less fibronectin in MIP-8a treated mice. This study suggests that FcαRI targeting could halt disease progression and lupus activation by selective inhibition of cytokine production, leucocyte recruitment and renal inflammation. Our findings provide a basis for the use of FcαRI as a molecular target for the treatment of lupus.

Surgical stress response after colorectal resection: a comparison of robotic, laparoscopic, and open surgery.

BACKGROUND: The perioperative immune status of colorectal robotic surgery (RS), laparoscopic surgery (LS), and open surgery (OS) patients has not been compared. Our aim was to evaluate perioperative stress and immune response after RS, LS and OS. METHODS: This prospective study included 46 colorectal surgery patients from the Department of Surgical Oncology of the University of Tokyo Hospital. Peripheral venous blood samples were obtained preoperatively and on postoperative days 1, 3, and 6. We evaluated expression of HLA-DR (marker of immune competence), C-reactive protein (CRP) levels, and lymphocyte subset counts (natural killers, cytotoxic T cells and helper T cells). RESULTS: Fifteen, 23, and 8 patients underwent RS, LS and OS, respectively. HLA-DR expression was the lowest on day 1 and gradually increased on days 3 and 6 in all the groups. There was no significant difference in postoperative HLA-DR expression between the RS and LS group. However, on day 3, HLA-DR expression in the RS group was significantly higher than in the OS group (p = 0.04). On day 1, CRP levels in the LS group were significantly lower than in the RS group (p = 0.038). There were no significant perioperative changes in the lymphocyte subset cell count between the three groups. CONCLUSIONS: Perioperative surgical stress, as evaluated by immunological parameters, was comparable between robotic and laparoscopic surgery and higher with open surgery. Robotic surgery may be an alternative to laparoscopic surgery, as a minimally invasive surgery option for colorectal cancer.

Negative regulation of inflammatory responses by immunoglobulin A receptor (FcαRI) inhibits the development of Toll-like receptor-9 signalling-accelerated glomerulonephritis.

Myeloid FcαRI, a receptor for immunoglobulin (Ig)A, mediates cell activation or inhibition depending on the type of ligand interaction, which can be either multivalent or monovalent. Anti-inflammatory signalling is triggered by monomeric targeting using anti-FcαRI Fab or IgA ligand binding, which inhibits immune and non-immune-mediated renal inflammation. The participation of Toll-like receptors (TLRs) in kidney pathology in experimental models and various forms of human glomerular nephritis has been discussed. However, little is known about negative regulation of innate-immune activation. In the present study, we generated new transgenic mice that express FcαRI(R209L) /FcRγ chimeric protein and showed that the monovalent targeting of FcαRI exhibited inhibitory effects in an in vivo model of TLR-9 signalling-accelerated nephritis. Mouse monoclonal anti-FcαRI MIP8a Fab improved urinary protein levels and reduced the number of macrophages and immunoglobulin deposition in the glomeruli. Monovalent targeting using MIP8a Fab attenuates the TLR-9 signalling pathway and is associated with phosphorylation of extracellular signal-related protein kinases [extracellular signal-regulated kinase (ERK), P38, c-Jun N-terminal kinase (JNK)] and the activation of nuclear factor (NF)-κB. The inhibitory mechanism involves recruitment of tyrosine phosphatase Src homology 2 domain-containing phosphatase-1 (SHP-1) to FcαRI. Furthermore, cell transfer studies with macrophages pretreated with MIP8a Fab showed that blockade of FcαRI signalling in macrophages prevents the development of TLR-9 signalling-accelerated nephritis. These results suggest a role of anti-FcαRI Fab as a negative regulator in controlling the magnitude of the innate immune response and a new type of anti-inflammatory drug for treatment of kidney disease.

The G1556S-type tuberin variant suppresses tumor formation in tuberous sclerosis 2 mutant (Eker) rats despite its deficiency in mTOR inhibition.

