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.

Bovine κ-casein inhibits human rotavirus (HRV) infection via direct binding of glycans to HRV.

Human rotavirus (HRV) is a major etiologic agent of severe infantile gastroenteritis. κ-Casein (κ-CN) from both human and bovine mature milk has been reported to have anti-HRV activity; however, the mechanism of this activity is poorly understood. The present study examined the molecular basis for the protective effect of bovine κ-CN derived from late colostrum (6-7 d after parturition) and from mature milk. Among the components of casein, κ-CN is the only glycosylated protein that has been identified. Therefore, we investigated whether the glycan residues in κ-CN were involved in the anti-HRV activity. Desialylated CN obtained by neuraminidase treatment exhibited anti-HRV activity, whereas deglycosylated CN obtained by o-glycosidase treatment lacked antiviral activity, indicating that glycans were responsible for the antiviral activity of CN. Furthermore, an evanescent-field fluorescence-assisted assay showed that HRV particles directly bound to heated casein (at 95°C for 30 min) in a viral titer-dependent manner. Although the heated κ-CN retained inhibitory activity in a neutralization assay, the activity was weaker than that observed before heat treatment. Our findings indicate that the inhibitory mechanism of bovine κ-CN against HRV involves direct binding to viral particles via glycan residues. In addition, heat-labile structures in κ-CN may play an important role in maintenance of κ-CN binding to HRV.

Skimmed, sterilized, and concentrated bovine late colostrum promotes both prevention and recovery from intestinal tissue damage in mice.

Bovine colostrum is a rich source of tissue repair and growth factors, and inhibits gastrointestinal injury induced by the side effects of nonsteroidal antiinflammatory drugs (NSAID), such as indomethacin. Nonsteroidal antiinflammatory drugs are drugs with analgesic and antipyretic effects, but in higher doses they have inflammatory effects. The pathogenesis of small intestinal damage caused by NSAID is unclear. The present study was performed to investigate the antiinflammatory effects of skimmed, sterilized, and concentrated bovine late colostrum on intestinal injury induced by side effects of NSAID, and then to identify the active ingredient in the colostrum for intestinal tissue. In Japan, the sale of bovine colostrum within 5 d after parturition is prohibited by law. Therefore, we focused on bovine late colostrum obtained from healthy lactating cows 6 to 7 d after parturition. Proliferation of small intestine epithelial cells was stimulated in mice fed the colostrum for 1 wk. With regard to indomethacin-induced enteropathy, both prefeeding and postfeeding with colostrum facilitated growth of the intestinal villi, indicating preventive and healing effects. Furthermore, to identify the active ingredient in the colostrum responsible for this effect, the casein and whey fractions were prepared from the colostrum and fed to normal mice. Only the colostrum casein fraction stimulated intestinal villus elongation, whereas the whey fraction and mature milk casein showed no such effect. Taken together, these observations indicate that the skimmed, sterilized, and concentrated bovine late colostrum, especially the casein fraction, could be used to treat the injurious effects of NSAID in the intestine and could be effective for treatment of other ulcerative conditions in the bowel, suggesting that the colostrum has therapeutic potential for intestinal inflammation.

Multiple-dose therapy with bovine colostrum confers significant protection against diarrhea in a mouse model of human rotavirus-induced gastrointestinal disease.

Rotavirus is the most important etiologic agent of severe gastroenteritis. Previously, we reported that skimmed and concentrated bovine late colostrum (SCBLC) obtained from normal unimmunized cows at 6 to 7d after parturition effectively prevented against human rotavirus (HRV)-induced severe gastroenteritis in vivo, when administered as a single dose 60 min before viral inoculation. In the present study, we examined the efficacy of multiple administrations of SCBLC at smaller dosages after viral inoculation in vivo. We demonstrate that multiple administrations within 24h after virus inoculation resulted in earlier recovery from diarrheal symptoms, in an administration frequency-dependent manner. Furthermore, we investigated whether isolated IgG anti-HRV activity in SCBLC was equivalent to that of IgG isolated from bovine mature milk as measured by in vitro activity assays. We found that IgG-containing fractions from SCBLC and mature milk exhibited approximately the same level of anti-HRV activity. We concluded that the SCBLC contains a high level of IgG against HRV-induced severe gastroenteritis, which will be possible to use in protective effects in immunocompromised hosts, such as children and the elderly. Multiple doses of SCBLC during the early stages of infection or lower dosage of SCBLC given as a single dose both resulted in relief of diarrheal symptoms.

