text:
Altered expression of protein tyrosine phosphatase, non-receptor type 22 isoforms in systemic lupus erythematosus
DOI: 10.1186/ar4440
© Chang et al.; licensee BioMed Central Ltd. 2014
Received: 28 June 2013
Accepted: 3 January 2014
Published: 17 January 2014
A C-to-T single nucleotide polymorphism (SNP) located at position 1858 of human protein tyrosine phosphatase, non-receptor type 22 (PTPN22) complementary DNA (cDNA) is associated with an increased risk of systemic lupus erythematosus (SLE). How the overall activity of PTPN22 is regulated and how the expression of PTPN22 differs between healthy individuals and patients with lupus are poorly understood. Our objectives were to identify novel alternatively spliced forms of PTPN22 and to examine the expression of PTPN22 isoforms in healthy donors and patients with lupus.
Various human PTPN22 isoforms were identified from the GenBank database or amplified directly from human T cells. The expression of these isoforms in primary T cells and macrophages was examined with real-time polymerase chain reaction. The function of the isoforms was determined with luciferase assays. Blood samples were collected from 49 subjects with SLE and 15 healthy controls. Correlation between the level of PTPN22 isoforms in peripheral blood and clinical features of SLE was examined with statistical analyses.
Human PTPN22 was expressed in several isoforms, which differed in their level of expression and subcellular localization. All isoforms except one were functionally interchangeable in regulating NFAT activity. SLE patients expressed higher levels of PTPN22 than healthy individuals and the levels of PTPN22 were negatively correlated with the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index (SLICC-DI).
The overall activity of PTPN22 is determined by the functional balance among all isoforms. The levels of PTPN22 isoforms in peripheral blood could represent a useful biomarker of SLE.
PTPN22 is a non-receptor type protein tyrosine phosphatase expressed mainly in hematopoietic cells [1]. It contains a basic bipartite nuclear localization signal (NLS) in its N-terminus, which is followed by a conserved protein tyrosine phosphatase (PTP) domain. An inhibitory domain inhibiting its phosphatase activity is located immediately after the PTP domain [2]. Its C-terminal half is relatively less conserved, with the exception of four proline-rich domains. Its physiological function is still not fully understood. PTPN22 has been shown to attenuate the strength of T cell receptor (TCR) signals by interacting with Lck, Csk, and Vav [3–6]. PTPN22-deficient mice developed age-dependent splenomegaly due to hyperactivation of lymphocytes, and knockdown of PTPN22 in human T cells with small interfering RNA (siRNA) led to enhanced TCR-mediated nuclear factor-kappa B (NF-κB) activity [7, 8]. PTPN22 is present in both the cytoplasm and nucleus of macrophages [9]. Its nuclear localization requires the NLS proximal to the PTP domain. The expression of PTPN22 is further induced in alternatively activated macrophages through a STAT6-dependent mechanism. Cytoplasmic PTPN22 suppresses the polarization of classically activated macrophages, whereas nuclear PTPN22 promotes the differentiation of alternatively activated macrophages [9].
Alternative splicing is an evolutionary conserved mechanism enabling a cell to produce proteins of different function from a single gene. A large body of evidence has indicated that the process of alternative splicing is correlated with disease activity or is even pathogenic in some autoimmune diseases [10–12]. At least two isoforms of PTPN22 have been reported. Lyp2, of which the sequence was deduced from two complementary DNA (cDNA) fragments, lacks the three most C-terminal proline-rich domains [1], whereas isoform 2, tentatively called PTPN22.2 for the purpose of discussion, splices out exons 10 and 11. However, it is unclear whether Lyp2 and PTPN22.2 are functionally distinct from the full-length PTPN22 (tentatively called PTPN22.1). We have also identified a novel isoform of PTPN22, called PTPN22.6, which lacks nearly the entire PTP domain [13]. Unlike PTPN22.1, overexpression of PTPN22.6 actually increased NFAT-dependent luciferase activity. More importantly, PTPN22.6 can act as a dominant negative mutant of PTPN22.1 in regulating cytokine production in Th cells, suggesting that the overall activity of PTPN22 can be influenced by the relative levels of its isoforms.
Several genome-wide association studies have linked PTPN22 to autoimmune diseases. A C-to-T single nucleotide polymorphism, which is located at position 1858 of PTPN22 cDNA and converts an arginine to a tryptophan, is associated with a higher risk of rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and type 1 diabetes but reduces the risk of Crohn's disease [7, 14–17]. Despite these observations, it is still unclear whether the expression of PTPN22 in patients with autoimmune diseases differs from that of healthy individuals, and how this would occur. In addition, the functional impact of the C1858T SNP is still controversial and appears to be complicated. The conversion from arginine to tryptophan resulted in both gain and loss of function of PTPN22 in T cells in different studies [18, 19]. We also found that the R-to-W conversion in the context of PTPN22.1 resulted in a gain of function of PTPN22 and a reduction of interleukin (IL)-2 production in T cells. In contrast, the R-to-W conversion in the context of PTPN22.6 led to a loss of function of PTPN22 and overproduction of IL-2 [13]. Thus, the functional impact of the C1858T SNP is isoform dependent. Furthermore, the C1858T SNP is associated with a loss-of-function phenotype in resting macrophages but a gain of function in classically activated macrophages [9]. How these C1858T SNP-associated functional changes affect the risk of autoimmune diseases is largely unknown. We have previously shown that the transcript level of PTPN22.6 but not total PTPN22 in peripheral blood is correlated with the 28-joint disease activity score with three variables including C-reactive protein (DAS28-CRP3) scores in patients with RA [13]. However, it is unclear whether the expression of PTPN22 and its isoforms is altered in SLE patients and whether the level of PTPN22 isoforms also is correlated with disease activity of SLE.
Here we report the identification of additional isoforms of human PTPN22. We examined the expression of the PTPN22 isoforms in primary human T cells and macrophages. We further compared the expression of PTPN22 isoforms in the peripheral blood of 15 healthy donors and 49 SLE patients, and correlated the expression pattern of PTPN22 isoforms with clinical features of lupus.
Human samples
Forty-nine individuals with SLE fulfilling the 1997 American College of Rheumatology revised classification criteria [20] were previously recruited from the Lupus Center of the Brigham and Women's Hospital (BWH) for the Lupus Biobank. Fifteen control samples from healthy individuals without SLE or related connective tissue disease were obtained from the PhenoGenetic project at the same hospital. All study subjects consented to participate in studies conducted through the Lupus Biobank and the PhenoGenetic project, and agreed to the publication of results derived from such studies. Demographics were collected in addition to the following data from all SLE cases: 1) age at SLE onset; 2) SLE manifestations and organ involvement; 3) concurrent anti-double-stranded DNA antibody titer, C3 and C4. The treating rheumatologist performed a history and physical examination for SLE disease activity (Safety on Estrogens in Lupus Erythematosus National Assessment-SLE Disease Activity Index; SELENA-SLEDAI) [21], and history and chart review to complete an SLE organ damage assessment (SLICC-DI) [22]. Whole blood samples in PAX gene tubes (Qiagen, Germantown, MD, USA) were obtained from all subjects by standard antecubital venipuncture and stored at -80C at the Broad Institute of MIT and Harvard as separated whole blood or extracted RNA. All aspects of the study were approved by the partners' Institutional Review Board.
Preparation of human peripheral blood mononuclear cells (PBMC) and helper T (Th) cells
PBMC were isolated from buffy coats. Th cells were enriched from PBMC with CD4 Microbeads (120-000-440, Miltenyi Biotec, Auburn, CA, USA). Macrophages were prepared from PBMC according to a published protocol [23].
Cell culture and medium
Human primary Th cells and Jurkat cells were cultivated in RPMI-1640. In some experiments, Th and Jurkat cells were stimulated with 2.5 μg/ml of plate-bound anti-CD3 (Hit3a, Cat. # 300314, BioLegend, San Diego, CA, USA) and 2 μg/ml of soluble anti-CD28 (CD28.2, Cat. # 302914, BioLegend) in the presence of IL-2 (50 unit/ml) for an indicated amount of time. Human colonic adenocarcinoma cell line HT-29 cells (HTB38, ATCC) and human embryonic kidney cells 293 T were cultivated in McCoy's 5A medium (Gibco, Grand Island, NY, USA) and Dulbecco's modified Eagle's medium (DMEM), respectively. Primary human macrophages were stimulated with lipopolysaccharide (LPS) (1 μg/ml, InvivoGen, San Diego, CA, USA) along with human interferon-gamma (hIFN-γ) (50 ng/ml, PeproTech, Rocky Hill, NJ, USA) for 24 hours for M1 polarization or hIL-4 (25 ng/ml, PeproTech) along with hIL-13 (25 ng/ml, PeproTech) for 24 hours for M2 polarization.
