Mizoribine (MZR) is a selective inhibitor of the inosine monophosphate dehydrogenase – a key enzyme in the de novo pathway of guanine nucleotides – that was developed in Japan. Clinically, MZR has been successfully used without any serious adverse effects
for the long-term treatment of young patients with lupus nephritis.[1-3] Besides its immunosuppressive effects, MZR has recently been reported to suppress the progression of histologic chronicity in selected patients with lupus nephritis and immunoglobulin A (IgA) nephropathy.[1-4] Moreover, some experimental reports described that MZR attenuates tubulointerstitial fibrosis in NVP-LDE225 purchase rat models of unilateral ureteral obstruction, non-insulin-dependent diabetes and peritoneal fibrosis via suppression of macrophage infiltration of the interstitium.[5-7] Also, we recently confirmed a significant suppression of intraglomerular macrophage infiltration accompanied with significant suppression of the chronicity indices following MZR treatment in a patient with proliferative lupus nephritis. These laboratory
and clinical observations suggest another beneficial mechanism of action of MZR from the histologic standpoint in the treatment of lupus nephritis. Since most of the oral dose of MZR is excreted unchanged in urine, the learn more drug is thought to expose directly to residual renal cells. Thus, it is important to examine the direct effects of MZR against inflamed residual renal cells. Glomerular mesangial cells (MCs) have been reported to produce a wide variety of proinflammatory molecules that play an important role in immune and inflammatory reactions in the kidney, and MCs itself are thought to play a pivotal role in the pathogenesis of renal diseases.
Interestingly, it has been reported that the implication of ‘psuedoviral’ immunity as a novel disease concept of lupus U0126 concentration nephritis, that is, the detection of self-nucleic acid particles resembling viral particles by toll-like receptors (TLRs) results in the activations of the downstream signalling cascades and subsequent type I interferons (IFNs) production. In this context, we have examined the TLR3 signalling cascades treated with polyinosinic-polycytidylic acid (poly IC), a synthetic analogue of viral dsRNA, that makes ‘pseudoviral’ infection in cultured human MCs, and found that the activation of mesangial TLR3 upregulated the expression of functional molecules including monocyte/macrophage chemoattractants: CC chemokine ligand (CCL) 2 (or monocyte chemoattractant protein-1 [MCP-1]), CCL5 (or regulated on activation, normal T-cell expression and secretion [RANTES]), CXC ligand 10 (CXCL10) (or IFN-γ-induced protein 10 [IP-10]), fractalkine (or CX3CL1), and neutrophil chemoattractant: interleukin (IL)-8 (or CXCL8), in cultured human MCs.
We found that morphological features of fibrosis in this disease are largely depending on the anatomical location wherein the lesion developed. Interstitial fibrosis located at intracapsule in 1, subcapsule in 3, cortex in 3, perivasculature in 5, perinerve in 2 cases and medulla in no case. The components of extra cellular matrices in the fibrosis are followings. In perivascular and perineural lesions, collagen type I (67%), III (100%) and VI (100%) were the major components,
while collagen type IV (27%) and V (0%) were scant. In subcapsular and cortical lesions, collagen type III (83%), IV (32%) and VI (50%) were the major components, although collagen type I (14%) and V (0%) were less dominant. Three cases revealed storiform fibrosis and all distributed only in the cortex. Storiform fibrosis was negative for collagen type Selleckchem Target Selective Inhibitor Library I. Fibronectin accumulated between collagen Trichostatin A fibers and increased as stage advanced. In conclusions, renal pathology in IgG4-related kidney disease reveals several distinct morphology, useful to discriminate TIN from other causes. Interstitial fibrosis mainly distributes along perivasculature, whereas storiform fibrosis is formed only in the cortex. The main components of interstitial fibers may be dependent on the locations
which are formed of interstitial fibrosis in IgG4-RKD. SAEKI TAKAKO Department of Internal Medicine, Nagaoka Red Cross Hospital, Japan IgG4-related kidney disease (IgG4-RKD) is a comprehensive term for renal lesions associated with IgG4-related disease (IgG4-RD). The most dominant feature of
IgG4-RKD is plasma cell-rich tubulointerstitial nephritis (TIN) with increased IgG4-positive plasma cells and fibrosis (namely IgG4-related TIN), although some glomerular lesions such as membranous nephropathy are sometimes evident concurrent with IgG4-related TIN. Clinical features: IgG4-RKD shows a striking male predominance (73–87%) and the average patient age is about 65 years. Systemic symptoms are relatively mild and the condition usually comes clinically apparent when renal 4��8C dysfunction and/or renal radiographic abnormalities occur. Most patients have accompanying IgG4-related extra-renal lesions such as sialadenitis, lymphadenopathy or type 1 autoimmune pancreatitis. Although nearly half of all patients with IgG4-RKD have proteinuria (and some have hematuria), it is mild in the majority. Nephrotic range proteinuria is rarely detected, except when glomerular lesions are also present. Kidney function varies from normal to renal failure, and the development of renal dysfunction also varies from relatively acute to slowly progressive. Serology usually demonstrates high levels of serum IgG and IgG4. A high level of serum IgE and hypocomplementemia are also frequent features. Although antinuclear antibodies and rheumatoid factor are often positive, anti-DNA, anti-SS-A and anti-SS-B antibodies are usually negative.