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.

Effect of nitric oxide on mouse clonal osteogenic cell, MC3T3-E1, proliferation in vitro.

Nitric oxide (NO) is a very small lipophilic molecule which rapidly diffuses and reaches the cytoplasmic components, and results in the activation of diverse biological function. It has been already reported that cultured osteoblasts synthesize NO in response to proinflammatory cytokines and lipopolysaccaride. In terms of the action of NO on bone metabolism, cytokine-induced NO by osteoblast inhibits bone resorption through inducing the apoptosis of osteoclast progenitor cells and suppressing the osteoclast activity. Also, NO synthase (NOS) inhibitor, NG-monomethyl-L-arginine is reported to induce a dose-dependent inhibitory effect on the proliferation of osteoblast-like cell lines MG63 and ROS 17/2.8, which indicate that NO may stimulate cell proliferation. On the other hand, cytokine-induced NO is reported to reduce osteoblast activity significantly in high concentration, as was evidenced by inhibition of DNA synthesis, cell proliferation, alkaline phosphatase activity, and osteocalcin production. Thus, the effect of NO on osteoblast activities is still controversial. In the present study, S-nitroso-N-acetyl-dl-penicillamine(SNAP), NO donor enhanced DNA synthesis of MC3T3-E1 in vitro. This activation seems to be mediated by NO directly because specific NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) partially attenuated the osteoblast proliferation induced by SNAP. On the other hand, the guanylate cyclase inhibitor, LY83583, failed to abolish the effect of SNAP on DNA synthesis of osteoblasts and 8-bromo cyclic guanosine 3',5'-monophosphate(cGMP), substituting for the accumulation of intracellular cGMP in osteoblasts, did not enhance the incorporation of 3H-thymidine(3H-TdR). It is, then, suggested that osteoblast proliferation might be enhanced by NO independently apart from the activation of cytoplasmic guanylate cyclase and cGMP-dependent mechanisms.

Human milk bile-salt-stimulated lipase is extremely reactive with the monoclonal antibody 1CF11 which recognizes a human-specific carbohydrate antigen.

1CF11 (Kanamaru, Y. et al.; Biochem. Biophys. Res. Commun., 249, 618-623, 1998) is a monoclonal antibody obtained after being raised in a mouse by injection of human milk MUC1 mucin as the antigen. Its reactivity was found to be unique in that it only reacts with a carbohydrate epitope shared by glycoproteins in human secretions, while its chemical nature is still unknown. Since a glycoprotein of Mr 135,000 (135K) in human milk was found to react extremely strongly with this antibody, we intended in this study to isolate the glycoprotein by a combination of various chromatographic techniques and identify it. It is a human milk bile-salt-stimulated lipase. By comparison of its immunoreactivity and glycan structures so far reported with those of lactoferrin from human milk, it is suggested that the epitope recognized by mAb ICF11 could be a human-specific novel glycan.

Unique occurrence of the 1CF11 carbohydrate epitope in primate saliva.

We examined a large number of individual human and animal saliva samples for the reactivity with ICF11, a mouse monoclonal antibody previously produced for the characterization of human milk mucin and apparently recognizing a certain carbohydrate antigenic structure shared by various human glycoproteins in secretions. The results obtained here confirm the unique occurrence of ICF11 epitope in each and every saliva sample from humans and Old world monkeys as well, though a vast variety was observed among individual saliva samples in the immunological reactivity with ICF11.