Western blotting
Whole-cell extract was obtained by lysing cells with lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% TritonX-100, 0.5% DOC, 0.1% SDS, and 1 mM EDTA) containing 0.5 mM PMSF and complete protease inhibitor cocktail (Roche, Basel, Switzerland). Cytoplasmic and nuclear extracts were prepared by washing cells with cold phosphate- buffered saline (PBS) and resuspending them in hypotonic lysis buffer (10 mM HEPES, pH7.9, 1 mM MgCl2, 10 mM KCl, 0.1% TritonX-100, 20% Glycerol, 0.5 mM PMSF, and protease inhibitors) on ice for 10 minutes. The supernatant, corresponding to cytoplasmic fraction, was collected by centrifugation at 12,000 g for 10 minutes. The nuclear pellet was washed with hypotonic lysis buffer and then resuspended in hypertonic lysis buffer (10 mM HEPES, pH7.9, 400 mM NaCl, 1 m EDTA, 0.1% TritonX-100, 20% Glycerol, 1 mM PMSF, and protease inhibitors) and then incubated on ice for 20 minutes. Nuclear extract was collected by centrifugation. Protein extract was analyzed by immunoblot. The following antibodies were used: human PTPN22 antibody AF3428 (R & D Systems, Minneapolis, MN, USA), Hsp90 α/β (sc-7947) and Lamin B (sc-6216) antibody (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA), Oct1 (sc-232, Santa Cruz Biotechnology, Inc.), and FLAG antibody (F3165, Sigma-Aldrich, St Louis, MO, USA).
Plasmid, transfection and luciferase assay
cDNAs encoding PTPN22.1 and PTPN22.8 were amplified directly from Jurkat cells with primers 5′-CGGGATCCTTGCTCTGCAGCATGGACCAAAGA-3′ and 5′-GACGTCGACGCGTTTAAATATTCCAAGTTGGTGGT-3′. cDNA clones BC071670 (PTPN22.2) and BC017785 (PTPN22.7) were purchased from Open Biosystems (Huntsville, AL, USA). cDNA clones AK3030124 (PTPN22.4), AK310698 (PTPN22.5), and AK310570 (PTPN22.6) were obtained from the NITE Biological Resource Center (Chiba, Japan). All cDNA fragments were cloned into an N-terminal FLAG-tag expression vector pCMV-Tag 2B (Stratagene, La Jolla, CA, USA). Transfection of 293 T cells was performed with Effectene Transfection Reagent (Cat. # 301427, Qiagen). Transfection of Jurkat cells was performed with electroporation with a Gene Pulser II (Bio-Rad, Hercules, CA, USA) set at 374 V/1050 μF. In all NFAT luciferase assays, Jurkat cells were transfected with 5 μg of 3xNFAT-Luc, 0.5 μg pTK-Renilla, and 10 μg of pCMV-Tag 2B expression vectors; rested for 48 hours; and then stimulated with anti-CD3 for 6 hours. Luciferase activity was determined with a Dual-Luciferase™ Reporter Assay System (Promega, Madison, WI, USA). Firefly luciferase activity was then normalized against Renilla luciferase activity obtained from the same sample. 3xNFAT-Luc and pTK-Renilla lucifease vectors were described previously [24].
Real-time PCR and non-quantitative PCR
Total RNA was prepared using a Trizol Plus kit (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed on 1 ug of total RNA using the QuantiTect Reverse Transcription kit (Qiagen). Real-time PCR analysis was performed using the Brilliant SYBR Green QPCR kit according to the manufacturer's protocol (Stratagene) on a MX-3000P apparatus (Stratagene). The cycling conditions are: one cycle of 95C for 10 minutes and 40 cycles of 95C for 30 seconds, 56C for 1 minute, and 72C for 1 minute. The following primer pairs were used: 'total' 5′-GCAGAAGTTCCTGGATGAG-3′ and 5′-TCAGCCACAGTTGTAGGATAG-3′; Lyp2 5′-GTGGAACATCTGAACCAAAG-3′ and 5′-AGCCAAGAGAAATTTTTACCTG-3′; PTPN22.2 5′-CCAAGAGGATGACAGTGTTC-3′ and 5′-CTTTGCTTGACTCCCATCTTTTA-3′; PTPN22.5/6 5′-ATCTGTAATTCTTGCCCACC-3′ and 5′-CTCTTCAGTAAAATAACACACATACC-3′; PTPN22.6 5′-TTTGCCCTATGATTATAGCCG-3′ and 5′-GTTCTCAGGAATTATAAGGACACT-3′; PTPN22.7/8 5′-TCTACAACCCTCCTGGACT-3′ and 5′-TTTCAGCTTCCTTTCCCATT-3′; Ets-1(p51) 5′-CTCCTATGACAGCTTCGACT′3′ and 5′-ATCTCCTGTCCAGCTGATAA-3′; β-actin 5′-GTGACAGCAGTCGGTTGGAG-3′ and 5′-AGGACTGGGCCATTCTCCTT-3′. All real-time PCR reactions were done in duplicate and the transcript levels were normalized against those of β-actin.
siRNA transfection
One million Jurkat cells were resuspended with 400 ul Opti-mem I containing 400 pmole of siRNA and subjected to electroporation with a Gene Pulser II (Bio-Rad) set at 250 V/400 uF. Human PTPN22 ON-TARGETplus SMARTpool siRNA (L-008066-00-0005), ON-TARGETplus non-targeting siRNA (D-001810-01-05), and siLyp2 (sense: 5′-UGGAAGAAUGGUUCGUUAAUU-3′; anti-sense: 5′-UUAACGAACCAUUCUUCCAUU-3′) were purchased from Dharmacon/Thermo Scientific (Lafayette, CO, USA).
Statistical analysis
Identification of additional PTPN22 isoforms
In addition to the published PTPN22 isoforms, we identified several cDNA sequences corresponding to three additional spliced variants of human PTPN22 in the NCBI Gene database (Figure 1A). AK303124 is the product of an out-of-frame splicing between exons 4 and 9. It contains two open reading frames: one of 135 amino acid residues and the other starting at a methionine of exon 9 and corresponding to the last 563 amino acid residues of the full-length PTPN22. AK310698 lacks exon 21 but includes at its C-terminus eight novel amino acid residues encoded by the genomic sequence immediately 3′ to exon 20. BC017785 splices out exons 6 and 8 to 19. We tentatively named these three novel isoforms PTPN22.4, PTPN22.5, and PTPN22.7. We also amplified a novel isoform PTPN22.8 (GenBank accession number JN084012), which lacks exon 6, directly from Jurkat T cells. We were able to confirm the presence of each of the unique or shared spliced junctions in human primary T cells with real-time PCR and DNA sequencing (Figure 1A and data not shown). In addition, we were able to amplify the transcript of each of the isoforms except Lyp2 in its entirety with PCR directly from Jurkat cells. Several attempts to amplify the entire Lyp2 were unsuccessful. The counterpart of PTPN22.2 is also present in rhesus monkeys and chimpanzees, according to NCBI Gene database, suggesting that these alternative splicing events are evolutionarily conserved.
Not all of the isoforms can be expressed efficiently in mammalian cells. We replaced the initiating methionine of each isoform with a FLAG tag and expressed the FLAG-fused PTPN22 proteins in 293 T cells. We found that PTPN22.1 and Lyp2 were expressed more efficiently than PTPN22.2, PTPN22.5, PTPN22.6, and PTPN22.8 (Figure 1B). No protein product of PTPN22.4, either starting from the methionine in exon 1 (4.1) or exon 9 (4.2), or of PTPN22.7 was detected, suggesting that PTPN22.4 and PTPN22.7 are non-productive. We therefore excluded these two isoforms from subsequent functional analyses.
Despite the difference in the protein level, the transcript level of each isoform in transfected cells was very comparable when measured with real-time PCR using a pair of primers targeting the FLAG/PTPN22 fusion junction that is common to all isoforms (data not shown). As the transcripts of these FLAG-PTPN22 proteins contain the same Kozak sequence, initiating ATG, and 3′ polyadenylation tail, which are embedded in the expression vector, we postulated that the difference in the protein level was due to a difference in protein stability instead of translational efficiency. To test this hypothesis, we compared the protein stability between PTPN22.1 and PTPN22.6, which was chosen as an example because it was the least expressed but detectable isoform. We treated transfected cells with cycloheximide to stop protein synthesis and then compared the degradation rate of FLAG-PTPN22.1 and FLAG-PTPN22.6. PTPN22.1 protein was indeed more stable than PTPN22.6 (Figure 1C).
We previously used a PTPN22.6-specific antibody to demonstrate the presence of endogenous PTPN22.6 protein in human T cells. In addition, an anti-human polyclonal PTPN22 antibody, which was raised against S306-S684 of PTPN22.1 and should recognize all isoforms, detected one dominant broad protein band centering around 110 kD and several smaller protein species in Jurkat human T cells but not in HT-29 adenocarcinoma cells, which do not express PTPN22 (Figure 1D and [13]). The levels of almost all of the protein species were reduced by PTPN22 siRNA but not by scrambled siRNA. The commercially available PTPN22 siRNA recognizes four different RNA sequences of PTPN22 and very likely affects the expression of all isoforms. Therefore, the 110 kD broad protein band, which was reduced by approximately 80% by the PTPN22 siRNA, is very likely composed of PTPN22.1, PTPN2.2, PTPN22.5, and PTPN22.8, which have comparable molecular weight. The smaller protein species very likely include Lyp2 and/or yet-to-be-discovered isoforms. However, there is no other isoform-specific antibody, and it is still unclear whether the other non-full-length PTPN22 proteins also exist in human cells. To further confirm their existence, we designed a single siRNA targeting the 3′ end of Lyp2. This siRNA is expected to suppress the expression of Lyp2 and any yet-to-be-discovered isoforms sharing the 3′ end with Lyp2, but not the other isoforms shown in Figure 1A. Indeed, expression of the siLyp2 in Jurkat cells led to the disappearance of the smaller PTP22 protein species including one with a molecular weight comparable to that of Lyp2 (Figure 1D, the left panel), but had a negligible effect on the level of the dominant 110 kD protein band (Figure 1D, the right panel). This result strongly indicates that, in addition to PTPN22.6, endogenous Lyp2 protein is also present in human T cells.
