Studies to explore angiotensin II (Ang II) and its downstream signaling pathways via Rho guanine nucleotide exchange factors (RhoGEFs) and RhoA signaling are crucial to understanding the mechanisms of smooth muscle contraction leading to hypertension. This study aimed to investigate the Ang II–induced expression of RhoGEFs in vascular smooth muscle cells (VSMCs) of spontaneously hypertensive rats (SHRs) and to identify the possible regulator associated with hypertension.
Cultured VSMCs of the aorta from SHRs and Wistar-Kyoto (WKY) rats were treated with or without Ang II or Ang II plus Ang II type 2 receptor antagonists. The expression levels of RhoGEF messenger RNA (mRNA) and protein were determined. To evaluate the changes of aortic ring contractile force in response to Ang II, a nonviral carrier system was adopted to deliver the leukemia-associated RhoGEF (LARG) small interfering RNA via nanoparticles into aortic rings.
The baseline mRNA levels of three RhoGEFs in cultured VSMCs of WKY rats did not increase with age, but they were significantly higher in 12-week-old SHRs than in 5-week-old SHRs. Expression levels of LARG mRNA were higher in SHRs than in age-matched WKY rats. The baseline LAGR protein of 12-week-old SHRs was about four times higher than that of WKY rats of the same age. After Ang II–stimulation, LAGR protein expression was significantly increased in 12-week-old WKY rats but remained unchanged in 12-week-old SHRs. LARG small interfering RNA was successfully delivered into aortic rings using nanoparticles. LARG knockdown resulted in 12-week-old SHRs showing the greatest reduction in aortic ring contraction.
There were differences in age-related RhoGEF expression at baseline and in response to Ang II–stimulation between SHRs and WKY rats in this study. Nanotechnology can assist in studying the silencing of LARG in tissue culture. The findings of this study indicate that LARG gene expression may be associated with the genesis of hypertension in SHRs.
Rho guanine nucleotide exchange factor; leukemia-associated Rho guanine nucleotide exchange factor; Wistar-Kyoto rats; nanoparticle delivery; hypertension
Leukocyte infiltration plays an important role in the pathogenesis and progression of myositis, and is highly associated with disease severity. Currently, there is a lack of: efficacious therapies for myositis; understanding of the molecular features important for disease pathogenesis; and potential molecular biomarkers for characterizing inflammatory myopathies to aid in clinical development.
In this study, we developed a simple model and predicted that 1) leukocyte-specific transcripts (including both protein-coding transcripts and microRNAs) should be coherently overexpressed in myositis muscle and 2) the level of over-expression of these transcripts should be correlated with leukocyte infiltration. We applied this model to assess immune cell infiltration in myositis by examining mRNA and microRNA (miRNA) expression profiles in muscle biopsies from 31 myositis patients and 5 normal controls.
Several gene signatures, including a leukocyte index, type 1 interferon (IFN), MHC class I, and immunoglobulin signature, were developed to characterize myositis patients at the molecular level. The leukocyte index, consisting of genes predominantly associated with immune function, displayed strong concordance with pathological assessment of immune cell infiltration. This leukocyte index was subsequently utilized to differentiate transcriptional changes due to leukocyte infiltration from other alterations in myositis muscle. Results from this differentiation revealed biologically relevant differences in the relationship between the type 1 IFN pathway, miR-146a, and leukocyte infiltration within various myositis subtypes.
Results indicate that a likely interaction between miR-146a expression and the type 1 IFN pathway is confounded by the level of leukocyte infiltration into muscle tissue. Although the role of miR-146a in myositis remains uncertain, our results highlight the potential benefit of deconvoluting the source of transcriptional changes in myositis muscle or other heterogeneous tissue samples. Taken together, the leukocyte index and other gene signatures developed in this study may be potential molecular biomarkers to help to further characterize inflammatory myopathies and aid in clinical development. These hypotheses need to be confirmed in separate and sufficiently powered clinical trials.
Myositis; Genomics; Leukocyte infiltration; Type 1 interferon; miR-146a
The roles of microRNAs (miRNAs) as important regulators of gene expression have been studied intensively. Although most of these investigations have involved the highly expressed form of the two mature miRNA species, increasing evidence points to essential roles for star-form microRNAs (miRNA*), which are usually expressed at much lower levels. Owing to the nature of miRNA biogenesis, it is challenging to use plasmids containing miRNA coding sequences for gain-of-function experiments concerning the roles of microRNA* species. Synthetic microRNA mimics could introduce specific miRNA* species into cells, but this transient overexpression system has many shortcomings. Here, we report that specific miRNA* species can be overexpressed by introducing artificially designed stem-loop sequences into short hairpin RNA (shRNA) overexpression vectors. By our prototypic plasmid, designed to overexpress hsa-miR-146b-3p, we successfully expressed high levels of hsa-miR-146b-3p without detectable change of hsa-miR-146b-5p. Functional analysis involving luciferase reporter assays showed that, like natural miRNAs, the overexpressed hsa-miR-146b-3p inhibited target gene expression by 3′UTR seed pairing. Our demonstration that this method could overexpress two other miRNAs suggests that the approach should be broadly applicable. Our novel strategy opens the way for exclusively stable overexpression of miRNA* species and analyzing their unique functions both in vitro and in vivo.