Interestingly, the grafting of purified TEC from embryos of NOD mice to newborn C57BL/6 nude mice results in the development of insulitis, suggesting GSI-IX mouse a functional anomaly in TEC from NOD mice cells . During negative selection, developing T cells interact with thymic epithelium- and bone marrow-derived antigen-presenting cells (APCs), in particular thymic medullary dendritic cells. Thus, aberrant negative selection results essentially from anomalies affecting thymic APCs. Like the majority of ubiquitous or organ-specific autoantigens, several islet β cell antigens involved in T1D, such as
glutamic acid decarboxylase (GAD) and proteins of the insulin family, are expressed promiscuously in the thymus to be presented to thymocytes during education [60,61]. The decreased expression of these antigens can disturb the negative selection
of autoreactive T lymphocytes, which may predispose to the development of autoimmunity. In humans, susceptibility to T1D is associated with a polymorphism in the 5′ region of the insulin gene, which influences the rate of expression of peptides derived from insulin by APCs in the thymus. The protective allele is associated with a high level of thymic expression of insulin and the susceptibility allele to a low level . NOD mice which express neither the pro-insulin 2 nor the islet-cell antigen 69 (ICA69) in the thymus develop diabetes rapidly [62,63], as in BioBreeding Diabetes Prone (BBDP) BAY 80-6946 in vivo rats, which do not express type 2 insulin-like growth factor (Igf2) in thymus . Furthermore, depletion of Ins2 expression in medullary TEC is sufficient to break central tolerance and induce anti-insulin autoimmunity and rapid diabetes
onset in mouse . Interestingly, intrathymic transplantation of pancreatic islet cells reduces autoimmunity towards β cells and prevents diabetes development in NOD/Lt mice . Thus, the thymus could also play a role in acquired tolerance and may be a potential candidate in the therapeutics of autoimmune diseases. Negative selection might also be affected owing to antigen-processing defects. A defect of peptide presentation can result from the weak affinity of TCR for unstable MHC–peptide PRKACG complexes and/or from a defect in antigen processing by proteases of thymic APCs [58,67]. Major defects in the architecture of the thymic stroma found in animal models of diabetes are also thought to contribute to a defect in negative selection [58,67]. In NOD mice, for example, medullar TEC are present in the cortex, and large areas devoid of TEC and expression of MHC molecules are observed in the thymus . Multiple thymocyte migration-related abnormalities have also been observed in the NOD mouse thymus .
