Suppression of collagen‐induced arthritis by oral administration of transgenic rice seeds expressing altered peptide ligands of type II collagen

Rheumatoid arthritis (RA) is an autoimmune disease associated with the recognition of self proteins secluded in arthritic joints. We previously reported that altered peptide ligands (APLs) of type II collagen (CII256‐271) suppress the development of collagen‐induced arthritis (CIA). In this study, we generated transgenic rice expressing CII256‐271 and APL6 contained in fusion proteins with the rice storage protein glutelin in the seed endosperm. These transgene products successfully and stably accumulated at high levels (7–24 mg/g seeds) in protein storage vacuoles (PB‐II) of mature seeds. We examined the efficacy of these transgenic rice seeds by performing oral administration of the seeds to CIA model mice that had been immunized with CII. Treatment with APL6 transgenic rice for 14 days significantly inhibited the development of arthritis (based on clinical score) and delayed disease onset during the early phase of arthritis. These effects were mediated by the induction of IL‐10 from CD4⁺ CD25^? T cells against CII antigen in splenocytes and inguinal lymph nodes (iLNs), and treatment of APL had no effect on the production of IFN‐γ, IL‐17, IL‐2 or Foxp3⁺ Treg cells. These findings suggest that abnormal immune suppressive mechanisms are involved in the therapeutic effect of rice‐based oral vaccine expressing high levels of APLs of type II collagen on the autoimmune disease CIA, suggesting that the seed‐based mucosal vaccine against CIA functions via a unique mechanism.     

Antibodies against the CB10 fragment of type II collagen in rheumatoid arthritis

Antibodies against intact type II collagen (CII) are a feature of rheumatoid arthritis (RA) but have limited diagnostic value. Here we assess whether either of the two major cyanogen bromide fragments of CII, namely CB10 or CB11, are more sensitive substrates for the detection of antibodies in RA. Cleavage of bovine CII with cyanogen bromide yielded CB10 and CB11; these were purified by column chromatography for use in an enzyme-linked immunosorbent assay. Serum antibodies were measured in patients with RA, psoriatic arthritis (PsA), osteoarthritis (OA) and blood donors. Results were compared with those using intact CII. Antibodies against CB10 were found in as many as 88% of 96 patients with long-standing RA, but only 12% of 33 patients with PsA, 6% of 34 patients with OA and 3% of 93 control sera. Lower frequencies for these diseases were obtained on testing for antibodies against CB11: 50%, 6%, 21% and 2%, respectively. The sensitivity of detection in RA of antibodies against CB10 compared with antibodies against intact CII (88% versus 24%) was not at the expense of specificity, which remained high at 94%. The much higher frequency of antibodies against CB10 in RA than in other rheumatic diseases or control sera indicates that CB10 is clearly a more sensitive substrate than the intact collagen molecule and, combined with other assays (rheumatoid factor, anti-cyclic citrullinated peptide [anti-CCP]), might comprise a panel with a highly reliable predictive value. Moreover, our findings should encourage renewed interest in the role of collagen autoimmunity in the pathogenesis of RA.     

Development and evaluation of transgenic rice seeds accumulating a type II-collagen tolerogenic peptide

Type II collagen (CII) in joint cartilage is known to be a major auto-antigen in human rheumatoid arthritis. Several animal model- and clinical-studies on tolerance-based immunotherapy for the arthritis have been conducted by administrating synthetic immunodominant peptides through an oral route. In the present study, to produce a tolerogenic peptide with therapeutic potential in transgenic rice plants, a gene construct producing glutelin fusion protein with tandem four repeats of a CII_250–270 peptide (residues 250–270) (GluA-4XCII_250–270) containing a human T-cell epitope was introduced with a selection marker, hygromycin phosphotransferase gene (hygromycin-resistance gene) (hph), by co-transformation. Several transgenic plants with high and stable expression of gluA-4XCII _250–270 , but no hph, were selected based on both DNA and protein analyses. The GluA-4XCII_250–270 fusion proteins were detected as both precursor and processed forms mainly in a glutelin fraction of rice endosperm protein extracts and in protein-body rich fractions prepared by density gradient ultracentrifugation. The amount of accumulated CII_250–270 peptide was immunochemically estimated to be about 1 μg per seed. Feeding DBA/1 mice the transgenic rice seeds (25 μg of the peptide per mouse a day) for 2 weeks showed tendencies lowering and delaying serum specific-IgG2a response against subsequent and repeated intraperitoneal-injection of type II collagen. Taken these together, the CII-immunodominant peptide could effectively be produced and accumulated as a glutelin-fusion protein in the transgenic rice seeds, which might be useful as pharmaceutical materials and functional food for prevention and therapy for anti-CII autoimmune diseases like human rheumatoid arthritis.     