T cell epitopes can be used for the accurate monitoring of avian influenza virus (AIV) immune responses and the rational design of vaccines. No T cell epitopes have been previously identified in the H5N1 AIV virus nucleoprotein (NP) in chickens. For the first time, this study used homology modelling techniques to construct three-dimensional structures of the peptide-binding domains of chicken MHC class Ι molecules for four commonly encountered unique haplotypes, i.e., B4, B12, B15, and B19. H5N1 AIV NP was computationally parsed into octapeptides or nonapeptides according to the peptide-binding motifs of MHC class I molecules of the B4, B12, B15 and B19 haplotypes. Seventy-five peptide sequences were modelled and their MHC class I molecule-binding abilities were analysed by molecular docking. Twenty-five peptides (Ten for B4, six for B12, two for B15, and seven for B19) were predicted to be potential T cell epitopes in chicken. Nine of these peptides and one unrelated peptide were manually synthesized and their T cell responses were tested in vitro. Spleen lymphocytes were collected from SPF chickens that had been immunised with a NP-expression plasmid, pCAGGS-NP, and they were stimulated using the synthesized peptides. The secretion of chicken IFN-γ and the proliferation of CD8+ T cells were tested using an ELISA kit and flow cytometry, respectively. The significant secretion of chicken IFN-γ and proliferation of CD8+ T lymphocytes increased by 13.7% and 11.9% were monitored in cells stimulated with peptides NP89–97 and NP198–206, respectively. The results indicate that peptides NP89–97 (PKKTGGPIY) and NP198–206 (KRGINDRNF) are NP T cell epitopes in chicken of certain haplotypes. The method used in this investigation is applicable to predicting T cell epitopes for other antigens in chicken, while this study also extends our understanding of the mechanisms of the immune response to AIV in chicken.
Glucocorticoid (GC) therapy remains important in improving the prognosis of patients with systemic lupus erythematosus (SLE). However, some patients do not achieve an effective response with GC treatment, creating an obstacle to the remission of SLE. Identification of the underlying mechanisms responsible for steroid resistance can be significant. Macrophage migration inhibitory factor (MIF) arouses our interest because of its reciprocal relationship with GCs. In the present study, we investigated for the first time whether MIF correlated with steroid resistance in SLE and explored potential mechanisms of action.
Sixty-two patients with SLE (40 steroid sensitive and 22 steroid resistant) and 21 normal controls were recruited. Serum levels of MIF were measured by ELISA. Cytosolic MIF and IκB expression in peripheral blood mononuclear cells (PBMCs) were determined by western blotting. The electrophoretic mobility shift assay was assessed by NF-κB in nuclear aliquots. Gene silencing was applied to reduce expression of MIF in PBMCs in steroid-resistant patients. PBMCs obtained from steroid-sensitive patients were treated with recombinant human MIF of different concentrations.
MIF levels in serum and PBMCs were higher in steroid-resistant patients compared with steroid-sensitive patients and controls. In contrast to the steroid-sensitive group, NF-κB levels were significantly higher and IκB levels lower in steroid-resistant patients. After MIF gene silencing, IκB levels in cells from steroid-resistant patients were increased. In steroid-sensitive patients, a decrease in IκB levels and an increase in NF-κB expression from baseline were detected in PBMCs treated with a higher concentration of recombinant human MIF. Treatment with recombinant human MIF did not regulate expression of IκB and NF-κB in PBMCs from patients treated with an anti-MIF monoclonal antibody.
Our results indicated that MIF may play a role in the formation of steroid resistance in SLE by affecting the NF-κB/IκB signaling cascade. As a regulator of glucocorticoid sensitivity, MIF may be a potential target for steroid sparing.
Previous genome wide association study conducted in a population of European ancestry identified rs4963128, a KIAA1542 SNP 23kb telomeric to IRF7, in strong association with SLE. This study was undertaken to investigate whether genetic polymorphism within IRF7 is a risk factor for the development of SLE.
We genotyped one KIAA1542 SNP rs4963128 and one IRF7 SNP rs1131665 (Q412R) in an Asian population (cases vs. controls: 1302 vs.1479) to assess their association with SLE using custom-designed Beadstation Infinium II platform (Illumina). Subsequently, rs1131665 was further genotyped in independent panels of Chinese (528 vs.527), European American (EA) (446 vs.461) and African American (AA) (159 vs.115) by Taqman genotyping assay to seek confirmation of association in various ethnic groups. Luciferase reporter assay was used to assess the effect of Q412R polymorphism on the activation of IRF7.
Consistent association of rs1131665 (Q412R) with SLE was identified in Asian, EA and AA populations (case vs. control: 2435 vs. 2582; Pmeta = 6.18×10−6, OR = 1.42[1.22–1.65]). Expression of IRF7 412Q risk allele resulted in a 2-fold increase in ISRE transcriptional activity compared with expression of IRF7 412R (P = 0.0003), suggesting IRF7 412Q confers elevated IRF7 activity and may therefore affect downstream IFN pathway.
We showed that the major allele of a nonsynonymous SNP rs1131665 (412Q) in IRF7 confers elevated IRF7 activation and predisposes to the development of SLE in multiple ethnic groups. This result provides direct genetic evidence supporting IRF7 may be a risk gene for human SLE.