Constitutive phosphorylation and nuclear localization of Smad3 are correlated with increased collagen gene transcription in activated hepatic stellate cells.

Hepatic stellate cells (HSC) are the main producers of type I collagen in fibrotic liver, and transforming growth factor-beta (TGF-beta) plays critical roles in stimulating collagen gene expression in the cells mainly at the level of transcription. We have previously identified an upstream sequence of alpha2(I) collagen gene (COL1A2) that is essential for its basal and TGF-beta-stimulated transcription in skin fibroblasts and HSC. We designated this region the TGF-beta-responsive element (TbRE). Recently Smad3, an intracellular mediator of TGF-beta signal transduction, has been shown to bind to the TbRE and stimulate COL1A2 transcription when overexpressed in skin fibroblasts. In the present study, we demonstrate increased transcription of COL1A2 and plasminogen activator inhibitor-1 (PAI-1) genes and low response to TGF-beta in an activated HSC clone derived from a cirrhotic liver. Western blot analyses indicated constitutive phosphorylation of Smad3 in the cells. Immunofluorescence studies revealed that, in contrast to Smad2 that translocated from the cytoplasm to the nucleus upon TGF-beta treatment, Smad3 and Smad4 were present in the nucleus irrespective of ligand stimulation. Increased COL1A2 and PAI-1 gene transcription in the cells was not affected by overexpression of inhibitory Smad7. Altogether, the results correlate abnormality in TGF-beta/Smad signaling with pathologically accelerated collagen gene transcription in activated HSC.

Identification of novel hypocholesterolemic peptides derived from bovine milk beta-lactoglobulin.

This study was designed to clarify the mechanisms of hypocholesterolemic action of beta-lactoglobuline tryptic hydrolysate (LTH) and to identify the novel hypocholesterolemic peptide derived from LTH by screening using Caco-2 cells and animal studies. Serum and liver cholesterol levels were significantly lower in rats fed LTH than in those fed casein tryptic hydrolysate (CTH). The present study suggests that the inhibition of micellar solubility of cholesterol which causes the suppression of cholesterol absorption by a direct interaction between cholesterol mixed micelles, and LTH in the jejunal epithelia is part of the mechanism underlying the hypocholesterolemic action of LTH. Though no one could trace the hypocholesterolemic peptide to any protein origin, we identified, for the first time, a novel hypocholesterolemic peptide, Ile-Ile-Ala-Glu-Lys (IIAEK). Surprisingly, the present study provides the first direct evidence that a new hypocholesterolemic peptide derived from beta-lactoglobuline can powerfully influence serum cholesterol levels and exhibit a greater hypocholesterolemic activity in comparison with that of medicine, beta-sitosterol, in animal studies.

Blockade of TGF-beta signaling in T cells prevents the development of experimental glomerulonephritis.

Anti-glomerular basement membrane (GBM) Ab-induced glomerulonephritis (GN) at late stage is thought to be mediated by T cells. However, signaling pathways of T cells that are involved in the development of anti-GBM Ab-induced GN are unclear. We have recently established transgenic mice expressing Smad7, an inhibitor of TGF-beta signaling, in mature T cells, where signaling by TGF-beta was blocked specifically in T cells. In this study, we showed that anti-GBM Ab-induced GN was suppressed in several measures in the transgenic mice including the severity of glomerular changes, proteinuria, renal function, and CD4 T cell infiltration into the glomeruli without down-regulation of CD62 ligand (CD62L) (L-selectin) expression on CD4 T cells. Furthermore, treatment with the soluble fusion protein of CD62L and IgG enhanced anti-GBM Ab-induced GN. These findings indicated that blockade of TGF-beta signaling in T cells prevented the development of anti-GBM Ab-induced GN. Because CD62L on T cells appears to be inhibitory for the development of anti-GBM Ab-induced GN, persistent expression of CD62L on CD4 T cells may explain, at least in part, the suppression of anti-GBM Ab-induced GN in the transgenic mice. Our findings suggest that the development of anti-GBM Ab-induced GN requires TGF-beta/Smad signaling in T cells.