Anti-citrullinated collagen type I antibody is a target of autoimmunity in rheumatoid arthritis

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, but its autoimmune mechanisms are not clearly understood. Recently, anti-citrullinated peptide antibodies have been specifically observed in sera of RA patients. Furthermore, we identified RA-susceptible variant in a gene encoding citrullinating enzyme, peptidylarginine deiminase type 4 (PADI4). Therefore, we hypothesized that proteins which are modified in RA synovium by PADI4 act as autoantigens. Subsequently, we obtained human collagen type I (huCI) as one of the autoantigens using a RA synoviocyte cDNA library by immunoscreening. We also investigated that the levels of anti-citrullinated huCI were significantly higher in RA patient sera than in normal control sera with high specificity (99%) and positively correlated with the levels of anti-cyclic citrullinated peptide (anti-CCP) antibodies. We concluded that huCI is a novel substrate protein of PADIs and that citrullinated huCI is a candidate autoantigen of RA.     

Evaluation of the cholesterol influence in type II collagen-induced arthritis in DBA/1J mice: an autoradiographic study

In order to verify the cholesterol influence in RA severity in DBA/1J mice, we quantified the cholesterol present in the knee joints of normal (N) and with collagen II induced arthritis (CIA). Forty male DBA/1J mice, were divided in normal (n=20) and CIA group (n=20). Mice in CIA group were injected with 100 μg of collagen II emulsified in Freund's complete adjuvant. Sixteen DBA/1J (8 N and 8 CIA) received an injection of 2.96 × 10⁶ Bq of 3H-cholesterol and were anesthetized and sacrificed. Semi-fine sections were covered with LM-1 emulsion, exposed for six weeks and developed. Collagen induced edema, erythema and dysfunction of knee joints in CIA group. Radioactive cholesterol was located more on the synovial membrane, where we found the greatest density of silver grains, significantly ( P <0.0001) higher in group CIA vs. controls (61±2.3 × 18±0.7). We conclude that the cholesterol deposits on the synovial membrane is related to CIA severity.     

Antihypertensive activity of transgenic rice seed containing an 18-repeat novokinin peptide localized in the nucleolus of endosperm cells

Novokinin (Arg-Pro-Leu-Lys-Pro-Trp, RPLKPW) is a new potent antihypertensive peptide based on the sequence of ovokinin (2-7) derived from ovalbumin. We previously generated transgenic rice seeds in which eight novokinin were fused to storage protein glutelins (GluA2 and GluC) for expression. Oral administration of these seeds to spontaneously hypertensive rats (SHRs) reduced systolic blood pressures at a dose of 1 g seed/kg of SHR. Here, 10- or 18-tandem repeats of novokinin with an endoplasmic reticulum (ER) retention signal (Lys-Asp-Glu-Leu, KDEL) at the C terminus were directly expressed in rice under the control of the glutelin promoter containing its signal peptide. Only small amounts of the 18-repeat novokinin accumulated, and it was unexpectedly deposited in the nucleolus. This abnormal intracellular localization was explained by an endogenous signal for nuclear localization. The GFP reporter protein fused to this sequence targeted to nuclei by a transient assay using onion epidermal cells. Transgenic seed expressing the 18-repeat novokinin exhibited significantly higher antihypertensive activity after a single oral dose to SHR even at one-quarter the amount (0.25 g/kg) of the transgenic rice seed expressing the fusion construct; though, its novokinin content was much lower (1/5). Furthermore, in a long-term administration for 5 weeks, even a smaller dose (0.0625 g/kg) of transgenic seeds could confer antihypertensive activity. This high antihypertensive activity may be attributed to differences in digestibility of expressed products by gastrointestinal enzymes and the unique intracellular localization. These results indicate that accumulation of novokinin as a tandemly repeated structure in transgenic rice is more effective than as a fusion-type structure.     