In vitro metabolism of methadone was investigated in cytochrome P450 (CYP) Supersomes and phenotyped human liver microsomes (HLMs) to reconcile past findings on CYP involvement in stereo-selective metabolism of methadone. Racemic methadone was used for incubations; (R)-and (S)-methadone turnover and (R)- and (S)-EDDP formation were determined using chiral liquid chromatography-tandem mass spectrometry. CYP Supersome activity for methadone use and EDDP formation ranked CYP2B6>3A4>2C19>2D6>2C18, 3A7>2C8, 2C9, 3A5. After abundance scaling, CYP3A4, 2B6 and 2C19 accounted for 63–74, 12–32 and 1,4–14% of respective activity. CYP2B6, 2D6 and 2C18 demonstrated a preference for (S)-EDDP formation; CYP2C19, 3A7 and 2C8 for (R)-EDDP; 3A4 none. Correlation analysis with 15 HLMs supported the involvement of CYP2B6 and 3A. The significant correlation of S/R ratio with CYP2B6 activity confirmed its stereo-selectivity. CYP2C19 and 2D6 inhibitors and monoclonal antibody (mAb) did not inhibit EDDP formation in HLM. Chemical and mAb inhibition of CYP3A in high 3A activity HLM reduced EDDP formation by 60–85%; inhibition of CYP2B6 in 2B6 high activity HLM reduced (S)-EDDP formation by 80% and (R)-EDDP formation by 55%. Inhibition changed methadone metabolism in a stereo-selective manner. When CYP3A was inhibited, 2B6 mediated (S)-EDDP formation predominated; S/R stereo-selectivity increased. When 2B6 was inhibited (S)-EDDP formation fell and stereo-selectivity decreased. The results confirmed the primary roles of CYPs 3A4 and 2B6 in methadone metabolism; CYP2C8 and 2C9 did not appear involved; 2C19 and 2D6 have minimal roles. CYP2B6 is the primary determinant of stereo-selective metabolism; stereo-selective inhibition might play a role in varied plasma concentrations of the two enantiomers.
Selective immunoglobulin A deficiency (IgAD) is the most common primary immunodeficiency in Caucasians. It has previously been suggested to be associated with a variety of concomitant autoimmune diseases. In this review, we present data on the prevalence of IgAD in patients with Graves disease (GD), systemic lupus erythematosus (SLE), type 1 diabetes (T1D), celiac disease (CD), myasthenia gravis (MG) and rheumatoid arthritis (RA) on the basis of both our own recent large-scale screening results and literature data. Genetic factors are important for the development of both IgAD and various autoimmune disorders, including GD, SLE, T1D, CD, MG and RA, and a strong association with the major histocompatibility complex (MHC) region has been reported. In addition, non-MHC genes, such as interferon-induced helicase 1 (IFIH1) and c-type lectin domain family 16, member A (CLEC16A), are also associated with the development of IgAD and some of the above diseases. This indicates a possible common genetic background. In this review, we present suggestive evidence for a shared genetic predisposition between these disorders.
Background and Aim
Calcium has been proposed as a mediator of the chemoprevention of colorectal cancer (CRC), but the comprehensive mechanism underlying this preventive effect is not yet clear. Hence, we conducted this study to evaluate the possible roles and mechanisms of calcium-mediated prevention of CRC induced by 1,2-dimethylhydrazine (DMH) in mice.
For gene expression analysis, 6 non-tumor colorectal tissues of mice from the DMH + Calcium group and 3 samples each from the DMH and control groups were hybridized on a 4×44 K Agilent whole genome oligo microarray, and selected genes were validated by real-time polymerase chain reaction (PCR). Functional analysis of the microarray data was performed using KEGG and Gene Ontology (GO) analyses. Hub genes were identified using Pathway Studio software.
The tumor incidence rates in the DMH and DMH + Calcium groups were 90% and 40%, respectively. Microarray gene expression analysis showed that S100a9, Defa20, Mmp10, Mmp7, Ptgs2, and Ang2 were among the most downregulated genes, whereas Per3, Tef, Rnf152, and Prdx6 were significantly upregulated in the DMH + Calcium group compared with the DMH group. Functional analysis showed that the Wnt, cell cycle, and arachidonic acid pathways were significantly downregulated in the DMH + Calcium group, and that the GO terms related to cell differentiation, cell cycle, proliferation, cell death, adhesion, and cell migration were significantly affected. Forkhead box M1 (FoxM1) and nuclear factor kappa-B (NF-κB) were considered as potent hub genes.
In the DMH-induced CRC mouse model, comprehensive mechanisms were involved with complex gene expression alterations encompassing many altered pathways and GO terms. However, how calcium regulates these events remains to be studied.
Dysregulated cytokine action on immune cells plays an important role in the initiation and progress of systemic lupus erythematosus (SLE), a complex autoimmune disease. Comprehensively quantifying basal STATs phosphorylation and their signaling response to cytokines should help us to better understand the etiology of SLE.
Phospho-specific flow cytometry was used to measure the basal STAT signaling activation in three immune cell types of peripheral-blood mononuclear cells from 20 lupus patients, 9 rheumatoid arthritis (RA) patients and 13 healthy donors (HDs). A panel of 27 cytokines, including inflammatory cytokines, was measured with Bio-Plex™ Human Cytokine Assays. Serum Prolactin levels were measured with an immunoradiometric assay. STAT signaling responses to inflammatory cytokines (interferon α [IFNα], IFNγ, interleukin 2 [IL2], IL6, and IL10) were also monitored.
