The utility of IgG,IgM, and CD138 immunohistochemistry in the evaluation of autoimmune liver diseases

Primary biliary cirrhosis (PBC) and autoimmune hepatitis (AIH) present with distinct clinical features. The term “PBC–AIH overlap syndrome (OS)” has been adopted to describe the condition characterized by occurrence of both PBC and AIH, although this clinical entity is difficult to define. This study aimed to assess the utility of IgG, IgM, and CD138 immunohistochemistry in the evaluation of AIH, PBC, and OS. Immunohistochemistry was performed with anti-human IgG, IgM, and CD138 to detect specific plasma cells in the liver. Predominant IgG staining was observed in AIH (85.7 %), while equivocal (46.1 %) or predominant (38.5 %) IgM staining was observed in PBC. In OS, equivocal (20 %) or predominant (80 %) IgG staining was observed. The IgM/IgG ratio was significantly higher in PBC than in AIH or OS (P < 0.005). Histological findings revealed significantly higher IgM expression in PBC at cholangitis activity grades 2–3 compared to those at cholangitis activity grades 0–1. In contrast, a significantly higher IgG expression was observed in PBC at hepatitis activity and fibrosis grades 2–3 compared to those at hepatitis activity and fibrosis grades 0–1. Taken together, periportal plasmacytic infiltrates with variable immunohistochemistry patterns of IgG and IgM expression characterized different autoimmune liver diseases.     

Expression of human T cell immunoglobulin domain and mucin-3 on kidney tissue from immunoglobulin A nephropathy patients

The aim of this study was to evaluate the expression of human T cell immunoglobulin domain and mucin-3 (Tim-3) in renal tissue from patients with immunoglobulin A nephropathy (IgAN) and without IgAN and to evaluate the difference in Tim-3 expression between them. A total of 71 patients with IgAN as IgA group and 13 patients without IgAN as control group were enrolled in the present study. Patients in IgAN accepted percutaneous renal biopsy. We examined the expression of Tim-3 in renal tissue and the serological parameters in serum from all enrolled cases. The expression of Tim-3 and serological parameters were compared between the different groups. Positive staining of Tim-3 protein was seen in 94.3 % patients with IgAN (67 out of 71), but only 15.4 % (2 out of 13) in the cases without IgAN were positive staining of Tim-3. There were significant differences between two groups in almost all serological markers, which reflect IgAN activity. There was a nearly positive correlation between pathological manifestations and expression degree of Tim-3. High immuno-reactivity of Tim-3 was found to be significantly correlated with serological grade (p < 0.001) in IgA group, but there was no such phenomenon in control group. The results showed that there was the expression of Tim-3 in renal tissue from the patients with IgAN, but rarely expression in cases without IgAN. Expression of Tim-3 was associated with the diseases’ activity.     

Decline of IgG pertussis toxin measured in umbilical cord blood,and neonatal and early infant serum

Maternal pertussis-specific antibodies are passively acquired by infants during pregnancy. An IgG pertussis toxin (IgG-PT) concentration of >20 U/ml is considered to protect neonates against pertussis. To evaluate the IgG concentration at birth and during the first two months of life, we examined the IgG-PT concentration in the umbilical cord blood and three times during the neonatal and early infant period. IgG-PT was measured by validated IgG-specific enzyme-linked immunosorbent assays (ELISA) in umbilical cord blood and in Guthrie card blood samples of umbilical cord blood in 2,790 children, born between 1 August 2006 and 1 December 2008. These measurements were comparable. All children with concentrations of IgG-PT >30 U/ml were included. IgG-PT was also measured in Guthrie card blood samples, when the neonates or early infants were 5 days, 1 month and 2 months old. The mean concentrations of IgG-PT were calculated. The mean concentration of IgG-PT in umbilical cord blood was 60.1 U/ml (LN 4.1; 0.6 SD; n?=?103). At the age of 5 days, 1 month and 2 months, the mean concentration of IgG-PT was 40.6 U/ml (LN 3.7; 0.5 SD; n?=?103), 20.7 U/ml (LN 3.0; 0.7 SD; n?=?62) and 16.7 U/ml (LN 2.8; 0.9 SD; n?=?61), respectively. Four percent of the neonates had a concentration of IgG-PT >30 U/ml in umbilical cord blood, which declined to levels around the concentration needed for protection against pertussis (>20 U/ml) in the first two months of life. Hence, it is of great importance to further investigate the safety of maternal immunisation during pregnancy to prevent life-threatening pertussis in newborns.     