Intrathecal infusion of recombinant FasL induces apoptosis of CNS-infiltrating inflammatory
cells, including T cells and macrophages, but does not exert cytotoxicity against CNS-resident cells, resulting in mitigated EAE manifestations . Elimination of infiltrating T cells in the CNS by Fas/FasL-mediated apoptosis is crucial for resolution of EAE [9, 18, 19], since FasL-deficient gld recipients develop prolonged PD0325901 ic50 EAE after adoptive transfer of myelin basic protein-reactive WT Fas+ T lymphocytes . The CNS-resident cell population which induces apoptosis of CD4+ T cells in EAE still remains to be identified. We hypothesize that astrocytes, which constitutively express FasL, may play a key role given that FasL-expressing astrocytes are in intimate contact with apoptotic T cells in EAE and can induce apoptosis of activated CD4+ T cells in vitro [21, 22]. Consistently, buy PD-0332991 our previous study also demonstrated that increased apoptosis of gp130-deficient astrocytes exacerbated EAE, partially due to an impaired elimination of CD4+ T cells from the CNS . However, in vivo evidence confirming that astrocytic FasL is involved in the induction of CD4+ T-cell apoptosis in EAE is still lacking. In order to determine whether FasL+ astrocytes are inducers of CD4+ T-cell apoptosis in EAE, we generated glial fibrillary acid protein (GFAP)-Cre FasLfl/fl mice that are deficient
of FasL selectively in astrocytes. We show in the present study that astrocytic FasL is crucial to terminate the autoimmune T-cell response in the CNS, which allows clinical recovery from EAE. We generated GFAP-Cre FasLfl/fl mice with selective FasL deletion in the CNS (Supporting
click here Information Fig. 1). Further PCR analysis of cultivated cells showed FasL deletion in astrocytes and to a minor extent in neurons (Fig. 1A). In contrast, microglia of GFAP-Cre FasLfl/fl as well as astrocytes, neurons, and microglia of FasLfl/fl control mice did not show deletion of FasL (Fig. 1A). To confirm astrocytic FasL deletion at the protein level, cell surface expression of FasL protein was analyzed by flow cytometry from cultivated astrocytes of GFAP-Cre FasLfl/fl and FasLfl/fl mice. As shown in Figure 1B, FasL expression was reduced on the surface of astrocytes from GFAP-Cre FasLfl/fl as compared to FasLfl/fl mice. Both GFAP-Cre FasLfl/fl mice and FasLfl/fl (control) mice were born in a normal Mendelian ratio and reached adulthood without any CNS defects. Collectively, these findings show that astrocyte-specific deletion of FasL was achieved in our newly generated GFAP-Cre FasLfl/fl mice, which did not show abnormalities under physiological conditions, thereby providing a useful tool for studying the function of astrocyte-specific FasL in experimentally induced models of CNS disorders.
The difference was considered statistically significant when P ≤ 0.05. Leica Microscopy system was used to take the picture, and magnification used was 40 with numerical aperture of the objectives, at temperature room. The slides were mounted using Vectashield mounting medium (Vector laboratories), and Alexa 488 fluorochrome was used to detect the positive signal (Invitrogen). As a first step, we designed recombinant adenovirus vectors containing ESAT-6 with and without calreticulin to determine whether calreticulin increased the immune response to the antigen. AdESAT-6 and AdCRT–ESAT-6 were created as described in the Materials and methods. Expression of ESAT-6 in both constructs was under the control of a cytomegalovirus promoter (Fig. 1A–C). The capacity of these constructs to express ESAT-6 was first verified by immunoblot RGFP966 in vitro analyses of HEK293 cells transfected with one of the recombinant vectors (data not shown). ESAT-6 protein expression was also demonstrated by immunofluorescence analysis of HEK293 cells transfected with AdESAT-6, AdCRT-ESAT-6 or AdLacZ (Fig. 1D). As shown in Fig. 1D, only cells transfected with
AdESAT-6 and AdCRT-ESAT-6 express ESAT-6. Therefore, our recombinant adenovirus constructs were proven to be capable of producing ESAT-6. To test the ability of AdCRT–ESAT-6 or AdESAT-6 to generate ESAT-6-specific cellular immune responses in vivo, mice Enzalutamide concentration were immunized by the intranasal route with the adenovirus constructs.
At 4 weeks post-vaccination, splenocyte cultures were prepared Cobimetinib order and restimulated with ESAT-6, and the resultant cytokine responses were analysed. It was found that while splenocytes from mice immunized with the antigen alone (AdESAT-6) showed no differences in cytokine production compared to splenocytes from LacZ-immunized mice (controls), there were significant inductions of IFN-γ and TNF-α (measured by ELISPOT and ELISA, respectively) in splenocytes from mice immunized with the antigen ESAT-6 fused to calreticulin (AdCRT–ESAT-6) (Fig. 2A,B). Taken together, these data demonstrate that immunization with ESAT-6 linked to calreticulin is an effective approach to generate potent immune responses. It has been previously shown that fusion of ESTA-6 with CFP-10 enhances the immune response. Hence, using the same strategy, we expressed a calreticulin–ESAT-6–CFP10 fusion protein (AdCRT–ESAT-6–CFP10) and compared its ability to induce a cytokine response against AdCRT–ESAT-6. The expression of the fusion protein was demonstrated by immunoblot analysis of lysates of cells transfected with the fusion vector using an anti-CFP10 polyclonal mouse antibody (Fig. 3A). While no reaction was observed in the uninfected HEK293 cell lysates, a single antibody-reactive band of approximately 90 kDa was detected in the AdCRT–ESAT-6–CFP10 cell lysates. The size of the reactive band correlated with the predicted size of the CRT-ESAT-6–CFP10 fusion protein.