We observed the basal activation of STAT3 in SLE T cells and monocytes, and the basal activation of STAT5 in SLE T cells and B cells. The SLE samples clustered into two main groups, which were associated with the SLE Disease Activity Index 2000, their erythrocyte sedimentation rate, and their hydroxychloroquine use. The phosphorylation of STAT5 in B cells was associated with cytokines IL2, granulocyte colony-stimulating factor (G-CSF), and IFNγ, whereas serum prolactin affected STAT5 activation in T cells. The responses of STAT1, STAT3, and STAT5 to IFNα were greatly reduced in SLE T cells, B cells, and monocytes, except for the STAT1 response to IFNα in monocytes. The response of STAT3 to IL6 was reduced in SLE T cells.
The basal activation of STATs signaling and reduced response to cytokines may be helpful us to identify the activity and severity of SLE.
Objectives. Recently, a non-synonymous (Gly307Ser) variant, rs763361, in the CD226 gene was shown to be associated with multiple autoimmune diseases (ADs) in European Caucasian populations. However, shared autoimmunity with CD226 has not been evaluated in non-European populations. The aim of the present study is to assess the association of this single nucleotide polymorphism (SNP) with ADs in non-European populations.
Methods. To replicate this association in non-European populations, we evaluated case–control association between rs763361 and coeliac disease (CED) samples from Argentina; SLE, RA, type-1 diabetes (T1D) and primary SS (pSS) from Colombia; and SLE samples from China and Japan. We genotyped rs763361 and evaluated its genetic association with multiple ADs, using χ2-test. For each association, odds ratio (OR) and 95% CI were calculated.
Results. We show that rs763361 is significantly associated with Argentinean CED (P = 0.0009, OR = 1.60). We also observed a trend of possible association with Chinese SLE (P = 0.01, OR = 1.19), RA (P = 0.047, OR = 1.25), SLE (P = 0.0899, OR = 1.24) and pSS (P = 0.09, OR = 1.33) in Colombians. Meta-analyses for SLE (using our three populations) and T1D (our population and three published populations) yielded significant association with rs763361, P = 0.009 (OR = 1.16) and P = 1.1.46 × 10−9 (OR = 1.14), respectively.
Conclusions. Our results demonstrate that the coding variant rs763361 in CD226 gene is associated with multiple ADs in non-European populations.
CD226; Autoimmunity; Latin-America; Asia
Systemic lupus erythematosus (SLE) is a complex autoimmune disease with a strong genetic predisposition, characterized by an upregulated type I interferon pathway. MicroRNAs are important regulators of immune homeostasis, and aberrant microRNA expression has been demonstrated in patients with autoimmune diseases. We recently identified miR-146a as a negative regulator of the interferon pathway and linked the abnormal activation of this pathway to the underexpression of miR-146a in SLE patients. To explore why the expression of miR-146a is reduced in SLE patients, we conducted short parallel sequencing of potentially regulatory regions of miR-146a and identified a novel genetic variant (rs57095329) in the promoter region exhibiting evidence for association with SLE that was replicated independently in 7,182 Asians (Pmeta = 2.74×10−8, odds ratio = 1.29 [1.18–1.40]). The risk-associated G allele was linked to reduced expression of miR-146a in the peripheral blood leukocytes of the controls. Combined functional assays showed that the risk-associated G allele reduced the protein-binding affinity and activity of the promoter compared with those of the promoter containing the protective A allele. Transcription factor Ets-1, encoded by the lupus-susceptibility gene ETS1, identified in recent genome-wide association studies, binds near this variant. The manipulation of Ets-1 levels strongly affected miR-146a promoter activity in vitro; and the knockdown of Ets-1, mimicking its reduced expression in SLE, directly impaired the induction of miR-146a. We also observed additive effects of the risk alleles of miR-146a and ETS1. Our data identified and confirmed an association between a functional promoter variant of miR-146a and SLE. This risk allele had decreased binding to transcription factor Ets-1, contributing to reduced levels of miR-146a in SLE patients.
Genome-wide association studies have identified quite a number of susceptibility loci associated with complex diseases such as systemic lupus erythematosus (SLE). However, for most of them, the intrinsic link between genetic variation and disease mechanism is not fully understood. SLE is characterized by a significantly upregulated type I interferon (IFN) pathway, and we have previously reported that underexpression of a microRNA, miR-146a, contributes to alterations in the type I IFN pathway in lupus patients. Here we identified a novel genetic variant in the promoter region of miR-146a that is directly related to reduced expression of miR-146a and is associated with SLE susceptibility. The risk allele of this variant confers weaker binding affinity for Ets-1, which is a transcription factor encoded by a lupus susceptibility gene found in recent GWAS. These findings suggest that reduced expression of Ets-1 and its reduced binding affinity to the miR-146a promoter both may contribute to low levels of this microRNA in SLE patients, which may contribute to the upregulated type I IFN pathway in these patients. To our knowledge, this is also the first piece of evidence showing association between a genetic variant in a promoter region of a miRNA gene and a human disease.
Human papillomavirus type 16 (HPV16) E7 is a viral oncoprotein believed to play a major role in cervical cancer. In this study, an antagonist peptide against HPV16E7 protein was first identified from screening the c7c phage display peptide library. The binding specificity and affinity of the selected peptide to HPV16E7 were tested by competitive enzyme-linked immunosorbent assay (ELISA). The antagonist peptide showed obvious anti-tumor efficacy both in cell lines and animal tumor models. Significant cell proliferation inhibition with high specificity was noted when HPV16-positive cells were treated with the peptide. This anti-tumor efficacy was resulted from overriding the activities of HPV16E7 and reactivating the pRb/E2F pathway, as shown by a series of experiments. Flow cytometry analysis revealed that the selected peptide induced G1 arrest in a dose-dependent manner. Competitive ELISA, pull down, and Co-IP experiments indicated that the selected peptide disrupted the interaction between HPV16E7 and pRb proteins both in vitro and in vivo. Luciferase reporter assay verified that transcription activities of E2F were suppressed by the peptide through restoration of pRb. RT-PCR and Western blot revealed that it reduced cyclins A, D1, and E1 expression, and led to HPV16E7 protein degradation, but pRb protein stabilization. The current study suggests that this specific peptide may serve as a potential therapeutic agent for HPV16-positive cervical cancer.