Differential RNAi responses of Nicotiana benthamiana individuals transformed with a hairpin-inducing construct during Plum pox virus challenge

Gene silencing and large-scale small RNA analysis can be used to develop RNA interference (RNAi)-based resistance strategies for Plum pox virus (PPV), a high impact disease of Prunus spp. In this study, a pPPViRNA hairpin-inducing vector harboring two silencing motif-rich regions of the PPV coat protein (CP) gene was evaluated in transgenic Nicotiana benthamiana (NB) plants. Wild-type NB plants infected with a chimeric PPV virus (PPV::GFP) exhibited affected leaves with mosaic chlorosis congruent to GFP fluorescence at 21 day post-inoculation; transgenic lines depicted a range of phenotypes from fully resistant to susceptible. ELISA values and GFP fluorescence intensities were used to select transgenic-resistant (TG-R) and transgenic-susceptible (TG-S) lines for further characterization of small interfering RNAs (siRNAs) by large-scale small RNA sequencing. In infected TG-S and untransformed (WT) plants, the observed siRNAs were nearly exclusively 21- and 22-nt siRNAs that targeted the whole PPV::GFP genome; 24-nt siRNAs were absent in these individuals. Challenged TG-R plants accumulated a full set of 21- to 24-nt siRNAs that were primarily associated with the selected motif-rich regions, indicating that a trans-acting siRNAs process prevented viral multiplication. BLAST analysis identified 13 common siRNA clusters targeting the CP gene. 21-nt siRNA sequences were associated with the 22-nt siRNAs and the scarce 23- and 24-nt molecules in TG-S plants and with most of the observed 22-, 23-, and 24-nt siRNAs in TG-R individuals. These results validate the use of a multi-hot spot silencing vector against PPV and elucidate the molecules by which hairpin-inducing vectors initiate RNAi in vivo.     

Temporal evolution of human autoantibody response to cytoplasmic rods and rings structure during anti-HCV therapy with ribavirin and interferon-α

Autoantibodies to inosine monophosphate dehydrogenase-2 (IMPDH2), an enzyme involved in de novo biosynthesis of guanine nucleotides, are observed in a subset of hepatitis C virus (HCV) patients receiving interferon alpha (IFN-α) plus ribavirin. Anti-IMPDH2 antibodies display a peculiar cytoplasmic “rod/ring” (RR) pattern in IIF-HEp-2. We examined the dynamics of anti-RR autoimmune response with respect to immunoglobulin isotypes, titer, avidity, and protein targets in 80 sequential samples from 15 HCV patients (plus 12 randomly selected anti-RR-positive, totalizing 92 samples) collected over an 18-month period, including samples collected before, during, and after IFN-α + ribavirin treatment. Immunoprecipitation showed reactivity with the 55 kDa IMPDH2 protein in 12/15 patients (80 %) and 11/15 (73 %) reacted with IMPDH2 in a sandwich ELISA. During treatment, anti-IMPDH2 autoantibodies hit their highest levels after 6–12 months of treatment and decreased post-treatment, while anti-HCV antibodies levels were stable over time. Anti-IMPDH2 IgM levels increased up until the sixth month of treatment and remained stable thereafter, while IgG levels increased steadily up to the twelfth month. Both IgG and IgM decreased during the post-treatment period. IgG avidity increased steadily up to the twelfth month of treatment. In conclusion, this study showed that the temporal kinetics of IFN-α + ribavirin-induced humoral autoimmune response to IMPDH2 exhibited a considerably delayed pace of increase in antibody levels and avidity as well as in isotype class switch in comparison with a conventional humoral response to infectious agents. These unique findings uncover intriguing differences between the autoimmune response and the immune response to exogenous agents in humans.     