Expression of PTPN22 isoforms in human PBMC
PTPN22 is also expressed in myeloid cells including macrophages. Macrophages can be divided into two major functional subsets: classically activated macrophages (M1 cells) and alternatively activated macrophages (M2 cells). We have recently shown that resting macrophages expressed a basal level of total PTPN22 and that the level stayed relatively unchanged in M1 cells [9]. In contrast, the expression of total PTPN22 was induced in M2 cells to a level three to four times higher than that of resting or M1 cells. To examine the expression of PTPN22 isoforms in macrophages, we quantified the transcript level of PTPN22 isoforms in macrophages from seven healthy donors. We found that the levels of Lyp2, PTPN22.2, PTPN22.5/6, PTPN22.6, and PTPN22.7/8 were very comparable among resting, M1, and M2 macrophages (Figure 2C). Thus, the increase in total PTPN22 observed in M2 cells mainly comes from PTPN22.1. Taken together, our data indicate that the level of PTPN22 isoforms varies significantly among cells types and in response to different stimuli.
Subcellular localization and function of PTPN22 isoforms
PTPN22.6 can act as a dominant negative variant of PTPN22.1. But the function of the other isoforms is still unclear. We therefore examined the effect of the other isoforms on NFAT-driven luciferase activity. As expected, overexpression of PTPN22.1 in Jurkat cells suppressed NFAT-dependent luciferase activity by approximately 50%. Interestingly, Lyp2, PTPN22.2, PTPN22.5, and PTPN22.8, despite missing parts of the PTP domain, also had the same effect (Figure 3B). There was no statistically significant difference among these isoforms even after adjustment for the protein level. In addition, a catalytic dead mutant of mouse PTPN22, which contains D195A and C227S mutations [4], had no impact on NFAT activity (Additional file 1), further indicating that these isoforms are still catalytic active. In contrast, expression of PTPN22.6 resulted in a subtle but statistically significant increase in NFAT activity. This result was reported before but was included for comparison [13].
Expression of PTPN22 isoforms in healthy and SLE populations
Demographic characteristics of the study subjects
With systemic lupus erythematosus
Number and gender
47 F and 2 M
Age (years)
46.2 ± 5.2
48.8 ± 12.5
9 Caucasians, 4 blacks, 1 Asians, and 1 Asian/Caucasian
36 Caucasians, 4 blacks, 4 Asians, and 5 Hispanics
The collection of PTPN22 isoforms has expanded significantly over the past few years. During the preparation of this manuscript, another isoform lacking exon 15 was deposited in the NCBI database as isoform 3 (tentatively called PTPN22.3), which is also present in chimpanzees [28]. The expression of PTPN22.3 in different types of hematopoietic cells and its effect on NFAT activity have yet to be examined. Despite the publication of the cloning of Lyp2, we would like to cautiously point out that the existence of this transcript has yet to be confirmed. The conclusion that this isoform exists is based on the identification of a cDNA fragment corresponding to its unique 3′ end. This isoform was then deduced from several overlapping cDNA fragments [1]. However, there is no full-length cDNA clone in the NCBI database matching Lyp2. In addition, we were unable to amplify the entire transcript of Lyp2 from Jurkat cells. Instead, we recently amplified an intact and novel transcript from Jurkat cells that shares the 3′ end with Lyp2 but contains a deletion in the PTP domain (data not shown). It is very likely that there are other yet-to-be-discovered isoforms sharing the 3′ end with Lyp2. This scenario is consistent with the observation that the siLyp2 suppresses the expression of several PTPN22 protein species (Figure 1D). Ultimately, mass spectrometry and/or isoform-specific antibodies will be needed to confirm the presence of non-full-length PTPN22 proteins in different types of immune cells.
With the exception of PTPN22.6, all isoforms examined in this study are functionally interchangeable in suppressing NFAT activity. However, the function of PTPN22 is still poorly understood. For example, we recently found that cytoplasmic PTPN22 suppresses M1 polarization, whereas nuclear PTPN22 promotes M2 polarization of macrophages [9]. PTPN22 is also expressed in neutrophils and NK cells, and its function in these cells is largely unknown. It is possible that the non-full-length isoforms, each missing a part (or parts) of the PTPN22 protein and some excluded from the nucleus, can also act as dominant negative mutants of PTPN22.1 in other functional readouts or immune cells. Thus, the overall activity of PTPN22 is determined by not only the total level of PTPN22 but also by the functional balance among all isoforms.
The functional balance of PTPN22 isoforms may critically influence the impact of the C1858T SNP. It is still poorly understood how the C1858T SNP increases the risk of SLE and RA but lowers the risk of Crohn's disease. There are conflicting data on the impact of this SNP on the responsiveness of human T cells to stimulation. We have previously shown that the effect of the C1858T SNP on cytokine production in Th cells is isoform dependent [13]. The R-to-W conversion in the context of PTPN22.1 further weakened NFAT activity and IL-2 production. In contrast, the R-to-W conversion in the context of PTPN22.6 enhanced IL-2 production. If the other non-full-length PTPN22 isoforms also have a function different from that of PTPN22.1, then the cumulative impact of the C1858T SNP can be complicated and highly dependent on the portfolio of PTPN22 isoforms. This scenario may explain the conflicting data described above.
Why do SLE patients express a higher level of PTPN22? The expression of PTPN22 is induced in activated T cells and M2 macrophages. Th and macrophages of patients with lupus are likely activated and express a higher level of PTPN22. The whole blood samples stored in the BWH Lupus Biobank did not allow separate quantification of PTPN22 levels in each type of blood cells. Some types of blood cells, such as T cells, may express more PTPN22 than other types of blood cells, such as macrophages. An expansion of the high PTPN22-expressing cells, but not necessarily an increase in their status of activation, may also contribute to the high level of PTPN22 observed in the whole blood of patients with SLE. Identifying the PTPN22-expressing cells responsible for the higher PTPN22 level in the peripheral blood of patients with SLE may shed light on the pathogenesis of this disease. It is also intriguing to notice that patients with SLE not only have a high level of PTPN22 but also have an altered portfolio of PTPN22 isoforms. They express more Lyp2 and PTPN22.2 at the expense of PTPN22.1 compared to healthy individuals. The clinical significance of such an alteration in the portfolio of PTPN22 isoforms has yet to be determined.
A recent paper by Ronninger et al. reported a trend suggesting that the combined transcript level of long PTPN22 isoforms including PTPN22.1, PTPN22.2, and PTPN22.3 was higher in PBMC of patients with RA than healthy controls [29]. Although this trend is consistent with the data shown in Figure 4, their data also showed a trend of lower Lyp2 in patients with RA. In addition, the ratio of long PTPN22 to Lyp2 was significantly higher in patients with RA. We, however, found that the level of Lyp2 was higher while the ratio of long PTPN22 to Lyp2 was lower in our patients with lupus. This discrepancy may originate from fundamental differences in the pathogenesis between RA and lupus. In addition, the primers and probe used to detect long PTPN22 isoforms in Ronninger's paper target their 3′ end and will not detect PTPN22.5, PTPN22.6, and other yet-to-be-discovered isoforms sharing the 3′ end with PTPN22.5 and PTPN22.6. The difference in primers used may also explain why the ratio of long PTPN22 to Lyp2 is much lower in Ronninger's study (1 to 1.5) than ours (20 to 30). Our calculated ratio is more consistent with the data shown in Figure 1D.
McKinney et al. recently showed that a higher level of PTPN22, along with a low level of ITGA and Notch1 in CD8 + T cells but not PBMC, was associated with a poor prognosis in SLE and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis [30]. The molecular explanation for this correlation is still lacking. The level of PTPN22 in McKinney's study was indirectly determined by gene chip, which is not specific to any of the PTPN22 isoforms. It will be interesting to know whether such an association could be observed in PBMC if the expression of each of the PTPN22 isoforms was measured. Given the McKinney's report, it is surprising to find in our study that the level of PTPN22 in peripheral blood was not correlated with SLE disease activity (SLEDAI), but actually was negatively correlated with SLICC-DI. SLICC-DI is usually associated with disease duration. However, we found no correlation between PTPN22 level and disease duration in our SLE population. It is possible that the negative correlation is attributed to damage to one organ. The SLICC-DI in our SLE population was relatively low. However, we did notice a trend suggesting a negative association between PTPN22 levels and damage to the musculoskeletal system (data not shown). More patients will be needed to establish such a negative association.
We do not have a biological explanation for the negative correlation between PTPN22 level and SLICC-DI at this moment. There was a substantial drop in the level of PTPN22 in patients with a SLICC-DI equal to or higher than three. We saw no obvious difference in the portfolio of PTPN22 isoforms in this small group of patients (N = 12) compared to the other patients with SLE. Deficiency of PTPN22 has been shown to lead to hyperactivation of lymphocytes and overexpression of inflammatory cytokines in macrophages [8, 9]. Thus, a reduction in the level of PTPN22 as detected in those 12 patients can be proinflammatory, thereby resulting in more organ damage. This hypothesis remains to be confirmed.