Cytochrome P-450 2E1 (CYP2E1) is an important member of the CYP superfamily, which is involved in the metabolism and activation of many low molecular weight toxic compounds. We tried to investigate the possible association of CYP2E1 tag single nucleotide polymorphisms (SNPs) with susceptibility to systemic lupus erythematosus (SLE) in a Chinese Han population.
The coding and flanking regions of the CYP2E1 gene were scanned for polymorphisms and tag SNPs were selected. A two-stage case-control study was performed to genotype a total of 876 SLE patients and 680 geographically matched healthy controls (265 cases and 288 controls in stage I and 611 cases and 392 controls in stage II). SLE associations of alleles, genotypes and haplotypes were tested by age and sex adjusted logistic regression. The gene transcription quantitation was carried out for peripheral blood mononuclear cell (PBMC) samples from 120 healthy controls.
Tag SNP rs2480256 was found significantly associated with SLE in both stages of the study. The "A" allele was associated with slightly higher risk (odds ratio (OR) = 1.165, 95% confidence interval (CI) 1.073 to 1.265, P = 2.75E-4) and "A/A" genotype carriers were with even higher SLE risk (OR = 1.464 95% CI 1.259 to 1.702, P = 7.48E-7). When combined with another tag SNP rs8192772, we identified haplotype "rs8192772-rs2480256/TA" over presented in SLE patients (OR 1.407, 95% CI 1.182 to 1.675, P = 0.0001) and haplotype "TG" over presented in the controls (OR 0.771, 95% CI 0.667 to 0.890, P = 0.0004). The gene transcription quantitation analysis further proved the dominant effect of rs2480256 as the "A/A" genotype showed highest transcription.
Our results suggest the involvement of CYP2E1 as a susceptibility gene for SLE in the Chinese population.
HIV-infected patients with opioid dependence often require opioid replacement therapy. Pharmacokinetic interactions between HIV therapy and opioid-dependence treatment medications can occur.
HIV-seronegative subjects stabilized on at least 3 weeks of buprenorphine/naloxone (BUP/NLX) therapy sequentially underwent baseline and steady-state pharmacokinetic evaluation of open-label, twice daily tipranavir 500 mg co-administered with ritonavir 200 mg (TPV/r).
Twelve subjects were enrolled and 10 completed the study. Prior to starting TPV/r, the geometric mean BUP AUC0-24h and Cmax were 43.9 ng▪hr/mL and 5.61 ng/mL, respectively. After achieving steady-state with TPV/r (≥7 days), these values were similar at 43.7 ng▪hr/mL and 4.84 ng/mL, respectively. Similar analyses for norBUP, the primary metabolite of BUP, demonstrated a reduction in geometric mean for AUC0-24h [68.7 to 14.7 ng▪hr/mL; ratio=0.21 (90% CI 0.19–0.25)] and Cmax [4.75 to 0.94 ng/mL; ratio=0.20 (90% CI 0.17–0.23)]. The last measurable NLX concentration (Clast) in the concentration-time profile, never measured in previous BUP/NLX interaction studies with antiretroviral medications, was decreased by 20%. Despite these pharmacokinetic effects on BUP metabolites and NLX, no clinical opioid withdrawal symptoms were noted. TPV steady-state AUC0-12h and Cmax decreased 19% and 25% respectively, and Cmin was relatively unchanged when compared to historical control subjects receiving TPV/r alone.
No dosage modification of BUP/NLX is required when co-administered with TPV/r. Though mechanistically unclear, it is likely that decreased plasma RTV levels while on BUP/NLX contributed substantially to the decrease in TPV levels. BUP/NLX and TPV/r should therefore be used cautiously to avoid decreased efficacy of TPV in patients taking these agents concomitantly.
pharmacotherapy; HIV/AIDS; pharmacokinetics; antiviral treatment; maintenance; opiates
The σB protein of Muscovy duck reovirus (DRV), one of the major structural proteins, is able to induce neutralizing antibody in ducks, but the monoclonal antibody (MAb) against σB protein has never been characterized.
Four hybridoma cell lines secreting anti-DRV σB MAbs were obtained, designated 1E5, 2F7, 4E3 and 5D8. Immunoglobulin subclass tests differentiated them as IgG2b (1E5 and 4E3) and IgM (2F7 and 5D8). Dot blot and western blotting assays showed that MAbs reacted with His-σB protein in a conformation-independent manner. Competitive binding assay indicated that the MAbs delineated two epitopes, A and B of σB. Immunofluorescence assay indicated that the four MAbs could specifically bind to Vero cells infected with DRV and σB was distributed diffusely in the cytoplasma of infected cells. MAbs had universal reactivity to all DRVs tested in an antigen-capture enzyme-linked immunosorbent assay.