The parasitic fauna of the European bison (Bison bonasus) (Linnaeus, 1758) and their impact on the conservation. Part 1 The summarising list of parasites noted

During the current century, 88 species of parasites have been recorded in Bison bonasus. These are 22 species of protozoa (Trypanosoma wrublewskii, T. theileri, Giardia sp., Sarcocystis cruzi, S. hirsuta, S. hominis, S. fusiformis, Neospora caninum, Toxoplasma gondii, Cryptosporidium sp., Eimeria cylindrica, E. subspherica, E. bovis, E. zuernii, E. canadensis, E. ellipsoidalis, E. alabamensis, E. bukidnonensis, E. auburnensis, E. pellita, E. brasiliensis, Babesia divergens), 4 trematodes species (Dicrocoelium dendriticum, Fasciola hepatica, Parafasciolopsis fasciolaemorpha, Paramphistomum cervi), 4 cestodes species (Taenia hydatigena larvae, Moniezia benedeni, M. expansa, Moniezia sp.), 43 nematodes species (Bunostomum trigonocephalum, B. phlebotomum, Chabertia ovina, Oesophagostomum radiatum, O. venulosum, Dictyocaulus filaria, D.viviparus, Nematodirella alcidis, Nematodirus europaeus, N. helvetianus, N. roscidus, N. filicollis, N. spathiger, Cooperia oncophora, C. pectinata, C. punctata, C. surnabada, Haemonchus contortus, Mazamastrongylus dagestanicus, Ostertagia lyrata, O. ostertagi, O. antipini, O. leptospicularis, O. kolchida, O. circumcincta, O. trifurcata, Spiculopteragia boehmi, S. mathevossiani, S. asymmetrica, Trichostrongylus axei, T. askivali, T. capricola, T. vitrinus, Ashworthius sidemi, Onchocerca lienalis, O. gutturosa, Setaria labiatopapillosa, Gongylonema pulchrum, Thelazia gulosa, T. skrjabini, T. rhodesi, Aonchotheca bilobata, Trichuris ovis), 7 mites (Demodex bisonianus, D. bovis, Demodex sp., Chorioptes bovis, Psoroptes equi, P. ovis, Sarcoptes scabiei), 4 Ixodidae ticks (Ixodes ricinus, I. persulcatus, I. hexagonus, Dermacentor reticulatus), 1 Mallophaga species (Bisonicola sedecimdecembrii), 1 Anoplura (Haematopinus eurysternus), and 2 Hippoboscidae flies (Lipoptena cervi, Melophagus ovinus). There are few monoxenous parasites, many typical for cattle and many newly acquired from Cervidae.     

Endogenous endothelin 1 mediates angiotensin II-induced hypertrophy in electrically paced cardiac myocytes through EGFR transactivation,reactive oxygen species and NHE-1

Emerging evidence supports a key role for endothelin-1 (ET-1) and the transactivation of the epidermal growth factor receptor (EGFR) in angiotensin II (Ang II) action. We aim to determine the potential role played by endogenous ET-1, EGFR transactivation and redox-dependent sodium hydrogen exchanger-1 (NHE-1) activation in the hypertrophic response to Ang II of cardiac myocytes. Electrically paced adult cat cardiomyocytes were placed in culture and stimulated with 1 nmol l^-1 Ang II or 5 nmol l^-1 ET-1. Ang II increased ~45 % cell surface area (CSA) and ~37 % [³H]-phenylalanine incorporation, effects that were blocked not only by losartan (Los) but also by BQ123 (AT₁ and ET_A receptor antagonists, respectively). Moreover, Ang II significantly increased ET-1 messenger RNA (mRNA) expression. ET-1 similarly increased myocyte CSA and protein synthesis, actions prevented by the reactive oxygen species scavenger MPG or the NHE-1 inhibitor cariporide (carip). ET-1 increased the phosphorylation of the redox-sensitive ERK1/2-p90^RSK kinases, main activators of the NHE-1. This effect was prevented by MPG and the antagonist of EGFR, AG1478. Ang II, ET-1 and EGF increased myocardial superoxide production (187?±?9 %, 149?±?8 % and 163.7?±?6 % of control, respectively) and AG1478 inhibited these effects. Interestingly, Los inhibited only Ang II whilst BQ123 cancelled both Ang II and ET-1 actions, supporting the sequential and unidirectional activation of AT₁, ET_A and EGFR. Based on the present evidence, we propose that endogenous ET-1 mediates the hypertrophic response to Ang II by a mechanism that involves EGFR transactivation and redox-dependent activation of the ERK1/2-p90^RSK and NHE-1 in adult cardiomyocytes.     