This paper is the first to examine and compare the expression, subcellular localization, and function of various isoforms of PTPN22, a gene that is strongly associated with several rheumatic diseases. Human PTPN22 can be expressed in several isoforms, and some of the isoforms are also present in the nucleus due to at least two essential nuclear localization signals. The expression profile of PTPN22 isoforms varies among cell types, and is altered in patients with lupus. In addition, the levels of total PTPN22 and one of the isoforms are negatively correlated with SLICC-DI scores. Future studies investigating the molecular basis of this negative correlation will provide important insight into the pathogenesis of SLE.
- cDNA:
complementary DNA
28-joint disease activity score with three variables including C-reactive protein
Dulbecco's modified Eagle's medium
- NF-κB:
nuclear factor-kappa B
nuclear localization signal
peripheral blood mononuclear cells
phosphate-buffered saline
polymerase chain reaction
protein tyrosine phosphatase
rheumatoid arthritis
safety on estrogens in lupus erythematosus national assessment-SLE disease activity index
- siRNA:
small inhibitory RNA
systemic lupus erythematosus
Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index
single nucleotide polymorphism
T cell receptor
- Th:
helper T.
We thank Tabatha Norton for her help in organizing the clinical samples and data. The statistical analyses of this work was conducted partly with support from Harvard Catalyst | The Harvard Clinical and Translational Science Center (National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institutes of Health Award 8UL1TR000170-05 and financial contributions from Harvard University and its affiliated academic health care centers). The content is solely the responsibility of the authors and does not necessarily represent the official views of Harvard Catalyst, Harvard University and its affiliated academic health care centers, or the National Institutes of Health.
This work is support by a grant from the Within Our Reach campaign of the American College of Rheumatology and an award from the Department of Defense (W81XWH-11-1-0492).
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This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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A Perfect Way to Find the Best Attorney in Miami
We have long been recognized by our clients as the leading referral service for finding the best attorney in Miami. We are proud of our largest and one-of-its-kind network of attorneys in all across the USA. Our more than 50 years of association with thousands of best-in-class legal attorneys can offer all our clients a comprehensive range of legal services, providing legal advice for car accident in Miami or any auto accident and car accident injuries, among others. We have been instrumental in finding the best and highly proficient Personal injury lawyer in Miami, Criminal lawyer in Miami, Bankruptcy lawyer in Miami, Dui attorney in Miami to our tens of thousands of satisfied clients in all the years of our existence in Miami.
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subset: commoncrawl


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SSI - server-side include
Server-side include (SSI) is a type of HTML comment that directs the Web server to dynamically generate data for the Web page whenever it is requested.
SSI Format
The basic format for a server-side include is as follows:
<!--#command tag="value"…>
Where #command can be any of various commands supported by the Web server. The simplest command is #include, which inserts the contents of another file. This is especially useful for ensuring that boilerplate components, such as headers and footers, are the same on all pages throughout a Web site. To change a boilerplate element, you need only modify the include file, instead of updating every individual Web page. SSIs can also be used to execute programs and insert the results. They therefore represent a powerful tool for Web developers.
SSI Standards
There is no official standard for SSIs, so every Web server is free to support different SSIs in different manners. However, many SSI commands, such as #include and #exec, have become de facto standards.
Web pages that contain SSIs often end with a .shtml extension, though this is not a requirement. The filename extension enables the Web server to differentiate those pages that need to be processed before they are sent to the browser.
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Like everything in technology, AI touches on so many other trends, like self-driving cars and automation, and Big Data and the Internet of Things... Read More »DevOp's Role in Application Security
As organizations rush to release new applications, security appears to be getting short shrift. DevSecOps is a new approach that holds promise. Read More »Slideshow: Easy Editorial SEO Tips to Boost Traffic
This slideshow reviews five easy on-page editorial SEO tips to help drive organic search engine traffic, including the page title, heading,... Read More »
Java is a high-level programming language. This guide describes the basics of Java, providing an overview of syntax, variables, data types and... Read More »Java Basics, Part 2
This second Study Guide describes the basics of Java, providing an overview of operators, modifiers and control Structures. Read More »The 7 Layers of the OSI Model
The Open System Interconnection (OSI) model defines a networking framework to implement protocols in seven layers. Use this handy guide to compare... Read More »


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Calder Security Ltd
Awards, business awards, security Wakefield
We are very pleased to have been shortlisted for 3 different business awards. We've been shortlisted for SME of the Year in the Wakefield Business Awards and our Contracts Manager, Arran Barton, has been shortlisted for Employee of the Year. We'll find out on 20th June if we've been successful. Having won Business of the Month in February in the Mid Yorkshire Business Awards, run by Eaton Smith and the Mid Yorkshire Chamber of Commerce, we go through to the Business of the Year Awards on 5th July. Fingers crossed!
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Calder Security makes the shortlist for 3 business awards
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B&B Kasa Kala
Find Hotel Rates
Recent Searches
Wego searches multiple travel sites so you can find and compare the best rates.
B&B Kasa Kala Hotel Reviews
Based on reviews from across the web.
B&B Kasa Kala Hotel Details
Check-in: 12:00
Check-out: 11:00
4 Quarti B&B is housed in a 16th-century building, just a 5-minute walk from the Cathedral. It offers free Wi-Fi throughout, elegantly decorated rooms with air conditioning, and an extensive typical Sicilian breakfast. The rooms are fitted with wooden furnishings and refined bed linens and curtains. Each is spacious and provided with a TV, soundproofed walls, and an en suite bathroom. Breakfast is continental style and it includes sweet pastries and typical Sicilian products, together with hot drinks. Palermo Centrale Train Station is just 1 km from the B&B 4 Quarti. Palermo Falcone Borsellino Airport is a 30-minute drive away.


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California residents are caught between following statewide watering restrictions and keeping up the appearance of their lawns amid a state drought. Wasteful watering can lead to $500 fines from the state, while brown lawns can also rack up $500 fines from local municipalities. An April law restricts HOAs from fining residents who follow conservation mandates.
Full Story:The Huffington Post


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Leadbelly (1976)
Directed by
Starring (View all)
Roger E. Mosley
Huddie Ledbetter
Huddie Ledbetter
Paul Benjamin
Wes Ledbetter
Wes Ledbetter
Madge Sinclair
Miss Eula
Miss Eula
Alan Manson
Captain Freeman
Captain Freeman
Albert Hall
Art Evans
Blind Lemon Jefferson
Blind Lemon Jefferson
James Brodhead
John Lomax
John Lomax
John Henry Faulk
Gov. Pat Neff
Gov. Pat Neff
Written by
Ernest Kinoy
11 votesThe Complete Music Biopics List (66 items)
list by Del Toro
Published 5 years, 12 months ago 1 comment
3 votesA film for each year of my life - 2.0 (50 items)
list by xxixii
Published 2 years, 9 months ago 1 comment
View all Leadbelly lists
Cover art, photos and screenshots
View all Leadbelly pictures
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text: Cache will have the luxury of returning an entirely intact offensive line for 2008. The group will be highlighted by mammoth 4th year starter Earnest Brewer. He measures in at 6' tall and 321 pounds. Those four years of experience will make him an invaluable resource of leadership on that line.


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Dear Raphael,
I work long hours in my job and have 2 kids who keep me busy at home. Except for a 20-minute walk at lunchtime, finding time to exercise is just not possible. Is there anything I can do at my desk during the day to get some type of workout for my muscles?
You're not alone. Not having time to exercise is the number one reason people give for not exercising. I do like the fact that you're walking 20 minutes at lunch time. This shows you're trying and really care about your health.
Don't worry. I'm providing 5 easy to perform movements that you can do at your desk while at work. The exercises will help to get your muscles tighter and keep your mobility intact.
If possible, try to do these exercises in succession. If you're unable to, then perform 2-3 exercises in a row and then continue the program when time permits. Ideally, I recommend doing the exercises twice per day while at work. However, it's designed for you to use whenever possible during the long work day.
Perform 12-15 repetitions for each exercise (excluding the Abdominal vacuum) and then repeat each an additional time. The exercises can be performed each day.
Abdominal vacuum
Starting position:
• Sit tall in your chair
• Start by exhaling absolutely every bit of air from your lungs.
• Pull the navel towards the spine as if I just told you to suck in your stomach. Continue to breathe lightly through your nostrils, but make sure you're pulling your navel in as tight as possible.
• You must hold the contraction very tight for at least 40 seconds.
• Benefit: In time, you'll notice the abdominal area pulled in flatter. This exercise provides benefit with no crunches or repetitious movement.
Seated office chair leg extension
Starting position:
• Sit on your office chair with your feet on the floor spaced about 12 inches apart.
• Contracting the quadriceps muscles, extend one leg (bending at the knee) until it is straight. The toes should be pointed up.
• Slowly return to the starting position.
• After completing the set on one side (12-15 reps), repeat on the other side.
Key points: • Exhale while lifting the leg.
• Inhale while returning to the starting position.
Water bottle alternating front shoulder raise
Starting position:
• Sit straight with your knees slightly bent and feet shoulder-width apart.
• Hold a water bottle in your right hand with your arm in front of you and your palms lightly resting on your thighs
• Contracting the front shoulder muscles, lift your right arm in front of you until it is slightly above shoulder height.
• Slowly return the right arm to the starting position stopping just short of the weight touching your legs.
• Raise and lower your left arm in the same manner.