High accumulation of bioactive peptide in transgenic rice seeds by expression of introduced multiple genes

We have developed a simple binary vector construction system for the simultaneous expression of multiple genes in plants. Up to three independent gene cassettes can be easily integrated into one binary vector using the MultiSite Gateway System. Using this system, we produced transgenic rice plants that accumulated high levels of the hypocholesterolaemic peptide lactostatin (IIAEK) in endosperm. Binary vectors were constructed that could accommodate up to three independent modified glutelin gene cassettes encoding multimer lactostatin in the variable regions. Eight construct permutations were used for rice transformation. We measured the accumulation of lactostatin expressed as a glutelin fusion protein in the mature seeds of 105 independent transgenic rice lines. A general correlation was observed between accumulation level and gene number in the vector constructs, indicating that a higher accumulation of lactostatin was obtained from transgenic rice plants containing the maximum number of gene inserts. These results indicate that this strategy is applicable for the selection of transgenic lines containing large amounts of bioactive peptides in rice seeds.     

Lipopolysaccharide accelerates collagen‐induced arthritis in association with rapid and continuous production of inflammatory mediators and anti‐type II collagen antibody

Collagen‐induced arthritis (CIA) is an animal model for rheumatoid arthritis (RA). Lipopolysaccharide (LPS) is known to accelerate CIA; however, the pathogenetic mechanisms are not yet fully understood. In this study, type II collagen (CII)‐immunized mice were found to have marked increases in degree of expression of mRNA of inflammatory mediators such as tumor necrosis factor alpha (TNF‐α), interleukin (IL)‐1β, and macrophage inflammatory protein‐2 (MIP‐2) in their arthritic paws and of serum anti‐CII antibody concentration before the onset of arthritis induced by LPS injection. The gene expression was rapid and continuous after direct activation of nuclear factor κB. The amounts of mRNA of TNF‐α, IL‐1β, and MIP‐2, as well as of matrix metalloproteinases and the receptor activator of nuclear factor κB ligand, increased with the development of arthritis, correlated positively with clinical severity and corresponded with histopathological changes. Moreover, anti‐TNF‐α neutralizing antibody inhibited the development of LPS‐accelerated CIA and a single injection of recombinant mouse TNF‐α induced increases in anti‐CII antibody concentrations, suggesting TNF‐α may contribute to the development of arthritis by both initiation of inflammation and production of autoantibodies. These data suggest that exacerbation of RA by LPS is associated with rapid and continuous production of inflammatory mediators and autoantibodies.     

Angiotensin II type 2 receptor correlates with therapeutic effects of losartan in rats with adjuvant‐induced arthritis