An identification key for the five most common species of Metastrongylus

Species of the Metastrongylus genus, the lung nematodes of pigs that require an intermediate host (earthworm) to complete their cycle, pose a potential risk to both livestock and humans. This parasite which can result in lung pathology and mixed infections with other pathogens (e.g. viruses) can be fatal to pigs. Although this genus is distributed worldwide, there are no classification keys for identifying this common parasite species. In this work, we take advantage of parasitological surveys of wild boar (Sus scrofa) in northern and central Spain and southern Poland to develop a morphological identification key for the five most common Metastrongylus species (Metastrongylus apri, Metastrongylus pudendotectus, Metastrongylus salmi, Metastrongylus confusus and Metastrongylus asymetricus). In addition, we provide the first record of M. confusus in Spain, probably unidentified until now due to the lack of appropriate identification keys. We hope that this user-friendly identification key will enable parasitologists and veterinary practitioners to avoid further misclassifications of Metastrongylus species.     

microRNA expression and its potential role in cardioprotection by ischemic postconditioning in pigs

Ischemic postconditioning (PoCo) reduces infarct size following myocardial ischemia/reperfusion. To protect, PoCo must be performed early during reperfusion, and causal cardioprotective signaling must occur then. The role of microRNA (miRNA) in PoCo is unclear. Anesthetized pigs were subjected to 60 min left anterior descending coronary artery (LAD) occlusion and 180 min reperfusion. Immediate full reperfusion (IFR, n?=?5) was compared to PoCo (four cycles of 60 s/60 s reperfusion/reocclusion, n?=?5). Transmural myocardial biopsies from the LAD territory were sampled at baseline, 60 min ischemia, 10 and 180 min reperfusion. RNA was isolated. The expression of 11 miRNAs, including muscle-specific (miRNA-1, -133a, -206, -208b, -214, and -499), fibrosis- (miRNA-21, -24, and -29b), neovascularization- (miRNA-92a), and inflammation-associated (miRNA-146b) candidates, was quantified using real-time PCR (RT-PCR). mRNA expression at baseline and 180 min reperfusion was quantified and validated (microarray and RT-PCR). PoCo reduced infarct size from 44.9?±?7.7 to 34.8?±?5.3 % of the area at risk. The expression of miRNA-1, -24, -29b, -133a, -146b, -208b, and -499 was increased at 10 min reperfusion with PoCo vs. IFR; however, that of miRNA-1, -24, -208b, and -499 was already increased at 60 min ischemia and probably reflects falsely positive results. Five mRNAs were different with PoCo vs. IFR. In silico analysis identified a tentative connection between three miRNAs and five mRNAs with the biological functions “cell death”, “inflammatory response” and/or “glucose metabolism”. If at all, only miRNA-29b, -133a, and -146b fulfill the minimal temporal requirements for a potential causal involvement in cardioprotection by PoCo.     

Early Systemic Sclerosis: Serum Profiling of Factors Involved in Endothelial,T-cell,and Fibroblast Interplay is Marked by Elevated Interleukin-33 Levels

Purpose

To assess the serum profile of factors involved in endothelial, T-cell, and fibroblast interplay in patients with Raynaud’s phenomenon (RP) associated with nailfold vodeocapillaroscopy (NVC) scleroderma findings and/or systemic sclerosis (SSc) marker autoantibodies, recently labeled as early SSc patients.

Methods

Serum levels of soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular adhesion molecule-1 (sVCAM-1), CCL2, CXCL8, IL-13, IL-33, and transforming growth factor-β (TGF-β) were measured in 24 early SSc patients, 48 definite SSc patients, and 24 osteoarthritis/fibromyalgia controls by multiplex suspension immunoassay. All SSc patients were investigated for the presence/absence of preclinical and clinical organ involvement, SSc marker autoantibodies, and NVC abnormalities.

Results

Serum sICAM-1, CCL2, CXCL8, and IL-13 were increased in all SSc patients as compared to controls, and paralleled the severity of the disease subset (early SSc < limited cutaneous SSc < diffuse cutaneous SSc; p?SSc patients as compared to both controls (p?SSc patients (p?SSc there were no differences in the investigated markers according to the functional and serological features assessed.

Conclusions

Our study suggests that an endothelial, T-cell and fibroblast activation can be present in patients with early SSc and it is associated with a distinct profile of circulating factors involved in the cross-talk of these cells. The marked increase of IL-33 in early SSc patients suggests new routes of investigation of cell-cell dynamics in target tissues predating overt disease manifestations, thus opening to new therapeutic approaches.     