We showed that in vitro treatment of spleen cells with recombinant guinea pig TNF-α (rgpTNF-α) and neutralizing anti-gpTNF-α anti-serum modulated antigen-specific T cell proliferation in guinea pigs [20,21]. Injection of anti-TNF antibody into bacille Calmette–Guérin
(BCG)-vaccinated and non-vaccinated guinea pigs following low-dose aerosol challenge with virulent M. tuberculosis resulted in splenomegaly in the BCG-vaccinated guinea pigs, while it augmented splenic granuloma organization in the non-vaccinated guinea pigs . Furthermore, direct intrapleural injection of anti-TNF antibody into guinea pigs with tuberculous pleuritis altered the inflammatory exudates by decreasing the proportions of macrophages and increasing the neutrophil and lymphocyte proportions . The purpose click here of this https://www.selleckchem.com/products/Y-27632.html study was to determine whether administration of rgpTNF-α into guinea pigs would mimic the effects as demonstrated in our in vitro studies and whether recombinant TNF-α would enhance immune responses induced by BCG vaccine. Our results indicate clearly that low doses of TNF-α, a major player in both innate and specific acquired immunity, could augment BCG vaccine-induced immunity in the guinea pig, a relevant model that mimics human tuberculosis in terms of tissue pathology, protection afforded by BCG vaccination and granuloma organization.
Random-bred Hartley strain guinea pigs weighing 250–350 g obtained from Charles River Breeding Laboratories, Inc. (Wilmington, MA, USA) were used for this study. The animals were housed individually in polycarbonate cages in a temperature- and humidity-controlled environment
with a 12-h light/12-h dark cycle. They were given commercial chow (Ralston Purina, St Louis, MO, USA) and tap water ad libitum. All procedures were reviewed and approved by the Texas A&M University Laboratory Animal Care Committee. Two groups of guinea pigs were vaccinated intradermally with 1 × 103 colony-forming units (CFU) of M. bovis BCG (Danish 1331 strain; Statens Seruminstitut, Copenhagen, Denmark) each in the left and right inguinal regions. The lyophilized vaccine was reconstituted with Sauton’s medium (Statens Seruminstitut) for injection. Beginning immediately after vaccination, the animals were injected intraperitoneally Cyclic nucleotide phosphodiesterase with either rgpTNF-α (25 µg/animal) or 1% bovine serum albumin (BSA) for a total of 12 injections given every other day. The recombinant TNF-α protein was expressed in a prokaryotic vector using the M15 Escherichia coli strain transformed with pQE-30/gpTNF-α. The functional properties of rgpTNF-α, including bioactivity, were determined by measuring the cytotoxicity on L929 cells and cytokine mRNA expression by real time-reverse transcription–polymerase chain reaction (RT–PCR) and the anti-mycobacterial activity of macrophages by metabolic labelling of M.
Recent thymic emigrant numbers were also reduced significantly in CVID patients, specifically in the PL, AC and OSAI subgroups; CVID patients with such complications treated with corticosteroids were Buparlisib datasheet excluded if they had received such therapy within 6 months of analysis. Together with the reduced CD4 naive T cells, reduced thymic emigrants suggest a lack of replenishment of the CD4 T cell pool by new thymically derived cells in CVID patients. Giovannetti et al.  also found that thymic output was reduced significantly in CVID patients, and associated this with a reduction in class-switch memory B cells, expansion
of CD21lo B cells, splenomegaly and granuloma. They also showed increased cell turnover as measured by Ki-67, particularly in the CD4 naive subset and increased apoptosis . We did not find such an association with CD21low B cells, although we found an association with PL for which granuloma is a criterion. Mouilott et al.  found a decrease in CD4 naive T cells which was accompanied by increased CD95+ expression, Dasatinib most pronounced in the PL and AC groups, while Iglesias et al.  found that CD4+CD45RA+ T cells, which contain predominantly naive CD4 T cells, had increased spontaneous apoptosis and CD95 expression in CVID
patients. Therefore, the reduction in naive CD4 T cells may, in part, be due to both reduced thymic output and increased cell turnover. Significant reductions in CD8 naive T cell numbers were seen in CVID patients compared to controls, particularly in the AC group. This has not been reported previously, and is likely to reflect the increases in terminally differentiated CD8 cells observed
in Metalloexopeptidase the PL and AC groups. Both CD4 and CD8 T cells in CVID patients, and most significantly in the AC, OSAI and PL groups, demonstrated a loss of the co-stimulatory molecules CD28 or CD27. This suggests T cell differentiation along an activation pathway. Other groups have observed increased activation in T cells of all CVID patients , as measured by CD38 and human leucocyte antigen D-related (HLA-DR) , particularly in patients with splenomegaly . The possibility of an infectious agent driving the clinical manifestations of lymphoproliferation observed in the PL subset of CVID patients has been suggested, but not established – a hypothesis supported by these T cell phenotypes. It has been suggested that cytomegalovirus (CMV) may play a role in the T cell abnormalities seen in CVID, as patients in one study had a 13-fold increased proportion of CMV-specific, functional T cells compared to aged-matched controls . CMV-specific CD8 T cells have the phenotype of CD45RA+CCR7-CD27- and the increase in CD8 T cells of this phenotype in the PL and AC subgroups of the CVID suggests that CMV or another similar infectious agent may be important [17,30].