The angiotensin II type 1 receptor (AT1R) blocker losartan ameliorates rheumatoid arthritis (RA) in an experimental model. In RA, AT2R mainly opposes AT1R, but the mechanism by which this occurs still remains obscure. In the present study, we investigated the role of AT2R in the treatment of rats with adjuvant-induced arthritis (AIA) by losartan. Adjuvant-induced arthritis rats were treated with losartan (5, 10 and 15 mg/kg) and methotrexate (MTX; 0.5 mg/kg) in vivo from day 14 to day 28. Arthritis was evaluated by the arthritis index and histological examination. Angiotensin II, tumour necrosis factor-α, and VEGF levels were examined by ELISA. The expression of AT1R and AT2R was detected by western blot and immunohistochemistry analysis. After stimulation with interleukin-1β in vitro, the effects of the AT2R agonist {"type":"entrez-protein","attrs":{"text":"CGP42112","term_id":"874777115","term_text":"CGP42112"}}CGP42112 (10⁻⁸–10⁻⁵ M) on the chemotaxis of monocytes induced by 10% foetal calf serum (FCS) were analysed by using Transwell assay. Subsequently, the therapeutic effects of {"type":"entrez-protein","attrs":{"text":"CGP42112","term_id":"874777115","term_text":"CGP42112"}}CGP42112 (5, 10 and 20 μg/kg) were evaluated in vivo by intra-articular injection in AIA rats. After treatment with losartan, the down-regulation of AT1R expression and up-regulation of AT2R expression in the spleen and synovium of AIA rats correlated positively with reduction in the polyarthritis index. Treatment with {"type":"entrez-protein","attrs":{"text":"CGP42112","term_id":"874777115","term_text":"CGP42112"}}CGP42112 inhibited the chemotaxis of AIA monocytes in vitro, possibly because of the up-regulation of AT2R expression. Intra-articular injection with {"type":"entrez-protein","attrs":{"text":"CGP42112","term_id":"874777115","term_text":"CGP42112"}}CGP42112 (10 and 20 μg/kg) ameliorated the arthritis index and histological signs of arthritis. In summary, the present study strongly suggests that the up-regulation of AT2R might be an additional mechanism by which losartan exerts its therapeutic effects in AIA rats.     

Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different hydrogels: influence of collagen type II extracellular matrix on MSC chondrogenesis

Bone marrow mesenchymal stem cells (MSCs) are candidate cells for cartilage tissue engineering. This is due to their ability to undergo chondrogenic differentiation after extensive expansion in vitro and stimulation with various biomaterials in three-dimensional (3-D) systems. Collagen type II is one of the major components of the hyaline cartilage and plays a key role in maintaining chondrocyte function. This study aimed at analyzing the MSC chondrogenic response during culture in different types of extracellular matrix (ECM) with a focus on the influence of collagen type II on MSC chondrogenesis. Bovine MSCs were cultured in monolayer as well as in alginate and collagen type I and II hydrogels, in both serum free medium and medium supplemented with transforming growth factor (TGF) beta1. Chondrogenic differentiation was detected after 3 days of culture in 3-D hydrogels, by examining the presence of glycosaminoglycan and newly synthesized collagen type II in the ECM. Differentiation was most prominent in cells cultured in collagen type II hydrogel, and it increased in a time-dependent manner. The expression levels of the of chondrocyte specific genes: sox9, collagen type II, aggrecan, and COMP were measured by quantitative "Real Time" RT-PCR, and genes distribution in the hydrogel beads were localized by in situ hybridization. All genes were upregulated by the presence of collagen, particularly type II, in the ECM. Additionally, the chondrogenic influence of TGF beta1 on MSCs cultured in collagen-incorporated ECM was analyzed. TGF beta1 and dexamethasone treatment in the presence of collagen type II provided more favorable conditions for expression of the chondrogenic phenotype. In this study, we demonstrated that collagen type II alone has the potential to induce and maintain MSC chondrogenesis, and prior interaction with TGF beta1 to enhance the differentiation.     