Tuberin, a tumor-suppressor protein produced by the tuberous sclerosis gene TSC2, downregulates the Rheb-mTOR-S6K pathway (mTOR axis). Comparison of the effects of human tuberin mutations, such as G1556S, suggests that pathways other than the mTOR axis might also be involved in the pathogenesis of tuberous sclerosis. Here we test this possibility using the rat G1556S-type mutation (GSM) and a transgenic Eker (Tsc2 mutant) rat system. Cells expressing GSM-tuberin failed to downregulate the mTOR axis. GSM-tuberin had an altered localization, which underlie its reduced ability to form a complex with hamartin, and a site-specific alteration in phosphorylation status indicating diverse regulation by Akt. GSM-transgenic (GSM-Tg) rats exhibited suppression of macroscopic renal tumors following N-ethyl-N-nitrosourea treatment. Intriguingly, rats with weaker GSM-Tg expression showed microscopic cystic and pre-tumorous lesions that were restricted in size and expansion, although they had hyper-phosphorylation of ribosomal protein S6. These results highlight a novel pathway involving tuberin that regulates tumor suppression independently of the mTOR inhibitory function. Identification of such a novel pathway will provide clear implications for generation of new therapeutic targets in the treatment of these tumors.

Severe hypoglycemia in a patient with anorexia nervosa.

OBJECTIVE: Severe hypoglycemia is an important but uncommon complication of anorexia nervosa. A 35-year-old woman showed severe hypoglycemia after a recovery from severe liver dysfunction due to malnutrition. METHODS: To reveal the cause of severe hypoglycemia, we measured plasma hormones and performed a 75g oral glucose loading test. RESULTS: Fasting serum adrenocorticotropic hormone, cortisol, growth hormone, somatostatin, and active ghrelin were elevated. Serum free triiodothyronine, leptin, and adiponectin were reduced. Plasma glucose fluctuated from 67 to 76 mg/dl after a 75g glucose ingestion, without hyperinsulinemia. Serum growth hormone, somatostatin, and active ghrelin levels were decreased after glucose ingestion. Plasma glucagon levels were increased and remained at high levels at 120 min, and glucose-dependent insulinotropic polypeptide (GIP) levels were continuously and remarkably increased after glucose ingestion. CONCLUSION: We observed a strongly reduced sensitivity in glucagon-induced hepatic glycogenolysis, and significantly elevated fasting and postprandial GIP levels, and a defective GIP-mediated glucagon secretion, in an anorectic patient with severe hypoglycemia.

Production of monoclonal antibodies against Chinemys reevesii turtle vitellogenin and their usage for comparison of biochemical and immunological characters of vitellogenins and yolk proteins of freshwater turtles.

Four hybridoma clones (ACV-1, -3, -4, and -5) were established for Chinemys reevesii (Reeves' turtle) vitellogenin (VTG) as a precursor protein of egg yolk and a biomarker of environmental pollution. Binding-inhibition experiments indicated that the epitopes of four mAbs were distinct. No binding of ACV-4 to C. reevesii VTG in the Western blot suggests that the epitope of ACV-4 would be dependent on the three-dimensional structure. ACV-1, -3, and -5 bound to C. reevesii VTG in the Western blot. The signal for ACV-1 and -5 disappeared by reduction of the VTG, suggesting that the construction of the epitopes for ACV-1 and -5 were dependent on the disulfide bridge in the VTG molecule. All four mAbs recognized Trachemys scripta and Mauremys japonica VTGs in the ELISA. The yolk proteins were tested for the binding of the mAbs in the Western blot. ACV-1 being capable to bind to the VTG in the reduced condition did not bind to any protein bands of the yolk. This indicates that ACV-1 recognizes a part of the VTG molecule that is not incorporated in the oocytes. Both ACV-3 and -5 bound to the 32- and 70-kDa yolk proteins. Since a mAb recognizes only one site (epitope) on a protein molecule, the 32-kDa protein originated from the 70-kDa one. An ELISA system using ACV-5 as the capture antibody and ACV-3 as the detecting antibody showed the lower detectable concentration (2 ng/mL) and a wide detectable range to 1000 ng/mL (R2=0.999). The system was used to determine serum VTG levels of juvenile turtles treated with estradiol-17beta or vehicle (corn oil). By the use of the mAbs described in this paper, basic and applied studies for turtle VTGs would be improved.