Histopathological analysis of the MSG biopsies from 48 patients with pSS showed a different degree of FLS, defined as focus score, for those with normal biopsy, or abnormal, as indicated in Table 1. Histopathological analysis of labial biopsies of 40 control subjects show different degrees of CS, as shown in Table 4. We observed that 40% of pSS
patients with FLS < 1 showed clonal IgH rearrangements compared with patients who had an abnormal biopsy (FLS ≥ 1), in some cases reaching 100%, as shown in Table 4. This difference was statistically significant (P < 0·01; χ2 test, 99% CI). In addition, we determined that 83·4% of the cases with pSS presented an oligo–monoclonal IgH rearrangement Rapamycin cost compared with 19% of the cases diagnosed with CS. There was a high correlation in control cases between the severity of CS and the presence of B cell clonality (Table 4). Seven VX-809 datasheet cases with severe CS showed B cell oligo–monoclonality compared with those diagnosed with mild to intermediate CS (87·5 versus 3·1%; P < 0·01; χ2 test). The biopsy was completely normal in only two cases and we did not detect a clonal IgH gene rearrangement by PCR (Table 4). Our results showed 58% and 79% of B cell clonality or oligoclonality, respectively, in the MSG of SS patients using FR3/LJH and FR2/LJH-VLJH primers. Similar results have been reported in the literature, where 77% of cases with NHL were PCR-positive, arguing that the
low detection of clonal B cells is due to partial rearrangements, inversions, somatic mutations or deletions
that can be missed by PCR . The addition of FR1c-LJH primers to our PCR analysis allowed a higher detection rate of SS cases, as reported previously by Aubin and co-workers . Therefore, the use of the three sets of primers diminished the false negative results and improved the detection rate in 86·7% of the SS patients (Table 3). Also, we observed that the addition of FR3 did not increase the number of positive cases, therefore the failure of FR3 or FR2 to detect clonality in some cases could be the acquisition of somatic mutations in the primer target sequences, due to mispriming during the PCR [11,12,15,26]. Another possibility is that the IgH gene rearrangement is related closely to the cellular triclocarban origin involved in the lymphoid pathology. In these cases, absence of clonality for the FR3-VLJH primers would indicate the presence of post-GC B cells or memory B cells in the salivary glands, characterized by cells bearing somatically hypermutated VH genes, as has been found in a series of studies in NHL and MALT [10,28,29]. It has been determined that patients with SS have a 16-fold increased risk of developing lymphoma [5,30]. Several studies have suggested that lympho-epithelial lesions in SS patients show a high presence of clonal expansion of B cells, as determined by molecular analysis of the IgH rearrangement, morphological or immunophenotypic determination.