白花蛇舌草对Ⅱ型胶原蛋白诱导的大鼠类风湿性关节炎的治疗作用

目的：探讨白花蛇舌草（HD）对Ⅱ型胶原蛋白诱导的大鼠类风湿性关节炎（CIA）的治疗作用。方法：取SD大鼠60只随机分为对照组10只、造模组50只,造模组于背部皮内注射Ⅱ型胶原乳剂建立CIA大鼠模型,采用大鼠关节炎评分法评价其模型是否复制成功。造模组再随机分为模型组、雷公藤多苷（GTW）6 mg/kg组、白花蛇舌草3、6、12 g/kg组,每组10只,每天灌胃给予相应的试剂。每周观察关节炎指数和痛阈,于连续干预28 d后,各组大鼠眼眶静脉取血,观察白细胞介素1β（IL-lβ）、肿瘤坏死因子-α（TNF-α）、前列腺素E₂（PGE₂）、核因子κB受体活化因子配体（RANKL）及护骨素（OPG）水平。结果：与对照组比较,模型组大鼠关节炎指数,血清IL-lβ、TNF-α、PGE₂、RANKL、OPG及RANKL/OPG水平均明显升高（P<0.05）,光照阈值明显降低（P<0.05）;与模型组比较,雷公藤多苷组、白花蛇舌草低、中、高剂量组关节炎指数,血清IL-lβ、TNF-α、PGE₂、RANKL、OPG及RANKL/OPG水平均明显降低（P<0.05）,光照阈值明显升高（P<0.05）。结论：白花蛇舌草可明显降低CIA模型大鼠关节炎指数,升高痛域阈值,下调IL-lβ、TNF-α、PGE₂、RANKL、OPG水平,从而有效的防治CIA。     

Localization of type II collagen in the lingual mucosa of rats during the morphogenesis of circumvallate papillae

Iwasaki, S., Aoyagi, H. and Yoshizawa, H. 2011. Localization of type II collagen in the lingual mucosa of rats during the morphogenesis of circumvallate papillae. —Acta Zoologica (Stockholm) 92 : 67–74. Immunoreactivity specific for type II collagen was recognized first in the mesenchymal connective tissue just beneath the circumvallate papilla placode in fetuses on E13. At this stage, most of the lingual epithelium was pseudostratified epithelium composed of one or two layers of cuboidal cells. However, the epithelium of the circumvallate papilla placode was composed of several layers of cuboidal cells. Immunoreactivity specific for type II collagen was detected mainly on the lamina propria just beneath the lingual epithelium of the rudiment of the circumvallate papilla in fetuses on E15 and on E17, and slight immunostaining was detected on the lamina propria around the rudiment. In fetuses on E19, immunoreactivity specific for type II collagen was widely and densely distributed on the connective tissue around the developing circumvallate papillae and on the connective tissue that surrounded the lingual muscle. Immunoreactivity specific for type II collagen was sparsely distributed on the lamina propria of central bulge. After birth, morphogenesis of the circumvallate papillae advanced gradually with the increase in size of the tongue. Immunoreactivity specific for type II collagen was distinctively distributed in the lamina propria around circumvallate papilla, in the central bulge, and in the connective tissue that surrounded the lingual muscle.     

Evaluating osteochondral defect repair potential of autologous rabbit bone marrow cells on type II collagen scaffold

The feasibility of using genipin cross-linked type II collagen scaffold with rabbit bone marrow mesenchymal stem cells (RBMSCs) to repair cartilage defect was herein studied. Induction of RBMSCs into chondrocytic phenotype on type II collagen scaffold in vitro was conducted using TGF-β 3 containing medium. After 3-weeks of induction, chondrocytic behavior, including marker genes expression and specific extracellular matrix (ECM) secretion, was observed. In the in vivo evaluation experiment, the scaffolds containing RBMSCs without prior induction were autologous implanted into the articular cartilage defects made by subchondral drilling. The repairing ability was evaluated. After 2 months, chondrocyte-like cells with lacuna structure and corresponding ECM were found in the repaired sites without apparent inflammation. After 24 weeks, we could easily find cartilage structure the same with normal cartilage in the repair site. In conclusion, it was shown that the scaffolds in combination of in vivo conditions can induce RBMSCs into chondrocytes in repaired area and would be a possible method for articular cartilage repair in clinic and cartilage tissue engineering.     