Analytical issues possibly affecting the performance of commercial human cytomegalovirus IgG avidity assays

In the majority of cytomegalovirus (CMV) immunoglobulin G (IgG) avidity assays, avidity determination can be performed on the entire CMV IgG measurable positive concentration range. However, in some exceptional samples with very low IgG levels, inappropriately low avidity indexes have been described. In this study, we addressed some possible causes and the clinical importance of these inappropriately low avidity indexes. We compared VIDAS (bioMérieux), Liaison (DiaSorin), and Architect (Abbott) CMV IgG avidity assays on 129 samples from patients with past CMV infections, focusing on samples with low IgG levels. Inappropriately low avidity samples were further evaluated using seven different urea-based IgG avidity assays. We confirmed that inappropriately low avidity indexes in samples with very low IgG levels occur, but are rare. We could show that this phenomenon is not confined to a single assay and that assays employing chaotropic agents are affected more frequently and profoundly. In situations where the CMV IgG avidity is performed on CMV immunoglobulin M (IgM)-negative samples, the avidity index should be interpreted cautiously in cases of very low CMV IgG levels, whatever the technique used.     

Increased levels of anti-heat-shock protein 60 (anti-Hsp60) indicate endothelial dysfunction,atherosclerosis and cardiovascular diseases in patients with mixed connective tissue disease

Heat-shock protein 60 (Hsp60) has been shown to provoke inflammation, and anti-Hsp60 may facilitate the development of atherosclerosis. In this study, we have investigated 30 patients with mixed connective tissue disease (MCTD) and assessed anti-Hsp60 and their relationship to cardiovascular diseases (CVD). Out of 30 patients with MCTD, 15 had CVDs. Anti-Hsp60 antibody was determined by enzyme-linked immunosorbent assay. Since endothelial dysfunction and accelerated atherosclerosis are characteristic to MCTD, a wide array of MCTD-, endothelial dysfunction- and CVD-associated parameters was investigated: serum lipid levels, paraoxonase activity (PON1), rich nuclear ribonucleoprotein U1 (anti-U1RNP), anti-endothelial cell antibodies, anti-cardiolipin and anti-β2-glycoprotein I antibody isotypes (anti-CL and anti-β2GPI), endothelin-1 (ET-1) levels, also intima–media thickness (IMT), a quantitative indicator of atherosclerosis. In MCTD, anti-Hsp60 antibody levels were significantly higher than in healthy individuals (p < 0.02). MCTD patients with CVD had significantly higher levels of anti-Hsp60 compared to MCTD without CVD (p = 0.001). Patients with MCTD had significantly lower high-density lipoprotein cholesterol (p = 0.02) and PON activity (p < 0.001), and significantly increased systolic (p < 0.0002) and diastolic (p < 0.001) blood pressure compared to healthy individuals. Anti-U1RNP levels (p < 0.002) and IMT were higher in patients compared to controls (p = 0.002). The CVD-positive MCTD patients had increased anti-Hsp60 (p < 0.0013), anti-CL IgG (p = 0.0005), ET-1 serum concentration (p < 0.05) and IMT levels (p < 0.001) compared to MCTD patients without CVD. Anti-Hsp60 showed a strong correlation with anti-oxLDL (r = 0.36, p = 0.01) and serum ET-1 (r = 0.62, p < 0.001) and negative correlation with PON activity (r = ?0.47, p = 0.01). Anti-Hsp60 indicates endothelial injury, CVD, and can function as a novel atherosclerotic risk factor, also a valuable diagnostic marker in patients with MCTD.     

Diagnostic potential of recombinant nonstructural protein 3B to detect antibodies induced by foot-and-mouth disease virus infection in bovines