DENV isolates passed serially from brain to brain led to increased neurovirulence and neurotropism in mice and a clear attenuation in human volunteers. However, viral encephalitis is not a major clinical symptom in human dengue disease, as nervous system involvement in DENV infections is
rare and few cases are reported. The IFN system is critical to the host antiviral response, which led to the use of AG129 mice, which are type I and II IFN-R-deficient 129 mice, immune deficient and highly susceptible. Intraperitoneal infection with the mouse-adapted neurotropic DENV-2 strain, New Guinea C, led to 100% lethality in AG129 mice, all of them presenting paralysis. The neuroinflammatory changes led to alterations in motor behaviour and muscle tone and strength in DENV-3-infected mice. The neuroinflammatory process was marked by up-regulation of the chemokines Epigenetics inhibitor CCL2, CCL5, CXCL1 and CXCL2, and of the cytokines TNF-α and IFN-γ, which occurs in parallel with increased leucocyte rolling and adhesion in meningeal vessels and infiltration of immune cells into the brain. In summary, even if these models were used to study antiviral compounds or behaviour, the major limitation involving immune-compromised mice is that paralysis is not a major clinical observation in DENV infection. Initial tropism studies using the
AG129 (IFN type I and II receptor-deficient) model demonstrated that clinical isolates from all four DENV serotypes replicate BGB324 nmr efficiently in spleen, lymph node, bone marrow and muscle. Negative-strand
viral RNA was detected in dendritic cells and macrophages of the lymph node and spleen. To develop an experimental model where viral encephalitis was not the major clinical observation, Shresta et al. infected AG129 mice intravenously with the DENV-2 strain PL046. Infected AG129 mice succumbed to DENV infection, RNA Synthesis inhibitor presenting increased levels of TNF-α and vascular leakage syndrome. AG129 mice are able develop cross-reactive and long-lasting antibody responses to DENV. Sequential DENV infection in AG129 mice results in decreased viral load of the second serotype and full protection against lethal infection. AG129 and other mouse strains have been used to study ADE by passive transfer of anti-DENV monoclonal antibodies, cross-reactive immune serum, or diluted homotypic serum before infection.[52, 53] Mortality was associated with vascular leakage syndrome, high levels of TNF-α and thrombocytopenia, similar to the clinical findings observed in DHF/DSS in humans. No memory response was observed in mice receiving passive transfer of serum or antibody. Hence, models of sequential DENV infection may be useful to study ADE in the presence of a cellular memory immune response.
3A). The MFG-E8 transcript that included the cryptic exon encoded an MFG-E8 protein that was truncated at the C2 domain (designated as C2del) (Fig. 3A). Studies on mouse and bovine MFG-E8 show that the C1/C2-homologous domains are required for binding to phosphatidylserine 7, 20. To characterize C2del, we prepared human rMFG-E8 using HeLa cell transformants that produced the transgene in a tetracycline-dependent manner. On SDS-PAGE, the purified C2del ran as a smeared band of approximately 50 kDa, which was significantly bigger than the 46-kDa wild-type MFG-E8 (Fig. 3B). This was unexpected considering that C2del had a truncation of 96 amino acids and contained
only one of three N-linked glycosylation sites present in the wild-type protein. The treatment of C2del with PNGase 5-Fluoracil manufacturer F reduced its molecular weight to 32.6 kDa (Fig. 3C), and a mutation of the remaining N-glycosylation site (Asn238) also reduced its molecular weight (data not shown). Neuraminidase treatment significantly reduced C2del’s molecular weight (Fig. 3D), indicating that it was sialylated. These results suggested that this C-terminal Bortezomib mouse truncation of human MFG-E8 caused it to be aberrantly glycosylated. We next examined the
ability of C2del to recognize apoptotic cells. As shown in Fig. 3E, C2del dose-dependently bound to phosphatidylserine. The dissociation constants (Kd) determined by Biacore for the wild-type and C2del MFG-E8 heptaminol were 1.1 and 8.0 nM, respectively. C2del supported phagocytosis with a bell-shaped dosage effect and the same dose dependency as the wild-type molecule (Fig. 3F). However, the ability of C2del to enhance the engulfment at the optimum concentration was consistently lower than that observed with the wild-type MFG-E8. As described above, C2del was aberrantly glycosylated, and in particular, sialylated. The sialylation of proteins is known to prolong their half-life in vivo21, 22. To examine whether this was true for C2del, the wild-type MFG-E8
and C2del proteins were injected into C57BL/6 mice, and their levels in serum were monitored by ELISA. As shown in Fig. 4A, when 12 pmol of the wild-type or mutant MFG-E8 was injected into the tail vein, about 20 pM wild-type MFG-E8 was found in the serum after 60 min, whereas the concentration of C2del was more than 1 nM at the same time point. These results suggested that C2del was sustained longer than the wild-type protein in the blood. We previously showed that excess MFG-E8 prevents the efficient engulfment of apoptotic cells and that some SLE patients carry a significantly increased level of MFG-E8 in their blood 15. Accordingly, the injection of wild-type MFG-E8 into mice induced the development of autoimmune diseases 16. Since C2del lasted longer in vivo than wild-type MFG-E8, we hypothesized that the administration of C2del might cause autoimmune disease in mice at a lower dose than the wild-type molecule. As shown in Fig.