The effect of TGF-beta1 and beta-estradiol on glycosaminoglycan and type II collagen distribution in articular chondrocyte cultures

Articular cartilage extracellular matrix (ECM) plays a crucial role in regulating chondrocyte functions via cell-matrix interaction, cytoskeletal organization and integrin-mediated signaling. Factors such as interleukins, basic fibroblast growth factor (bFGF), bone morphogenic proteins (BMPs) and insulin-like growth factor (IGF) have been shown to modulate the synthesis of extracellular matrix in vitro. However, the effects of TGF-beta1 and beta-estradiol in ECM regulation require further investigation, although there have been suggestions that these factors do play a positive role. To establish the role of these factors on chondrocytes derived from articular joints, a study was conducted to investigate the effects of TGF-beta1 and beta-estradiol on glycosaminoglycan secretion and type II collagen distribution (two major component of cartilage ECM in vivo). Thus, chondrocyte cultures initiated from rabbit articular cartilage were treated with 10ng/ml of TGF-beta1, 10nM of beta-estradiol or with a combination of both factors. Sulphated glycosaminoglycan (GAG) and type II collagen levels were then measured in both these culture systems. The results revealed that the synthesis of GAG and type II collagen was shown to be enhanced in the TGF-beta1 treated cultures. This increase was also noted when TGF-beta1 and beta-estradiol were both used as culture supplements. However, beta-estradiol alone did not appear to affect GAG or type II collagen deposition. There was also no difference between the amount of collagen type II and GAG being expressed when chondrocyte cultures were treated with TGF-beta1 when compared with cultures treated with combined factors. From this, we conclude that although TGF-beta1 appears to stimulate chondrocyte ECM synthesis, beta-estradiol fails to produce similar effects. The findings of this study confirm that contrary to previous claims, beta-estradiol has little or no effect on chondrocyte ECM synthesis. Furthermore, the use of TGF-beta1 may be useful in future studies looking into biological mechanisms by which ECM synthesis in chondrocyte cultures can be augmented, particularly for clinical application.     

Effect of epigallocatechin-3-gallate on the increase in type II collagen accumulation in cartilage-like MSC sheets

With the aim to increase type II collagen content in the scaffold-free cartilage-like cell sheet using human bone marrow mesenchymal stem cells, we examined the effect of epigallocatechin-3-gallate (EGCG) addition to the chondrogenic medium for the cell sheet culture. The addition of EGCG (10 μM) increased the content of type II collagen 2-fold, while the addition did not markedly change the expression level of the genes encoding type II collagen and Sox 9. The reactive oxygen species level in the cells in cell sheets was thought to be too low to suppress the accumulation of type II collagen. On the other hand, the addition of EGCG markedly decreased both the matrix metalloproteinase-13 concentration in the supernatant of cell sheet culture and the type II collagen degradation activity in that supernatant. Taken together, EGCG may enhance the accumulation of type II collagen by suppressing type II collagen degradation.     

Genetic stability of an Escherichia coli strain engineered to produce a novel therapeutic DNA vaccine encoding chicken type II collagen for rheumatoid arthritis

The development of therapeutic DNA vaccines capable of recovering immunological tolerance through the induction of both CD4 + CD25 + FoxP3 + regulatory and CD3 + CD8 + C28-suppressor T cells, and/or inhibition of both autoreactive CD4 + CD28+ type 1 T helper and autoantibody-producing B cells offers a promising new strategy for the treatment of rheumatoid arthritis. Previously, we developed pcDNA-CCOL2A1, a novel therapeutic DNA vaccine, which encodes the full-length chicken type II collagen sequence, and demonstrated that the efficacy of this vaccine for treating rheumatoid arthritis was comparable to that of the current “gold standard” treatment, methotrexate. In this study, we investigated the genetic stability of a strain engineered to produce the vaccine during continuous passage and long-term storage at different temperatures. By screening a panel of 12 strains, we identified a DH5α strain that exhibited high levels (12.30 ± 0.05 mg L⁻¹) of pcDNA-CCOL2A1 production after 15 h cultivation, and subsequently utilized this strain to establish a three-tier cells bank for future studies. Continuous passage of this strain for 100 inoculation times demonstrated that a higher percentage (>95%) of cells maintained the plasmid when cultivated under selective pressure (ampicillin) than under nonselective conditions, suggesting that the presence of antibiotics in the medium prevents the loss of the pcDNA-CCOL2A1 plasmid. Meanwhile, restriction digestion and gene sequencing analyses demonstrated that the pcDNA-CCOL2A1 vector remained stable, and that the plasmid sequence was conserved during this period. Lastly, the DH5α pcDNA-CCOL2A1 strain exhibited a high plasmid preservation (>90%) and high levels of plasmid production (9.05mg L⁻¹) after storage for 60 months at −80 °C. Furthermore the plasmid extracted from the DH5α pcDNA-CCOL2A1 strain after storage for 60 months at −80 °C was transfected to COS-7 cells, it can stably express the target protein chicken type II collagen. Conversely, this strain exhibited a complete loss of capability after 24 and 18 months storage at −20 °C and 4 °C, respectively. These findings will facilitate further pilot-scale testing, and even industrial-scale production, of the novel therapeutic vaccine pcDNA-CCOL2A1.     