Detection of antibodies to nonstructural proteins (NSP) of foot-and-mouth disease virus is the preferred diagnostic method to differentiate infected from vaccinated animals. In India, an endemic region practising preventive biannual vaccination, 3AB3 indirect ELISA (r3AB3 I-ELISA) has been employed as the primary screening test for serosurveillance. However, because of the variability observed in the immune response to the NSPs, the likelihood of detecting or confirming an infected animal is increased if an antibody profile against multiple NSPs is considered for diagnosis. In this study, all three copies of NSP 3B were expressed in a prokaryotic system to develop an indirect ELISA (r3B I-ELISA). At the decided cutoff of 40 percent positivity, the diagnostic sensitivity and specificity of the r3B I-ELISA were estimated to be 92.1 % (95 % CI: 89.0–94.5) and 98.1 % (95 % CI: 96.9–98.8), respectively, as compared to 97.04 % and 95.04 % for r3AB3 I-ELISA. Although r3B I-ELISA displayed lower sensitivity compared to the screening assay, which could possibly be attributed to additional relevant B-cell epitopes in the carboxy-terminal half of the 3A protein, the former achieved considerably higher specificity on repeatedly vaccinated animals. NSP antibodies could be detected from 10 to as late as 998 days postinfection in experimental calves. Substantial agreement in the test results (90.6 %) was found between the two ELISAs. The r3B I-ELISA, when used in conjunction with the r3AB3 I-ELISA as an integrated system, can potentially augment the efficiency and confidence of detection of infected herds against the backdrop of intensive vaccination.     

Histopathological characterization of ductal carcinoma in situ (DCIS) of the breast according to HER2 amplification status and molecular subtype

This study aimed to characterize ductal carcinoma in situ (DCIS) according to human epidermal growth factor receptor 2 (HER2) amplification status and molecular subtype. In addition, we performed a detailed HER2 and CEP17 copy number analysis and we assessed the impact of recent changes in the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines on HER2 immunohistochemical (IHC) scores in DCIS. Nuclear grade, extensive comedonecrosis, stromal architecture, stromal inflammation, and progesterone receptor (PR) expression were significantly associated with HER2 amplification status. In multivariate analysis, stromal inflammation and extensive comedonecrosis were the only two features that remained significantly related to HER2 amplification status. The recent changes in ASCO/CAP guidelines resulted in significant upgrading of HER2 IHC score. Remarkably, about one in five non-amplified DCIS presented a 3+ IHC score, regardless of the scoring method. The biological significance of this phenomenon is presently unknown. After categorization according to molecular subtype, luminal A DCIS mainly presented histopathological features associated with good prognosis, whereas luminal B/HER2+ and HER2+ categories displayed a more aggressive phenotype. Overall, our results demonstrate that HER2-amplified DCIS constitute a clearly distinct subgroup which is characterized by histopathological features associated with poor prognosis. Further studies are required to elucidate the biological significance of a 3+ IHC score in non-amplified DCIS, as well as its mechanism.     

An analysis and decision tool to measure cost benefit of newborn screening for severe combined immunodeficiency (SCID) and related T-cell lymphopenia

Severe combined immunodeficiency (SCID) is a group of syndromes resulting from genetic defects causing absence in T-cell and B-cell function, leading to serious and life-threatening infections. SCID is often fatal in the first 2 years of life if not identified and properly treated. While additional laboratory methods are being developed, the current T-cell receptor excision circle assay has proven to have outstanding specificity and sensitivity to accurately identify infants with SCID and other T-cell lymphopenia. The Jeffrey Modell Foundation (JMF) has a long history of advocacy and continues to promote newborn screening for SCID to be implemented in the United States and worldwide. Based on reports provided by California, New York, Texas, and Wisconsin on the results of their population based newborn screening programs, the overall incidence of SCID averaged 1:33,000 and T-cell lymphopenia averaged 1:6,600. JMF has developed a working algorithm or “decision tree”, validated by peer-reviewed scientific literature, to be used by Public Health Departments and Health Ministries in states, countries, and regions throughout the world. This decision tool allows for local or regional data to be applied to measure the threshold and economic impact of implementing newborn screening for SCID and T-cell lymphopenia.     

The Chemokine CX3CL1 (Fractalkine) and its Receptor CX3CR1: Occurrence and Potential Role in Osteoarthritis