• After completing 12-15 reps on the right side, switch to the left.
Water bottle biceps curl
Starting position:
• Sit upright, feet forward and your head a natural extension of your spine.
• Hold a water bottle in your right hand with the arms hanging down at your sides and palms facing your body.
• Keep your wrists straight throughout the exercise.
• Contracting the biceps muscles, bend your right arm at the elbow while turning your wrist until your palm is just short of touching your shoulder.
• Slowly return to the starting position stopping just short of the elbow fully extending.
• After 12-15 repetitions, repeat on the other side.
Key points:
• The upper arm should remain stationary throughout the exercise.
One-arm tricep kickback
Starting position:
• While seated, bend forward very slightly.
• Hold a water bottle in your right hand or just make a fist. Bend the elbow at a 90-degree angle pointing behind you with the upper arm not quite parallel to the floor.
• Contracting your triceps muscles, move your lower arm back until the bottle or fist is pointing away from your body to the rear of the room.
• Slowly return to the starting position.
• After completing the set on the right side, repeat on the left side.
Key point: • Your upper arm should remain stationary throughout the exercise.
Start a diet plan at tescodiets.com
Thanks to tescodiets.com who have provided this article.


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Tropical Fish Keeping - Aquarium fish care and resources (http://www.tropicalfishkeeping.com/forum.php)
- Beginner Freshwater Aquarium (http://www.tropicalfishkeeping.com/beginner-freshwater-aquarium/)
- - Good news for those in USA!!! (http://www.tropicalfishkeeping.com/beginner-freshwater-aquarium/good-news-those-usa-3376/)
Good news for those in USA!!!
Wal-Mart to cut pet fish sales | Practical Fishkeeping magazine
get lost wal-mart, target is way better. I dont like anything in wal-mart, targets prices are actually cheaper where i live and is much much much more organized.
But good, they are finally slowing down
Well that is good news. The fish at Wal-Mart do not look healthy, and the employees there know even less than the employees at PetSmart!! (and I used to be one of them, at PetSmart).
I must say that our walmart is not to bad on there fish department, i guess we got one out of a million.LOL the do misprice some of there fish.They are selling collumbien blue flame tetra's for $1.50 and at the petstores out of town they are selling for $3.50 each i think.But the staff is not informative one told us it was fine to put tiger barbs in with platy's and molly's she said "nah they won't bother any of your other fish they are very non-aggresive"
very non-aggressive?? lol hahahahahahahahaha
Good news ! :welldone:
Haha non aggressive? Thats too funny. But thats good news! Although I like the good price on fish bowls (until I get my new tank, bettas have to be in bowls, then they will turn into 'quarantine' bowls for new bettas) I found so many dead bettas =( and their fish look terrible!
That is good to hear in a way, but now i have lost 1 of the the 4 places i can get fish within 75 miles of me.
i have 7 fish stores withing 15 miles of me, i guess its a good thing living in a city :). Why dont you order online, the gas price makes up for the shipment cost
well i wouldnt order enough fish to make the expense of shipping worth it. Plus i have had horrid troubles with both fedex and usps lately claiming to not be able to find my house. I wouldnt wanna risk that when lives are at stake.
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This post contains references to products from one or more of our advertisers. We may receive compensation when you click on links to those products. For an explanation of our Advertising Policy, visit this page.
We're well into 2016, so now is the time to get your credit-card strategy for the year into place. With British Airways raising low-level redemptions in the US this month, an impending devaluation of American Airlines' award chart, and more news sure to come down the pike about the takeover of Starwood by Marriott, it's going to be an interesting year.
Update: Some offers mentioned below are no longer available. View the current offers here – Citi / AAdvantage Platinum Select World Elite MasterCard, British Airways Visa Signature Card, Southwest Rapid Rewards Premier Credit Card, Barclaycard Arrival Plus World Elite MasterCard, Marriott Rewards Premier Credit Card
With all that change in the air, it is more imperative than ever to keep up with developments in the world of points and miles, and to make sure you're maximizing your loyalty programs and the credit cards associated with them. By doing so, you'll have a much stronger, more cohesive award-travel strategy in place, and you'll be better insulated against any further changes or devaluations.
Each month, I compile a list of the top current travel rewards credit card offers out there. Many of the cards on this list remain consistent from month to month thanks to great benefits and relatively constant bonus opportunities and benefits, like those of the Chase Sapphire Preferred card and Platinum Card from American Express. However, the list does change, and rankings shift as new offers come out. For example, you'll notice that the Citi / AAdvantage Executive World Elite MasterCard makes an appearance thanks to its exciting new lounge-access benefit expansion, the Southwest Rapid Rewards Premier Card also earns a place here thanks to one of its periodic 50,000-point sign-up bonus offers and the Citi ThankYou Premier Card has lost its place here because of the change in its sign-up bonus last month.
This is not a list of every offer on the market. For that, I recommend checking out the TPG Hot Deals page where you'll find a comprehensive roundup of the best deals currently available, as well as specific hotel and airline credit cards that might interest you. However, if you're thinking of applying for a credit card now, this list features my top all-around choices of the moment. You can check back each month for updates and the latest and best offers out there.
Before you get started, take a look at these posts for advice on checking your credit score, applying for new cards and getting approved:
- How to Check Your Credit — Preparing for the Credit Card Application Process
- When to Cancel a Card — My Personal Card Inventory and Decision Process
- How Credit Card Applications Affect Your Credit Score
- 5 Things to Understand About Credit Before Applying for Cards
- Should I Be Concerned About A Credit Card Denial?
- Your FICO Score and Which Credit Cards Offer it for Free
- Credit Card Application Restrictions for the Major Issuers
- Avoiding Late Payments and Other Silly Credit Card Mistakes
- 5 Lesser-Known Things That Affect Your Credit Score
- How to Use the CardMatch Tool to get Even Better Sign-Up Offers
Here's the short list of cards, but continue reading below for more details on each of them.
1. Chase Sapphire Preferred
2. Southwest Rapid Rewards Premier Credit Card
3. Citi / AAdvantage Platinum Select World Elite MasterCard
4. Citi / AAdvantage Executive World Elite MasterCard
5. Citi Prestige
6. Citi Hilton HHonors Reserve
7. Platinum Card from American Express
8. Marriott Rewards Premier Credit Card
9. British Airways Visa
10. Barclaycard Arrival Plus World Elite MasterCard
Bonus Entry: Citi Double Cash
Here's an in-depth look at each of the cards listed above, including their benefits and reasons to apply now.
1. Chase Sapphire Preferred
Current Bonus: 50,000 Ultimate Rewards points when you spend $4,000 on the card in the first three months. You'll earn another 5,000 bonus points when you add an authorized user and make a purchase within the first three months.
Benefits: This card regularly ranks highly on my list of top travel credit cards thanks to 2 points per dollar on travel and dining, no foreign transaction fees, primary auto rental insurance and the ability to transfer points to 10 travel partners, including British Airways, Southwest, United, Hyatt and Marriott.
Annual Fee: $0 introductory annual fee the first year, then $95.
Reasons to get it: Since it was introduced a few years ago, the Sapphire Preferred card has been one of my all-around top travel credit cards. It's the card I use for most of my purchases thanks to the ability to earn 2 points per dollar on restaurant and travel purchases with no limit. It ranks as the top card this month because Chase recently raised the sign-up bonus from 40,000 points up to 50,000 points — a 25% increase. The program did lose Amtrak as a transfer partner in December. However, Ultimate Rewards still has 10 other travel transfer partners that I think more than pick up the slack.
The way I see it, this card's tremendous benefits still far outweigh those negatives. Not only is the sign-up bonus back up to 50,000 points, but you can score an extra 5,000 points just for adding an authorized user. The bonus-spending categories are very broad, including restaurants and a range of travel purchases, which makes this a top earner in my wallet. The card also offers primary rental car insurance instead of the usual secondary coverage offered by most travel credit cards, and its trip cancellation coverage is higher than its competitors' at $10,000. All that makes it a great choice for travel purchases and to use when you're on the road, either domestically or abroad.
For more details on the card and why I think it's worth applying for, check out these 5 reasons to get the Sapphire Preferred now. Then if you do get the card, be sure to check out this post on 5 things to do when you get the Chase Sapphire Preferred to make sure you maximize this card's outstanding benefits.
Beyond that, read these posts for more information on the card and the Ultimate Rewards program:
- 5 Reasons Chase Sapphire Preferred Should Be Your First Credit Card in 2016
- 5 Chase Sapphire Preferred Benefits You Might Not Know About
- 10 Fun Ways to Use 50,000 Ultimate Rewards Points
- One Year of Earning and Burning With The Chase Sapphire Preferred
- Redeeming Chase Ultimate Rewards Points for Maximum Value
- Ranking the Chase Ultimate Rewards Transfer Partners
- Redeeming Chase Ultimate Rewards Points for Maximum Value
- Ways to Maximize Each Ultimate Rewards Partner
- How Long Do Chase Ultimate Rewards Take To Transfer?
- Why All Award Travelers Should Earn Transferable Points
2. Southwest Rapid Rewards Premier Card
Current Bonus: 50,000 Rapid Rewards points after spending $2,000 in the first three months your account is open.
Benefits: Earn 2 points per dollar on Southwest Airlines, and with Rapid Rewards participating hotel and rental car partners. No foreign transaction fees. Earn 6,000 bonus points each year after your account anniversary. Earn 1,500 Tier-Qualifying Points per $10,000 you spend on the card each calendar year up to a maximum of 15,000 Tier-Qualifying Points per year.