Results of this research provide important information about the four monoclonal antibodies and therefore the MAbs may be useful candidate for the development of a MAb capture ELISA for rapid detection of DRVs. In addition, it showed that the σB protein was located in the cytoplasma of infected cells by immunofluorescence assay with MAbs. Virus isolation and RT-PCR are reliable way for detection of DRV infection, but these procedures are laborious, time consuming, and requiring instruments. These obvious diagnosis problems highlight the ongoing demand of rapid, reproducible, and automatic methods for the sensitive detection of DRV.
Systemic lupus erythematosus is a complex and potentially fatal autoimmune disease, characterized by autoantibody production and multi-organ damage. By a genome-wide association study (320 patients and 1,500 controls) and subsequent replication altogether involving a total of 3,300 Asian SLE patients from Hong Kong, Mainland China, and Thailand, as well as 4,200 ethnically and geographically matched controls, genetic variants in ETS1 and WDFY4 were found to be associated with SLE (ETS1: rs1128334, P = 2.33×10−11, OR = 1.29; WDFY4: rs7097397, P = 8.15×10−12, OR = 1.30). ETS1 encodes for a transcription factor known to be involved in a wide range of immune functions, including Th17 cell development and terminal differentiation of B lymphocytes. SNP rs1128334 is located in the 3′-UTR of ETS1, and allelic expression analysis from peripheral blood mononuclear cells showed significantly lower expression level from the risk allele. WDFY4 is a conserved protein with unknown function, but is predominantly expressed in primary and secondary immune tissues, and rs7097397 in WDFY4 changes an arginine residue to glutamine (R1816Q) in this protein. Our study also confirmed association of the HLA locus, STAT4, TNFSF4, BLK, BANK1, IRF5, and TNFAIP3 with SLE in Asians. These new genetic findings may help us to gain a better understanding of the disease and the functions of the genes involved.
In this study, we first conducted a genome-wide association study in a Hong Kong Chinese population, followed by replication in three other cohorts from Mainland China and a cohort from Thailand, which totaled 3,300 Asian patients and 4,200 ethnically and geographically matched controls. We identified novel variants in ETS1 and WDFY4 associated with SLE with genome-wide significance and confirmed the association of HLA locus, STAT4, BLK, IRF5, BANK1, TNFSF, and IRF5 with the disease. ETS1 encodes a critical transcription factor involved in Th17 and B cell development. Allelic expression study showed a significantly lower expression of ETS1 from the risk allele, which provided functional support to the genetic findings. WDFY4 is a huge protein with unknown function but is predominantly expressed in primary and secondary immune tissues, and a nonsynonymous SNP in this gene was found to be highly associated with SLE susceptibility. Our findings shed new light on the function of these genes as well as the mechanism of this devastating disease.
Cocaine withdrawal symptoms are thought to play a role in relapse; studies characterizing the symptomatology have yielded mixed findings. This study sought to examine the pharmacodynamic/pharmacokinetic profile of repeated high dose exposure to oral cocaine and characterize acute and protracted withdrawal in cocaine abusers. This study employed a repeated-dosing, single-blind design in which subjects (n=9), resided for 40 days on a closed ward. They were maintained for two 4-day cocaine exposure periods (Days 1-4 & Days 9-12, cocaine 175 mg, p.o.; 5 hourly doses [875 mg/day]) separated by a 4-day matched placebo exposure period (Days 5-8). After these 12 days, an additional period of 28 days of placebo maintenance followed (Days 13-40). Test sessions were conducted during each phase; measures of mood, drug effects, sleep, pharmacokinetics, and prolactin were collected throughout the study. The dosing regimen produced cocaine plasma concentrations (Cmax of 680 ng/mL) 2- to 3-fold higher than typically seen in acute dose studies. Prototypic psychostimulant effects, including subjective ratings of euphoric effects [liking, high, good effects] and significant cardiopressor effects, were sustained during the active dosing periods, corresponding to the rise and fall of plasma cocaine. Withdrawal-like symptoms (i.e., disruptions of sleep, increased ratings of anxiety, irritability, crashing) were observed within 24-hr after cessation of dosing. Cocaine reduced prolactin acutely, but no sustained alterations were observed for this measure or for other signs or symptoms during the 28-day abstinence period. These findings indicate that exposure to controlled high doses of cocaine produces modest symptoms consistent with cocaine withdrawal within hours of cessation of dosing but provide no evidence of symptoms persisting beyond 24 hours.
cocaine; withdrawal; pharmacokinetics; stimulants
Phagocytosis has been extensively examined in ‘professional’ phagocytic cells using pH sensitive dyes. However, in many of the previous studies, a separation between the end of internalization, beginning of acidification and completion of phagosomal-endosomal/lysosomal fusion was not clearly established. In addition, very little work has been done to systematically examine phagosomal maturation in ‘non-professional’ phagocytic cells. Therefore, in this study, we developed a simple method to measure and decouple particle internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in Madin-Darby Canine Kidney (MDCK) and Caco-2 epithelial cells.
Our method was developed using a pathogen mimetic system consisting of polystyrene beads coated with Internalin A (InlA), a membrane surface protein from Listeria monocytogenes known to trigger receptor-mediated phagocytosis. We were able to independently measure the rates of internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in epithelial cells by combining the InlA-coated beads (InlA-beads) with antibody quenching, a pH sensitive dye and an endosomal/lysosomal dye. By performing these independent measurements under identical experimental conditions, we were able to decouple the three processes and establish time scales for each. In a separate set of experiments, we exploited the phagosomal acidification process to demonstrate an additional, real-time method for tracking bead binding, internalization and phagosomal acidification.