Comparative analyses of serum vitellogenin levels in male and female Reeves' pond turtles (Chinemys reevesii) by an immunological assay.

A quantitative immunological method was developed for measuring serum vitellogenin levels of Reeves' pond turtles (Chinemys reevesii) to investigate the effects of endocrine disruptors on the freshwater ecosystem. Vitellogenin was induced by injecting estradiol-17beta into C. reevesii turtles (adult females, juvenile females, and males) and was purified from the turtle serum by EDTA-MgCl2 precipitation followed by gel filtration. Using a polyclonal antibody raised against C. reevesii vitellogenin, an enzyme-linked immunosorbant assay was established. The detectable range, recovery of vitellogenin, and coefficient of variation in this assay were 0.0040-1.0 microg.ml(-1), 85.3-109% and 3.4-11.5%, respectively. This assay was also applicable for measurement of the concentrations of vitellogenins from other species, Japanese pond turtles (Mauremys japonica) and red-eared turtles (Trachemys scripta). The serum vitellogenin concentration of 131 C. reevesii turtles captured at a Japanese local river was measured by the assay. In females, vitellogenin ranged from 0.10 microg.ml(-1) to 15,000 microg.ml(-1) with two peaks, 0.10-1.0 microg.ml(-1) (juveniles) and 1,000-10,000 microg.ml(-1) (adults). However, in males, it ranged from 0.10 microg.ml(-1) to 0.60 microg.ml(-1), showing one peak, 0.10-0.20 microg.ml(-1). Therefore, if relatively high concentrations of vitellogenin are detected in males or juvenile females, it is suggested that they would have been exposed to xenobiotic estrogens.

High-level expression of naked DNA delivered to rat liver via tail vein injection.

BACKGROUND: High levels of foreign gene expression in mouse hepatocytes can be achieved by rapid tail vein injection of a large volume of a naked DNA solution, the 'hydrodynamics-based procedure'. Rats are more tolerant of the frequent phlebotomies required for monitoring blood parameters than mice, and thus are better for some biomedical research. METHODS: We tested this technique for the delivery of a therapeutic protein in normal rats, using a rat erythropoietin (Epo) expression plasmid vector, pCAGGS-Epo. RESULTS: We obtained maximal Epo expression when the DNA solution was injected in a volume of 25 ml (approximately 100 ml/kg body weight) within 15 s. We observed a dose-response relationship between serum Epo levels and the amount of injected DNA up to 800 microg. Using quantitative real-time PCR, the vector-derived Epo mRNA expression was mainly detected in the liver. When a lacZ expression plasmid was injected similarly, beta-galactosidase was exclusively detected in the liver, mainly in hepatocytes. Toxicity attributable to the technique was mild and transient, as assessed by histochemical analysis. Epo gene expression and erythropoiesis occurred with Epo gene transfer in a dose-dependent manner, and persisted for at least 12 weeks, the last time point examined. Repeated administration of the plasmid DNA also effectively led to erythropoiesis. CONCLUSIONS: These results demonstrate that gene transfer into the liver via rapid tail vein injection can easily be achieved in the rat, which is more than 10 times larger than the mouse, and has significant value for gene function analysis in rats.

Dynamics of postprandial remnant-like lipoprotein particles in serum after loading of diacylglycerols.

BACKGROUND: To compare the effects of diacylglycerol (DAG) on postprandial lipid metabolism with triacylglycerol (TAG), we examined the differences in the dynamics of remnant lipoproteins after loading of DAG or TAG of the same fatty acid composition. METHODS: The subjects were comprised of 6 male volunteers who orally took creamed test meals prepared with either DAG or TAG at a dose of 30 g lipid/m(2) of body surface area in the early morning after fasting for at least 12 h. Blood was taken before and 2, 3, 4, 6 and 8 h after lipid loading. To quantify the amount of remnants, as the parameters, we used concentrations of cholesterol (RLP-C) and triacylglycerol (RLP-TG) in remnant-like lipoprotein particles (RLP) of serum. RESULTS: The serum triacylglycerol concentration was markedly increased 2 h after loading of DAG and TAG. Serum triacylglycerol concentrations at 2, 3 and 8 h after loading of DAG were significantly lower than those after loading of TAG. The serum RLP-C concentration was significantly lower 2, 3 and 8 h after loading of DAG than TAG. There was a trend toward less increase in the RLP-TAG in the DAG group. The area under the curve (AUC) of serum RLP-TAG after DAG loading was significantly lower than that following TAG loading. CONCLUSIONS: DAG might reduce the risk of coronary arteriosclerotic diseases by weakening the postprandial increase of RLP that is known to be closely correlated with atherosclerosis.