Regulatory effect of vasoactive intestinal peptide on the balance of Treg and Th17 in collagen-induced arthritis

Vasoactive intestinal peptide (VIP) is a well-known anti-inflammatory neuropeptide. The capacity of VIP can be exhibited through inhibiting inflammatory responses, shifting the Th1/Th2 balance in favor of anti-inflammatory Th2 immunity and inducing regulatory T cells (Tregs) with suppressive activity. In addition to pro-inflammatory Th1 response, Th17 are also believed to play important roles in the pathogenesis of rheumatoid arthritis (RA). In this study, we used collagen-induced arthritis (CIA) model in Wistar rats to investigate the role of VIP in the balance of CD4⁺ CD25⁺ Tregs and Th17 on RA. Data presented here showed that administration of VIP decreased incidence and severity of CIA. Disease suppression was associated with the upregulation of CD4⁺ CD25⁺ Tregs, downregulation of Th17- and Th1-type response and influence on the RANK/RANKL/OPG system. The results provide novel evidence that the therapeutic effects of VIP on CIA rats were associated with the balance of CD4⁺ CD25⁺ Tregs and Th17.     

Online immunoaffinity liquid chromatography/tandem mass spectrometry determination of a type II collagen peptide biomarker in rat urine: Investigation of the impact of collision-induced dissociation fluctuation on peptide quantitation

Proteolytic fragments of type II collagen, a major component of joint tissue, have recently been identified as biomarkers for osteoarthritis, a progressive disease associated with cartilage degeneration. A liquid chromatography/tandem mass spectrometry (MS/MS) assay that utilizes online immunoaffinity chromatography and column switching was developed in our laboratory for the neoepitope of type II collagen (NET2C). During method development, peptide collision-induced dissociation (CID) was found to be a significant source of assay variation, which exceeded 10% CV, despite the fact that a stable-isotope-labeled (SIL) internal standard was used to minimize imprecision. This phenomenon was studied in detail using peptides and associated SIL internal standards of varying lengths and amino acid compositions. Variability in peptide CID necessitated the monitoring of multiple MS/MS transitions to obtain acceptable assay precision. The assay was subsequently validated to measure NET2C concentrations in rat urine over the range of 0.1 to 10 ng/mL. The interday accuracy and precision ranged from 3.9 to 13.1 (%CV) and 10.7 to 5.3 (%RE), respectively, across the range of validated concentrations. A specific application of the assay is presented in which the role of estrogen deficiency in the development and progression of osteoarthritis was investigated. In this study, the effect of estrogen on lowering NET2C concentrations in urine in ovariectomized rats was demonstrated.     

Immunofluorescence localization of the regulatory subunit type II of cAMP-dependent protein kinase in PC12 and 3T3 cells in different proliferative states

Localization of the regulatory subunit of cAMP-dependent protein kinase type II was studied in proliferating and quiescent fibroblasts 3T3 and in a cell line of neural origin pheochromocytoma PC12. In actively proliferating PCl2 cells the regulatory subunit was found to be localized in the nucleus. Transition of these cells into a quiescent state was accompanied by a regulatory subunit translocation to the cytoplasm. In 3T3 cells the regulatory subunit was localized in the cytoplasm both in the quiescent and proliferating (though less actively than PC12 cells) states. Similar results were obtained both with monoclonal antibodies and with rabbit monospecific antiserum raised against the regulatory subunit type II from pig brain.