Using this method, we found that the time scales for internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion ranged from 23–32 min, 3–4 min and 74–120 min, respectively, for MDCK and Caco-2 epithelial cells. Both the static and real-time methods developed here are expected to be readily and broadly applicable, as they simply require fluorophore conjugation to a particle of interest, such as a pathogen or mimetic, in combination with common cell labeling dyes. As such, these methods hold promise for future measurements of receptor-mediated internalization in other cell systems, e.g. pathogen-host systems.
T-614 is a novel oral antirheumatic agent for the treatment of rheumatoid arthritis. Whether it has immunomodulatory or disease-modifying properties and its mechanism of action are largely undetermined.
Rats with collagen-induced arthritis (CIA) were treated with T-614 (5 and 20 mg/kg) daily. Animals receiving methotrexate (1 mg/kg every 3 days) and the nonsteroidal anti-inflammatory agent nimesulide (10 mg/kg per day) were used as controls. A combination therapy group was treated with both T-614(10 mg/kg per day) and methotrexate (1 mg/kg every 3 days). Hind paw swelling was evaluated and radiographic scores calculated. Serum cytokine levels were assessed by Bio-plex analysis. Quantitative PCR was used to evaluate expression of mRNA for interferon-γ, IL-4 and IL-17. Serum IL-17 and anti-type II collagen antibodies (total IgG, IgG1, IgG2a, IgG2b and IgM) were measured using ELISA.
Oral T-614 inhibited paw swelling and offered significant protection against arthritis-induced cartilage and bone erosion, comparable to the effects of methotrexate. CIA rats treated with T-614 exhibited decreases in both mRNA expression of IL-17 in peripheral blood mononuclear cells and lymph node cells, and circulating IL-17 in a dose-dependent manner. T-614 also reduced serum levels of tumor necrosis factor-α, IL-1β and IL-6. A synergistic effect was observed for the combination of methotrexate and T-614. In addition, T-614 (20 mg/kg per day) depressed production of anti-type II collagen antibodies and differentially affected levels of IgG2a subclasses in vivo, whereas IgM level was decreased without any change in the IgG1 level. Together, the findings presented here indicate that the novel agent T-614 has disease-modifying effects against experimental arthritis, as opposed to nimesulide.
Our data suggested that T-614 is an effective disease-modifying agent that can prevent bone/cartilage destruction and inflammation in in CIA rats. Combination with methotrexate markedly enhances the therapeutic effect of T-614.
Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease with a heterogeneous course and varying degrees of severity and organ damage; thus, there is increasing interest in identifying biomarkers for SLE. In this study we correlated the combined expression level of multiple interferon-inducible chemokines with disease activity, degree of organ damage and clinical features in SLE, and we investigated their roles as biomarkers.
Peripheral blood cells obtained from 67 patients with SLE patients, 20 patients with rheumatoid arthritis (RA) and 23 healthy donors were subjected to real-time PCR in order to measure the transcriptional levels of seven interferon-inducible chemokines (RANTES, MCP-1, CCL19, MIG, IP-10, CXCL11, and IL-8). The data were used to calculate a chemokine score for each participant, after which comparisons were performed between various groups of SLE patients and control individuals.
Chemokine scores were significantly elevated in SLE patients versus RA patients and healthy donors (P = 0.012 and P = 0.002, respectively). Chemokine scores were correlated positively with SLE Disease Activity Index 2000 scores (P = 0.005) and negatively with C3 levels (P < 0.001). Compared with patients without lupus nephritis and those with inactive lupus nephritis, chemokine scores were elevated in patients with active lupus nephritis, especially when their daily prednisone dosage was under 30 mg (P = 0.002 and P = 0.014, respectively). Elevated chemokine scores were also associated with the presence of cumulative organ damage (Systemic Lupus International Collaborating Clinics/American Society of Rheumatology Damage Index ≥ 1; P = 0.010) and the occurrence of anti-Sm or anti-RNP autoantibodies (both P = 0.021).
The combined transcription level of interferon-inducible chemokines in peripheral blood leucocytes is closely associated with disease activity, degree of organ damage, and specific autoantibody patterns in SLE. The chemokine score may serve as a new biomarker for active and severe disease in SLE.
This paper reports dissociation constants and “effective molarities” (Meff) for the intramolecular binding of a ligand covalently attached to the surface of a protein by oligo(ethylene glycol) (EGn) linkers of different lengths (n = 0, 2, 5, 10, and 20), and compares these experimental values with theoretical estimates from polymer theory. As expected, the value of Meff is lowest when the linker is too short (n = 0) to allow the ligand to bind noncovalently at the active site of the protein without strain, is highest when the linker (n = 2) is the optimal length to allow such binding to occur, and decreases monotonically as the length increases past this optimal value (but, only by a factor of approximately eight from n = 2 to n = 20). These experimental results are not compatible with a model in which the single bonds of the linker are completely restricted when the ligand has bound non-covalently to the active site of the protein, but are quantitatively compatible with a model that treats the linker as a random-coil polymer. Calorimetry revealed that enthalpic interactions between the linker and the protein are not important in determining the thermodynamics of the system. Taken together, these results suggest that the manifestation of the linker in the thermodynamics of binding is exclusively entropic. The values of Meff are, theoretically, intrinsic properties of the EGn linkers, and can be used to predict the avidities of multivalent ligands with these linkers for multivalent proteins. The weak dependence of Meff on linker length suggests that multivalent ligands containing flexible linkers that are longer than the spacing between the binding sites of a multivalent protein will be effective in binding, and that the use of flexible linkers with length somewhat greater than the optimal distance between binding sites is a justifiable strategy for use in the design of multivalent ligands.