An immature rat lymphocyte marker CD157: striking differences in the expression between mice and rats.

We have established a novel monoclonal antibody that recognises mouse and rat CD157, and uncovered striking differences in both the level and stage of expression of this antigen in the primary lymphoid organs between these two species. Unlike mouse, the majority of rat thymocytes express CD 157. SHR and WKY rats were the exception, having unusually low levels (similar to those of the mouse) of these cells. However, in both species, a subset of CD3- CD4- CD8- thymocytes exhibited high levels of CD157. Surprisingly, these CD157high cells temporarily upregulated MHC class I molecules in both species. Furthermore, a third of CD157high rat thymocytes were CD45RC+, a marker found on immature thymocytes with regenerative capacity. Examination of the bone marrow lymphoid population shows that the expression of rat CD157 is largely observed at the CD45R+ IgM- pre-B-II cell stage, and unlike mouse, extension of expression into the IgM+ immature B cell stage was marginal. Similar to CD157high immature thymocytes, these immature B cells also expressed high levels of MHC class I. With the exception of the LEC, SHR and WKY rat strains, which have three- to four-fold less CD157+ bone marrow myeloid cells, percentages of these cells are similar between these two species. Thus, marked differences in the level and stage(s) of CD157 expression on lymphoid cells in mouse and rat indicate that CD157 may not, as previously thought, have a direct role in T or B cell differentiation.

[Factors related to satisfaction level in daily life for older people].

The purpose of this study was to examine relations between satisfaction levels and life-style in daily life for older people. A questionnaire, based on 7 factors of life satisfaction level and 13 factors of life-style chosen after considering theoretical validity, was administered to 1,320 healthy people aged 60 or more in the community (665 males and 655 females). Remarkable gender and grade differences were confirmed in the "physical health" satisfaction level. Satisfaction level for "personal relations" related to the number of friends for both sexes and to volunteer activities for males. The influence of the life-style factor on satisfaction level was highest in physical health. The influence of the number of friends was high for each satisfaction level. It was inferred that there are many aspects of life-style backgrounds contributing to the satisfaction level of older people in the community, and individual satisfaction with daily life is affected by different life-style factors.

Macrophage- and neutrophil-dominant arthritis in human IL-1 alpha transgenic mice.

To study the effects of IL-1 alpha in arthritis, we generated human IL-1 alpha (hIL-1 alpha). Transgenic mice expressed hIL-1 alpha mRNA in various organs, had high serum levels of hIL-1 alpha, and developed a severe polyarthritic phenotype at 4 weeks of age. Not only bone marrow cells but also synoviocytes from knee joints produced biologically active hIL-1 alpha. Synovitis started 2 weeks after birth, and 8-week-old mice showed hyperplasia of the synovial lining layer, the formation of hyperplastic synovium (pannus) and, ultimately, destruction of cartilage. Hyperplasia of the synovial lining was due to the accumulation of macrophage-like cells expressing F4/80 molecules. hIL-1 alpha was widely distributed in macrophage- and fibroblast-like cells of the synovial lining cells, as well as synovial fluid monocytes. T and B cells were rare in the synovial fluid, and analysis of marker expression suggests that synoviocytes were directly histolytic and did not act as antigen-presenting cells. In the joints of these mice, we found elevated levels of cells of the monocyte/macrophage and granulocyte lineages and of polymorphonuclear neutrophils (PMNs), most of which expressed Gr-1, indicating that they were mature, tissue-degrading PMNS: Cultured synoviocytes and PMNs from these animals overexpress GM-CSF, suggesting that the hematopoietic changes induced by IL-1 and the consequent PMN activation and joint destruction are mediated by this cytokine.