Annual Fee: $69
Reasons to get it: Chase recently relaunched this increased sign-up bonus (normally, the offer is just 25,000 points). The card offers a solid overall return with a relatively low minimum-spending requirement, even with the annual fee due in the first year. That's why it's one of my top airline co-branded credit cards. I list Rapid Rewards points at 1.3 cents apiece in my most recent valuations, so the bonus alone is worth at least $650.
However, these points can be doubly lucrative if you can leverage this offer to earn the Southwest Companion Pass at 110,000 qualifying-points in a calendar year. The Companion Pass is essentially a two-for-one that allows you to bring a companion along on any flight and just pay the taxes on his or her ticket, including those you book with points.
The faster you earn the Companion Pass in 2016, the longer it's valid, since it's good not only for the calendar year in which you earn it, but the entire following year. So if you earn it right off the bat, it will be valid not only through 2016, but also through 2017. That could equate to nearly two years of half-priced travel! If you applied for this card and the Southwest Rapid Rewards Premier Business Card (which also currently offers a sign-up bonus of 50,000 points after you spend $2,000 on purchases in the first three months), you'd be almost all the way to a Companion Pass just by meeting both cards' minimum-spending requirements. And you'd have a ton of Southwest points to use as well.
For more information, see these posts:
- One Year of Earning and Burning with the Southwest Rapid Rewards Premier
- Earning Southwest Rapid Rewards Points Without Flying
- How Can I Earn Points for the Southwest Companion Pass?
- Maximize 50,000 Southwest Points on Flights to the Caribbean
- How Can I Get the Southwest Companion Pass for 2015/16?
3. Citi / AAdvantage Platinum Select World Elite MasterCard
Current Bonus: Earn 50,000 AAdvantage miles after spending $3,000 in the first three months.
Benefits: Free first checked bag and Group 1 priority boarding for you and up to four travel companions on American Airlines. 10% mileage refund on awards (up to 10,000 miles each year), and reduced-price awards. Two miles per dollar on American Airlines purchases, one mile per dollar on everything else.
Annual Fee: $95, waived the first year.
Reasons to get it: The current sign-up bonus of 50,000 miles is as high as the public bonus on this card usually gets (more commonly it offers 30,000 miles), and AAdvantage miles are among the most valuable mileage currencies out there right now (especially if you can score the bonus before the award-chart devaluation in March). I currently value them at 1.7 cents apiece, so the sign-up bonus alone is worth $850. If you maximize the 10% mileage refund on award redemptions, that's equivalent to another $170 in value annually. The card also includes benefits like 25% off in-flight purchases on American Airlines, as well as 2 miles for airfare purchases. All in all, this is a good offer and one to get in on sooner rather than later before we see more changes from American.
4. Citi / AAdvantage Executive World Elite MasterCard
Current Bonus: 50,000 AAdvantage miles when you spend $5,000 in the first three months.
Benefits: This card's greatest benefit is probably Admirals Club access, which is why the annual fee is at a premium level. However, the card recently expanded this benefit so that authorized users (of which you can add up to 10 per card account, so I got it for several TPG staffers) also get lounge access, greatly increasing its value proposition. It also offers a credit for a Global Entry or TSA PreCheck application, which equals another $100 in value. Apart from that, cardholders earn 2 miles per dollar spent on eligible American Airlines purchases, and can earn 10,000 elite-qualifying miles for spending $40,000 on the card every calendar year (January through December billing statement), which can be a nice shortcut to earning elite status. Cardholders also get priority boarding, 25% savings on eligible in-flight purchases, first checked bag free and concierge service, and the card comes with embedded Smart Chip technology.
Annual Fee: $450
Reasons to get it: While the sign-up bonus on this card has been as high as 100,000 miles in the past, you had to spend $10,000 in three months to get there, so this spending-bonus equation might be more in your wheelhouse. The expanded lounge benefits are a great selling point in my book because you can get up to 10 additional cards at no further cost. So you're basically getting lounge access for 10 people (and the guests they can bring) for the price of one. Many people seem to wonder whether to get the Citi Prestige or the Citi / AAdvantage Executive because the two cards offer similar benefits. The Executive card is still a great option for American Airlines loyalists who value elite status (or perks that mimic it), while the Prestige offers a broader range of benefits. Check out this post for a more thorough answer to that question, and see these tips on how to use 82,500 miles from the Citi / AAdvantage Executive Card.
5. Citi Prestige Card
Current Bonus: 40,000 ThankYou points when you spend $4,000 in the first three months.
Benefits: Earn 3 ThankYou points per dollar on air travel and hotel purchases, 2 points per dollar on dining and entertainment and 1 point per dollar on everything else. $250 air travel credit each year for things like airfare, baggage fees, lounge access and some in-flight purchases. Admirals Club access (when flying AA) and Priority Pass Select lounge membership. No foreign transaction fees. $100 Global Entry application fee waiver. Get your 4th night free at hotels with no blackout dates when you book four consecutive nights at any hotel via a personal travel advisor designated by MasterCard. Redeem your points for air travel at a rate of 1.33 cents apiece, or 1.6 cents apiece on American Airlines. Citi ThankYou Rewards now has 13 transfer partners, including Air France/KLM Flying Blue, Cathay Pacific Asia Miles, Singapore Airlines KrisFlyer, Virgin America and Hilton HHonors, among others.
Annual Fee: $450
Reasons to get it: This card has a much higher annual fee than its mid-tier counterpart, the Citi ThankYou Premier, but the Prestige comes with a slate of high-end benefits that make it potentially much more valuable. The annual $250 air travel credit equates to more than half of the annual fee, and you can even use it on airfare, which sets it apart from competitors like the Platinum Card from American Express. If you take advantage of the other great benefits (such as lounge access, a Global Entry fee waiver, the 4th night free and free rounds of golf), this card is a clear winner. But the real standout benefit as I see it is that fourth-night free perk. It recently saved TPG Editor-in-Chief Zach Honig $1,490 on a single hotel stay!
For more information on the Citi Prestige and Citi ThankYou Rewards, check out these posts:
- Maximizing Benefits with the Citi Prestige in 2016
- How I've Gotten $2,250 in Value from my Citi Prestige Card
- Maximizing Citi Prestige Benefits – A Reader Success Story
- Immediately Denied for the Citi Prestige? You're Not Alone!
- Is Amex Platinum or Citi Prestige Better for Lounge Access?
6. Citi Hilton HHonors Reserve
Current Bonus: Two weekend night certificates after spending $2,500 in the first four months. Each certificate is good for one weekend night (standard room, double occupancy) at most hotels and resorts within the Hilton Worldwide portfolio.
Benefits: Earn 10 HHonors points for every dollar spent on hotel stays within the Hilton Worldwide portfolio, 5 points for every dollar spent on airline and car rental purchases and 3 points for every dollar spent on all other purchases. Receive automatic Hilton HHonors Gold status and earn Diamond status with $40,000 in annual spending, no foreign transaction fees, SmartChip technology. Cardholders who spend $10,000 annually (starting on your annual fee date) get a free weekend night certificate on the account anniversary.
Annual Fee: $95
Reasons to get it: The free nights, automatic Gold status and the ability to earn Diamond status through spending all make this card a top choice for Hilton HHonors loyalists, but also for folks who can maximize its benefits and use those free weekend nights at high-end properties. The sign-up bonus alone can be worth thousands of dollars depending on where you redeem those free-night certificates. I do see the status as less of a benefit since Hilton (following Hyatt's lead) announced it would be offering status matches through March 2017, so there might be a glut of new elites, but it's still worth something in my book.
Another great benefit: If you spend $10,000 annually, you also get another free weekend night certificate, which can far outweigh the card's $95 annual fee, depending where you redeem it. Citi sometimes offers a limited-time $100 statement credit for your first Hilton stay within three months of account opening, but that extra incentive isn't available at the moment. If you aren't in a rush to apply, you might hold out to see if that offer shows up again, but this is a great card even without it.
For more information, check out these posts:
- Citi Hilton HHonors Reserve Card Review
- Maximizing Citi Hilton Reserve Free Nights
- Citi Hilton Honors Reserve Free Weekend Nights Valid at Category 1-10 Hotels
- Which Hilton Card Offers Elite Status
7. Platinum Card from American Express
Current Bonus: 40,000 Membership Rewards points when you spend $3,000 in the first three months. There are targeted offers through the CardMatch Tool, where you might be eligible for special bonuses like 100,000 points after spending $3,000 in the first three months, which we saw over the summer and into the fall.
Benefits: This card comes with an entire laundry list of valuable benefits, including a $200 annual airline rebate, access to Delta and Priority Pass lounges (as well as the Amex Centurion Lounges) and a Global Entry or TSA PreCheck application rebate. Just as important, you can transfer the Membership Rewards points it earns you to 20 airline and hotel partners.
Annual Fee: $450
Reasons to get it: Though the annual fee on this card is among the highest on the market, it has several valuable perks that you can learn more about in the posts I've linked to below. If you can maximize benefits like the $200 annual airline rebate, lounge access, free Hilton Gold status (which is fairly new) and free Starwood Preferred Guest Gold status, as well as the Fine Hotels & Resorts program and Global Entry or TSA PreCheck application fee credits, this card's perks can easily be far more valuable than the cost of the annual fee. The Platinum Card did lose some of its lounge benefits last year, but Amex is actively expanding the network of Centurion Lounges throughout the US and beyond, including the recently added lounges in Miami and Seattle, and one in Houston opening later this year — with an additional location possibly opening at LAX.