Chemokines are molecules able to induce chemotaxis of monocytes, neutrophils, eosinophils, lymphocytes and fibroblasts. The complex chemokine acts in many physiological and pathological phenomena, including those occurring in the articular cartilage. To date, chemokine CX3CL1 (fractalkine) is the only member of the CX3C class of chemokines with well-documented roles in endothelial cells. CX3CL1 is a unique chemokine that combines properties of chemoattractant and adhesion molecule. The main roles of CX3CL1 include promotion of leukocyte binding and adhesion as well as activation of the target cells. The soluble chemokine domain of CX3CL1 is chemotactic for T cells and monocytes. CX3CL1 acts via its receptor, CX3CR1, which belongs to a family of G protein-coupled receptors. Stimulation of CX3CR1 activates both CX3CL1-dependent and integrin-dependent migrations of cells with synergistically augmented adhesion. Genetic polymorphisms of CX3CR1 may significantly modify the biological roles of CX3CL1, especially in pathologic conditions. Osteoarthritis (OA) is the most common joint disease, affecting approximately 7–8 % of the general population. Development of OA is largely driven by low-grade local background inflammation involving chemokines. The importance of CX3CL1/CX3CR1 signalling in the pathophysiology of OA is still under investigation. This paper, based on a review of the literature, updates and summarises the current knowledge about CX3CL1/CX3CR1 in OA and indicates possible interactions with a potential for therapeutic targeting.     

Preservation of nitric oxide-induced relaxation of porcine coronary artery: roles of the dimers of soluble guanylyl cyclase,phosphodiesterase type 5, and cGMP-dependent protein kinase

Soluble guanylyl cyclase (sGC), phosphodiesterase type 5 (PDE5), and guanosine 3′,5′-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) are all dimeric. The present study was to determine the role of their dimeric status in nitric oxide-induced vasodilatation. In isolated porcine coronary arteries, after 20 h incubation with serum-free medium, serum-containing medium, or phosphate-buffered saline solution, the protein levels of the dimers of sGC, PDE5, and PKG were diminished while the monomer levels remained unchanged, associated with reduced cGMP elevation in response to DETA NONOate and decreased PDE5 activity; the activity of PKG was not significantly altered. DETA NONOate caused a greater relaxation in arteries incubated for 20 vs. 2 h. The relaxant response was largely abolished by 1H-[1, 2, 4]oxadiazolo[4,3-a]quinoxalin-1-one, an sGC inhibitor. Zaprinast, a PDE5 inhibitor, had no effect on relaxation caused by DETA NONOate of arteries incubated for 20 h but augmented the response incubated for 2 h. A greater relaxation to 8-bromo-guanosine 3′5′-cyclic monophosphate occurred in arteries incubated for 20 than for 2 h. The protein level of the dimers but not monomers of PDE5 was reduced by dithiothreitol and unaffected by hydrogen peroxide, accompanied with decreased PDE5 activity and reduced response to DETA NONOate. These results demonstrate that the dimeric but not monomeric status of sGC and PDE5 of coronary arteries are closely related to their activities. The preserved vasodilator response after 20 h incubation may result in part from a synchronous reduction of the dimer levels of sGC and PDE5 as well as an augmented response to cGMP.     

Expression,cloning, and IgE-binding of the full-length dust mite allergen Der f 8

Dermatophagoides farinae, a domestic mite species, produces some of the most potent allergens that contribute to allergy in China and worldwide. We sought to clone and express the group 8 allergen of D. farinae (Der f 8) to investigate its IgE-binding reactivity. The full-length cDNA encoding Der f 8 was generated by using RT-PCR and 5′ RACE, cloned into pCold-TF expression vector, confirmed by nucleotide sequencing, sub-cloned into pET-28b (+), and transfected into E. coli BL21 cells for expression. After purification by nickel affinity chromatography and identified by SDS-PAGE, the recombinant Der f 8 bound with sera from 40.9 % (9/22) of mite-allergic patients according to ELISA testing. Analysis of the recombinant DNA sequence revealed a 231 amino acid open reading frame encoding a protein with a derived molecular mass of 26.4 kDa and an isoelectric point of 6.84. The deduced amino acid sequence has nine phosphorylation sites, displaying strong homology with glutathione S-transferase, and its secondary structure comprises alpha helix (45.5 %), extended strand (11.3 %), and random coils (43.3 %). BLAST through the National Center for Biotechnology Information database and alignment identified similarity with group 8 allergens or glutathione S-transferases of Dermatophagoides pteronyssinus, Suidasia medanensis, Lepidoglyphus destructor, Glycyphagus domesticus, and Aleuroglyphus ovatus (64, 65, 53, 53, and 50 %, respectively). The first recombinant Der f 8 protein produced in full length successfully bound with patient IgE, demonstrating the importance of Der f 8 in mite allergy.