A high-Mr glycoprotein fraction from cow's milk potent in inhibiting replication of human rotavirus in vitro.

Rotavirus is the major cause of infectious diarrhea in infants and young children all over the world. We have found that a high-M(r) glycoprotein fraction from cow's milk is potent in inhibiting replication of human rotaviruses in vitro. Since the activity seems to be unique and specific, this fraction may be useful as a novel agent for treatment or prevention of rotavirus diarrhea.

Effects of hydroxyapatite on spontaneous mammary tumourigenesis and related parameters in SHN virgin mice.

Spontaneous mammary tumourigenesis in a high mammary tumour strain of SHN virgin mice was inhibited by the chronic ingestion of a diet containing hydroxyapatite [HAP: Ca5(OH)(PO4)3] at 2.5% or 5% up to 7 months of treatments, while not thereafter. The decrease in the excretion of urinary component levels associated with mammary tumour development was prevented in the experimental group given HAP, which resulted in the higher component levels in the experimental group than in the control of the tumourous mice. The treatment little affected body weight change, the pattern of the oestrous cycle, endocrine organ weights, glucose tolerance, serum levels of glucose and free fatty acid and serum superoxide dismutase activity. The results suggest that the inhibition by HAP of mammary tumourigenesis is at least partly ascribed to its prevention of a decreased metabolic turnover as reflected by the higher excretion of urinary components in tumourous mice given HAP.

A monoclonal antibody that recognizes a common carbohydrate epitope shared by various glycoproteins in human secretions.

A monoclonal antibody (mAb; a mouse IgM referred to as 1CF11) recognizing various human glycoproteins was obtained. While the immunoreaction of glycoproteins from human secretions including milk, saliva, and bronchus was demonstrated as a typical dose-responded S-shaped reaction curve on ELISA, no reaction was detected with milks and sera of animal origin as well as human serum. In the constituting polypeptides of the human milk secretory IgA molecule, only the secretory component was recognized by this mAb. Among various chemical treatments of the purified human milk lactoferrin (Lf), only either periodate or mild alkaline treatment abolished the immunoreactivity of the glycoprotein. A recombinant human Lf was not immunoreactive. Finally, the immunoreactive fragments were isolated from human milk Lf, which remained reactive with PAS reagent while lacking the previously reported N-glycans. These results strongly suggest that the mAb 1CF11 recognizes a new glycan O-glycosidically linked to glycoproteins in human secretions.

Passive immunizations of suckling mice and infants with bovine colostrum containing antibodies to human rotavirus.

After immunizing 8-month pregnant Holstein cows with the human rotavirus MO strain, cow colostrum containing neutralizing antibody to four different serotypes of human rotavirus, designated Rota colostrum, was obtained. Oral inoculation of human rotavirus MO strain into 5-day-old BALB/c mice causes gastroenteritis characterized by diarrhea. Using this small animal model, passive protection of suckling mice against human rotavirus infection was achieved with the use of Rota colostrum. Rota colostrum completely protected against rotavirus infection, but purified IgG and IgA obtained from Rota colostrum were unable to protect against infection. After grouping randomly 20 infants from a baby care center, 10 infants received 20 ml of Rota colostrum per day for 2 weeks and 10 control infants did not. Rotavirus-associated diarrhea developed in 7 of 10 infants in the control group. None of the three infants in the every day recipient group of Rota colostrum had such symptoms, and one of three infants in the every other day recipient group developed rotavirus-induced diarrhea. All four infants who received Rota colostrum after symptoms appeared developed diarrhea. Oral administration of Rota colostrum seems to be an effective and safe means of preventing diarrhea caused by human rotavirus infection.