For more information, check out these posts:
- My Experience Getting Refunded for the Global Entry Application Fee With My Amex Platinum Card
- Maximizing the Amex Platinum $200 Annual Airline Rebate
- Is Amex Platinum Worth It If I'm Not Near Centurion Lounges?
- The Best American Express Membership Rewards Redemptions
- Is the Amex Platinum Card Worth the $450 Annual Fee?
8. Marriott Rewards Premier Credit Card
Current Bonus: 50,000 Marriott Rewards points when you spend $2,000 on purchases in the first three months.
Benefits: Enjoy 1 Free Night Stay at a Category 1-5 location every year after your account anniversary date. For every dollar spent, you'll earn 5 points at 4,000 Marriott locations; 2 points on airline tickets purchases directly with the airlines, at car rental agencies & restaurants and 1 point everywhere else. 15 nights of elite credit per year after approval and after your account anniversary (enough for Silver status) and the ability to earn additional elite nights at a rate of one per $3,000 in spending per calendar year. Smart Chip technology.
Annual Fee: $0 the first year, then $85 thereafter.
Reasons to get it: Though the bonus on this card regularly goes up to 70,000-80,000 points, you might want to consider it now for a few reasons. First, with the impending acquisition of Starwood, it might be good to have some Marriott points in your back pocket while we see how the two chains' loyalty programs will be merged. Second, this card automatically confers 15 nights of credit toward elite status each year, enough for automatic Silver status. Marriott also announced a new scheme that lets members redeem points for status if they failed to requalify this year, so a handy points bonus from this card might be just what you need. Especially if you were Platinum and can get it back since, thanks to Marriott's RewardsPlus partnership with United, Marriott Platinum members can get automatic United Silver status. The anniversary free night at a Category 1-5 property can be worth well over the $85 annual fee. Finally, if you're a Southwest flyer, if you take advantage of Marriott's Hotel + Air packages and transfer Marriott points for an award stay and airline points with Southwest, those points will count toward Companion Pass qualification — a potentially huge value.
- Is the Marriott Rewards Premier Credit Card Worth It?
- One Year of Earning and Burning with the Marriott Visa
- 9 Marriott Properties that Make Awesome Award Redemptions
- Finding Value with Marriott's Cash + Points Awards
- How Much is Marriott Rewards Elite Status Worth?
9. British Airways Visa Signature Card
Current Bonus: A total of 100,000 Avios. Earn 50,000 bonus Avios after you spend $2,000 on purchases within the first three months of account opening. Plus, earn an additional 25,000 bonus Avios when you spend $10,000 on purchases for a total of 75,000 bonus Avios. You'll also earn another 25,000 bonus Avios when you spend a total of $20,000 on purchases within your first year for a total of 100,000 bonus Avios.
Benefits: Earn 3 Avios for every dollar spent on British Airways purchases and 1 for every dollar spent on all other purchases. 10% off British Airways ticket purchases with CARDOFFERU cardholder code through the end of 2016. No foreign transaction fees. Every calendar year you make $30,000 in purchases on your British Airways Visa card, you'll earn a Travel Together Companion Ticket good for the rest of the year and the following.
Annual Fee: $95
Reasons to get it: Though this card ranks ninth on this list thanks to that high sign-up bonus, there are a few things to keep in mind when applying. First, you have to spend $20,000 in order to get the full 100,000-Avios bonus. Second, British Airways' Avios program underwent a major devaluation in April that brought some huge increases in redemption requirements for most premium-cabin and partner awards. BA also recently raised its lowest-level 4,500-Avios awards within the US to 7,500 Avios, meaning awards booked on American Airlines using BA Avios are now more expensive. The bonus categories on the British Airways Visa also changed in May so you earn more points per dollar spent on BA airfares, but fewer Avios on all other purchases, which is a major consideration.
However, there are still a lot of good reasons to get the card, including the fact that most economy awards did not increase in price (and some even decreased thanks to off-peak pricing). Avios can still offer some pretty good value on short-haul flights, including on BA's Oneworld partner American Airlines within the US, as well as Cathay Pacific flights in Asia, LAN flights in South America and Qantas flights in Australia. Recently, BA even offered a few discounted award redemptions of up to 40% off.
One of the major reasons you might consider this card now, though, is the fact that British Airways and Amex also decreased the transfer ratio of Membership Rewards to Avios by 20% in October. That makes Membership Rewards points a less attractive option for BA redemptions, but using this card to earn Avios without having to transfer from Amex a much better option for some flyers.
If you purchase flights on the airline, you can get some tremendous value from this card's 10% discount on BA fares. Plus, the ability to spend your way to a companion award ticket can be worth thousands of dollars if you strategize your spending and awards.
For more information on how to maximize British Airways Avios, check out these posts:
- 7 Ways to Get Great Value From the British Airways Visa
- Lesser-Known British Airways Visa Perks
- British Airways Avios and What to Do After the Devaluation
- Best Ways to Redeem British Airways Avios
- How to Redeem British Airways Avios Without Huge Fees
10. Barclaycard Arrival Plus World Elite MasterCard
Current Bonus: 40,000 miles after spending $3,000 in 90 days.
Benefits: Earn 2 miles per dollar spent on all purchases, and receive 5% of your miles back when you redeem for travel. Chip and Pin technology. No foreign transaction fees.
Annual Fee: $89, waived the first year.
Reasons to get it: It might surprise some to see this card back on my list due to some recent changes to it and its benefits, such as lowering the travel redemption points refund from 10% to 5%, raising the minimum point redemption from 5,000 to 10,000 and the elimination of the tourist attraction category for redemptions. However, I like having a card that can cover the in-between costs of travel, from Uber to bed and breakfasts to even private jets. Plus, 2.11% on travel redemptions is slightly better than the 2% cash back I can usually get on other cards like the Capital One Venture.
For more information on this card, check out these posts:
- Review of the Barclaycard Arrival Plus World Elite MasterCard
- Travel Bonus Category and Redemption Options
- Top 10 Ways To Maximize Barclaycard Arrival Miles
Bonus Entry: Citi Double Cash
Current Bonus: None
Benefits: Earn 1% cash back on purchases, with no limit on earnings. Earn another 1% cash back on payments you make toward charges that appear on your current monthly statement, so long as you pay at least the minimum amount due. 0% Intro APR on Balance Transfers for 18 months. After that, the variable APR will be 13.99% – 23.99% based on your creditworthiness. Get a pass on your first late fee. Other standard Citi benefits like Price Rewind.
Annual Fee: $0
Reasons to get it: This card does not offer a sign-up bonus, but I still think it's a tremendous asset to have in your wallet. Though some folks might benefit more from the Chase Freedom and Discover it cards thanks to their 5% rotating cash-back bonus categories, if you are looking for a card that gives you a good, solid cash back rate on all purchases, this might be a better option for you. That simplicity makes it a great starter card, and a useful one for earning cash-back that you can put toward travel expenses for which you cannot normally redeem points. For more, check out this review of the card.
With great transfer partners like United and Hyatt, 2x points on travel & dining and a 50,000 point sign up bonus, the Chase Sapphire Preferred is a great card for those looking to get into the points and miles game.
- Earn 50,000 bonus points after you spend $4,000 on purchases in the first 3 months from account opening. That's $625 toward travel when you redeem through Chase Ultimate Rewards®
- Named Best Credit Card for Flexible Travel Redemption - Kiplinger's Personal Finance, July 2016
- 2X points on travel and dining at restaurants worldwide & 1 point per dollar spent on all other purchases.
- Earn 5,000 bonus points after you add the first authorized user and make a purchase in the first 3 months from account opening
- No foreign transaction fees
- 1:1 point transfer to leading airline and hotel loyalty programs
- Get 25% more value when you redeem for airfare, hotels, car rentals and cruises through Chase Ultimate Rewards. For example, 50,000 points are worth $625 toward travel
- No blackout dates or travel restrictions - as long as there's a seat on the flight, you can book it through Chase Ultimate Rewards
Editorial Disclaimer: Opinions expressed here are author's alone, not those of any bank, credit card issuer, airlines or hotel chain, and have not been reviewed, approved or otherwise endorsed by any of these entities.
Disclaimer: The responses below are not provided or commissioned by the bank advertiser. Responses have not been reviewed, approved or otherwise endorsed by the bank advertiser. It is not the bank advertiser's responsibility to ensure all posts and/or questions are answered.


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TheLiberalOC mourns our friend Dot De Leon who has been battling leukemia for several months now. In spite of receiving a devastating prognosis several months ago, Dot remained committed to living every day and every moment with grace, courage and class. She loved her friends, Disneyland, and Democrats. She is a co-host for a February…
Tag Archive for Democrats
Happy New Year
by Dan Chmielewski •
So who said non-election years aren't exciting. We're not looking back at 2013 but looking ahead to so much more. The mid-term elections that will bring about one new Congressional rep from OC and hopefully a new Democratic majority in the House. John Campbell steps down and maybe someone can finally beat Dana Rohrabacher who…
DPOC lists priorities for 2014
by Dan Chmielewski •
Hot off the press from the Democratic Party of Orange County's chairman Henry Vandermeir on the party's priorities for 2014. He's spot on. Since being elected Chair of the Democratic Party of Orange County nearly a year ago we have made great strides in moving our Party forward. From building our volunteer base to recruiting…
Orange County Young Dems Strive To Make A Difference
by Joel Smith •
The Orange County Young Democrats (OCYD) are "the official voice for young people in the Democratic Party of Orange County." While many clubs around Orange County represent specific areas, OCYD represents the youth voice of Democrats from all over Orange County.