Using oligonucleotide microarray, many IFN-inducible genes have been found to be highly expressed in peripheral blood mononuclear cells (PBMCs) from most patients with systemic lupus erythematosus (SLE). Among these IFN-inducible genes, IFN-induced protein with tetratricopeptide repeats 4 (IFIT4) is a novel gene whose function is unknown.
In this study we examined the role played by IFIT4 in monocyte differentiation and the correlation between IFIT4 expression and the clinical manifestation of SLE. To this end, we used plasmid transfection, flow cytometry, mixed leucocyte responses, ELISA, quantitative RT-PCR and Western blotting.
We found that both IFIT4 mRNA and protein expression levels were significantly higher in PBMCs and monocytes from SLE patients than in those from healthy control individuals. IFIT4 expression was positively correlated with antinuclear antibodies, anti-double-stranded DNA, and anti-Sm auto-immune antibodies in SLE. Patients with SLE exhibiting higher expression of IFIT4 had a higher prevalence of leucopenia, thrombocytopenia and C3/C4 decrease. IFIT4 protein was localized exclusively to the cytoplasm, and it was significantly upregulated by IFN-α in normal PBMCs. To determine the role played by IFIT4 in monocyte differentiation, the monocytic cell line THP-1 was transfected with pEGFP-IFIT4 expression plasmid and stimulated with granulocyte-macrophage colony-stimulating factor/IL-4 to generate IFIT4-primed dendritic cell-like cells (DCLCs). IFIT4-primed DCLCs acquired morphological characteristics of dendritic cells more quickly, with greater resemblance to dendritic cells, as compared with DCLCs primed with pEGFP-C1 control plasmid trasfection. Furthermore, they exhibited higher expressions of CD40, CD86, CD80, HLA-DR and CD83, along with lower expression of CD14; increased IL-12 secretion; and an increased ability to stimulate T-cell proliferation. In addition, IFIT4-primed DCLCs enhanced IFN-γ secretion (about 2.4-fold) by T cells compared with controls.
Our findings suggest that IFIT4 might play roles in promoting monocyte differentiation into DCLCs and in directing DCLCs to modulate T-helper-1 cell differentiation; these actions might contribute to the autoimmunity and pathogenesis of SLE.
The function of synaptotagmin as a Ca2+ sensor in neurotransmitter release involves Ca2+-dependent phospholipid binding to its two C2 domains, but this activity alone does not explain why Ca2+ binding to the C2B domain is more critical for release than Ca2+ binding to the C2A domain. Synaptotagmin also binds to SNARE complexes, which are central components of the membrane fusion machinery, and displaces complexins from the SNAREs. However, it is unclear how phospholipid binding to synaptotagmin is coupled to SNARE binding and complexin displacement. Using supported lipid bilayers deposited within microfluidic channels, we now show that Ca2+ induces simultaneous binding of synaptotagmin to phospholipid membranes and SNARE complexes, resulting in an intimate quaternary complex that we name SSCAP complex. Mutagenesis experiments show that Ca2+ binding to the C2B domain is critical for SSCAP complex formation and displacement of complexin, providing a clear rationale for the preponderant role of the C2B domain in release. This and other correlations between the effects of mutations on SSCAP complex formation and their functional effects in vivo suggest a key role for this complex in release. We propose a model whereby the highly positive electrostatic potential at the tip of the SSCAP complex helps to induce membrane fusion during release.
synaptotagmin; SNAREs; Ca2+ binding; phospholipid binding; neurotransmitter release; microfluidic channels
Plasma levels of cocaine (COC) and two of its principle metabolites, benzoylecgonine (BE) and ecgonine methyl ester (EME) were determined by liquid chromatography–tandem mass spectrometry (LC/MS/MS) in samples collected up to 3 h after a subcutaneous injection of cocaine (10 mg/kg) on six different days between days 4 and 24 postpartum, a period of dramatic change in the endocrine state of the female rat. Locomotor activity was measured in the same animals during this period using automated animal activity monitors. Additional measures in males provide a link to existing literature. We found that plasma levels of cocaine and its metabolites, as well as their respective time courses, are remarkably uniform across the postpartum period in female rats, as are the effects of cocaine on locomotor activity. Data from males show accord with prior published values. COC and BE, but not EME levels, were higher in males, and the time courses of COC and BE levels after injection varied somewhat between postpartum females and males; however, neither baseline nor cocaine-induced locomotor activity differed between postpartum females and males. We conclude that in the postpartum rat, there are no significant differences in the peripheral processing or general accessibility of cocaine to the brain to activate motor systems across the postpartum period. These data are critical to our understanding of differences in the reward salience of cocaine across the postpartum period and in other adult rat models [Mattson BJ, Williams S, Rosenblatt JS, Morrell JI. Comparison of two positive reinforcing stimuli: pups and cocaine throughout the postpartum period. Behav Neurosci 2001;115:683–94, Mattson BJ, Williams SE, Rosenblatt JS, Morrell JI. Preferences for cocaine- or pup-associated chambers differentiate otherwise behaviorally identical postpartum maternal rats.
Cocaine; Subcutaneous; Benzoylecgonine; Ecgonine methyl ester; Locomotor activity; Postpartum; Plasma; Female; Male; Rat