Thousands attend Naturalization Ceremony in Anaheim
by Joel Smith •
The Anaheim Convention Center played host Wednesday to over 1,400 immigrants who were naturalized and sworn in as American citizens. In addition to the new citizens, the convention center was also crowded with family and friends who waited outside for their loved ones.
Welcome to OC, Republicans; You Know Your Party is in Big Trouble Right?
by Dan Chmielewski •
The California GOP Convention is set this weekend for Anaheim and I'm not sure where to send flowers for the wake. The party is clearly on life support. Governor Jerry Brown's progressive agenda and the complete Democratic Control in the legislature have turned California's gloomy outlook under a Republican governor and millions of dollars in…
Fleischman's Editorial on Pensions; Dumb and Dumber
by Dan Chmielewski •
In his Voice of OC commentary Jon Fleishman tries to paint the serious issue of unfunded actuarially accrued liability (UAAL) into a political box of Republican vs. Democrat, Conservative vs. Liberal and Unions vs.Taxpayers. Both Democrats and Republicans agree that responsible recognition of pension liabilities and their funding is necessary.


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Devin McCourty as a captain and other things to look for in Patriots-Dolphins opener
|09.12.11 at 1:11 pm ET
The Patriots take the field tonight in Miami Gardens as a team bound and determined to learn from the last two seasons that ended prematurely in postseason losses on their home turf to the Ravens and Jets, respectively.
They'll do so with three new captains – Devin McCourty, Logan Mankins and Matthew Slater– joining the established leadership of Tom Brady, Vince Wilfork and Jerod Mayo.
McCourty, in his second season, provided leadership in his rookie season out of Rutgers simply by the way he played.
He became just the fourth player in Patriots history to make the Pro Bowl as a rookie, joining Mike Haynes (1976), John Stephens (1988) and Curtis Martin (1995).
His seven interceptions in 2010 fell just one shy of Haynes' Patriots rookie team record. One of McCourty's seven picks came against a quarterback he'll be facing at Sun Life Stadium tonight – Chad Henne.
Some other morsels to chew on in advance of tonight's game:
* Tom Brady is 12-6 career vs. the Dolphins, with five of the six losses coming in the same stadium he'll be playing in this evening. His only loss at home to the Dolphins came on Jan. 1, 2006, when he played just the first quarter. Brady is 4-5 all-time in South Florida, including the only shutout loss of his career, 21-0, on Dec. 10, 2006 – also a Monday night.
* The Patriots and Dolphins have met 15 times in prime time, with the two teams tied 5-5 when meeting on Monday night.
* When these two AFC East rivals meet on Monday night, you can almost always bet it will be in South Florida. Nine of the previous 10 have come in Miami Gardens, including last year's 41-14 Patriots win.
* As Brady pointed out this week, if it weren't for the special teams last year in Miami, the game could've gone the other way. The Patriots received two blocked kicks and a pick-6 from Patrick Chung. The Patriots scored on a blocked field goal, kick-off return, interception, rushing touchdown and receiving touchdown, becoming the first team in NFL history to score a TD five different ways.
* The Dolphins still lead the all-time series 50-41 but the Patriots lead the playoff series, 2-1, and have won seven of the last 10 encounters, including a series sweep in 2010.
* Since Bill Belichick took over the Patriots in 2000, the Dolphins have given him the most trouble in the division, as he has just a 13-9 (.591) record. Also consider: Since 2001, the Patriots own a 48-14 record against AFC East foes, the best intra-divisional mark of any NFL team. Exactly half of those 14 losses have come against the Dolphins as the Pats have gone 13-7. The Pats are 14-6 against the Jets in that span and 19-1 against the Bills.
* Another player to keep an eye on tonight is Wes Welker. In seven career games against his former team, Welker has caught 55 passes for 683 yards and two TDs. He is averaging 7.8 catches and 98 receiving yards per game against the Fish. He has three career 100-yard games against Miami, all in South Florida. He totaled 138 yards on Oct. 21, 2007, 120 yards on Nov. 23, 2008 (with Matt Cassel) and 167 yards on 10 catches on Dec. 6, 2009.
* The Dolphins will have a point to prove after matching a franchise record for home futility in 2010. They went 1-7 on home turf, tying the 2007 team that went 1-15, with that one win coming against the Ravens. In 2010, their only home win came against the Titans, 29-17, on Nov. 14.
Be sure to follow WEEI.com throughout the evening as Patriots beat writer Chris Price will be on the scene in Miami Gardens while Mike Petraglia, Kirk Minihane and Paul Flannery will break down the game during and after the 2011 season opener.
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Listing Courtesy of Paul Anderson of Coldwell Banker Anchor-Pentwater
Listing information
- Status: Active
- Foreclosure: No
- Estimated Annual Taxes: $1,862
- Subdivision: O11 - Pentwater Weare Golden Hart
- Style: Office
- Stories: 0
- Total Square Footage: 453 ($132.23 per sq. foot)
- County: Oceana
- Year Built: 1983
- New Construction: No
- Lot Area: 0.01
- School District: Pentwater
Additional Features Bath Common Area
Air Conditioning Wall Unit
Auction or For Sale For Sale
Below Grade None
Business Includes Real Estate
Construction Type Block, Concrete, Frame/Wood
Driveway Paved
Exterior Material Brick/Stone
Heat Source Electric
Heat Type Space Heater
Other Road Frontage - 0.00, Zoning - 210 Comm.
Property Sub-Type Office
Purchase Terms Avail Cash/Conventional
Roofing Rubber
Sale Conditions None
Street Type Paved, Public
Tenant Pays All Common Area Maintena, Electric, Prop Taxes/Assess
Uses Office, Service
Util Avail at St Broadband, Cable, Electric, Public Sewer, Public Water, Telephone
Utilities / Sewer Public
Utilities Attached Cable, Electric, Telephone
Water Type Public
Waterfront Options: Water Access Y/N No
Waterfront Options: Waterfront Y/N No
Listing Courtesy of Paul Anderson of Coldwell Banker Anchor-Pentwater


text: OverviewBack to top Trimedica - Alka Max Liquid Drops for pH Balance - 1 oz. Item# :68109UPC# :744665006822 Brand:Trimedica Size/Form:1 oz. Ship Weight:0.13 Servings:60 Dosage:10 Drop(s) TriMedica - AlkaMax Liquid Drops for pH Balance 1 oz. (30 mL) TriMedica - AlkaMax Liquid Drops for pH Balance provides a welcome alkaline addition to your diet. Add to a glass of water, or add to coffee, tea or other beverages for a more alkaline, less acid drink. What Is pH and pH Balance?pH = Potential Hydrogen When you measure your alkalinity with a pH strip you are measuring how much hydrogen your body is unable to convert back into an alkaline state. When you have too much potential hydrogen, your body produces acid bases that lead to acidic conditions such as occasional fatigue, heartburn, acid indigestion and even more serious conditions. Taking antacids merely reduces the hydrochloric acid levels your body depends upon for digestion. AlkaMax addresses acidic conditions by using powerful alkaloid minerals that convert potential hydrogen back into a state of alkalinity at the cellular level without eliminating the healthy acid that your body needs for digestion. AlkaMax is a natural antacid that not only helps neutralize excess stomach acid and indigestion, but actually reduces acid buildup throughout your entire body! Unlike other antacids that only treat short-term symptoms of indigestion and heartburn, AlkaMAX gets to the underlying source of acid buildup, by promoting better long-term acid/alkaline (pH) balance all through your system.AlkaMAX is the first natural supplement line that provides a unique patent-pending blend of essential nutrient alkaline boosters that help quench the burning acid damage that can destroy your health and your youthful looks and vigor. We all want to reduce stress and eat better, but with our hectic modern lifestyles, it's almost impossible to stay balanced. So start giving your body the alkaline boost it needs to balance your pH and combat excess acid damage with easy, convenient and effective AlkaMAX capsules or powder! HistoryFounded in 1993, TriMedica International Inc. is a unique natural supplement manufacturer that focuses on delivering unique, innovative, highest quality natural health products throughout the world. TriMedica's team of professionals is dedicated to providing extraordinary, high-quality health products and information that reflect the company's belief in the profound benefits that can be realized when people embrace a natural and healthy lifestyle. Each product in the TriMedica line represents careful in-depth research and painstaking quality control. Their products are continuously tested and retested for quality assurance, and frequently greatly exceed the industry's highest expectations and standards. At TriMedica they focus on bioavailability as a key feature of their products, offering easily assimilable liquid and capsule mediums that provide the highest ratios of maximum absorption and potency, rather than excipient-filled tablets, which limit both absorption and effectiveness. Headquartered in Tempe, Arizona, TriMedica's 36,000 sq. ft. manufacturing plant utilizes strict GMP manufacturing guidelines, quality control testing and state of the art manufacturing equipment to produce highest quality, safe and effective natural health products.