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Video: Vector – Oja (Drugs & Friends)
Video Vector Oja (Drugs & Friends) Mp4 Download
Vector the Viper unlocks another intellectual track titled "Oja" (Drugs & Friends).
'Oja' (Drugs & Friends) a track off the coming EP of #Vector titled #VibesBeforeTeslim #Oja. The talented rapper shares a great message about the abuse of Drugs.
"Oja" is a marketplace where anyone is welcome to experience the buying and selling of ideas during the journey of self discovery in life. We don't have the authority to judge anyone, but we should be willing to take on the responsibilities that come with our actions.
This record is a subject matter of Peer Influence on an individual… Listen!
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Video: Teni – Billionaire
Video Teni Billionaire Mp4
Dr Dolor Entertainment vocalist, Teni drops the TRENDING video of her latest song hit titled "Billionaire", prod. by Pheelz.
The visual featured the famous IG comedian, Broda Shaggi and coordinated by raving cinematographer, TG Omori.
Thutmose x Rema – Love In The Morning
Thutmose x Rema Love In The Morning Mp3
American-English rapper/singer, Thutmose links up with Mavin's superstar, Rema for the music video of their new joint "Love In The Morning".
Thutmose just released his newest masterpiece titled "Don't Wake Me". Off the body of work is this smashing tune with Remmy boy (Bad Commando) – "Love In The Morning". Avedon is the producer behind the production of this addictive song that can lure a lady into something deep. Rema the Nigerian sweet boy with a special Indiana sound blessed this track with a good vibe.
The video was Shot in London, Enjoy!
Video: Davido – Risky ft. Popcaan
Video Davido Risky ft. Popcaan Mp4 Download
Shortly after the audio, DMW Boss, Davido premieres the music video to his latest titled "Risky" featuring Jamaican dancehall artist, Popcaan.
However, "Risky" serves as the lead single on Davido's forthcoming project, "A Good Time" album which is due for release in few weeks.
Song produced by DMW record producer, Speroach Beatz; video shot in London and directed by Meji Alabi for JM Films.
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OO Programming In Java - Lecture 20 (3/17/13)
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This lecture covers examples in Java for Exception Handling and File I/O. Another text editor is demonstrated. A program the goes to a URL and downloads the ...
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There are wealthy men that really hide their wealth. They also chose social environments where crass materialism is discouraged or carefully avoid women that make displays of conspicuous consumption or who live beyond their means. Wealthy men may also really look for women that have real accomplishments of their own, even if those are not financial in nature. There are couples in which _both_ partners are wealthy and they agree from the start to keep their finances separate.
You also have men that do background checks on women they may be interested in and see what kind of pattern emerge: hard core serial "gold diggers" are pretty easily spotted. I have an old friend who retired with a net worth in the upper 5%. When he was actively dating, big thing he wanted to establish on the first date with a woman : did she have means to support herself that she was reasonably happy with? If that was not the case, there probably would not be a second date.
There are also wealthy men that very pointedly avoid anything in the way of emotional attachment: they may use prostitutes or even seek out "gold diggers", use them for sexual gratification and cast them aside rather rapidly. Sometimes that extends beyond women that are mercenary into women that just want a little security.
I saw one case in which a rather wealthy man was getting women to risk serious prison time to help make him more wealthy. I don't think all of those women were really interested in "money only" but they got dazzled by a guy that was really good at projecting an attractive image and acting supremely "confident".
I've also seen men that set up their assets in trusts so that a woman that got involved with them would have trouble attaching any real assets.
Wealthy men can also afford good legal advice. That may mean offering some kind of formal agreement early on in a relationship that limits exposure of their assets to a palimony lawsuit or offering marriage but with an accompanying prenup that limits their exposure.
Men who are rich, good looking and socially prominent may find themselves literally overwhelmed with relationship options, at least ones based on mercenary or superficial interest. A lot of that interest may really be pretty shallow and unsatisfying. Years ago, I had a housemate who looked like he stepped out of GQ. He had stalkers. This was not something I expected when we became housemates. I hate to think what his life might have been like if fame and money were added to the equation.
I know some folks who were involved in Microsoft early on. I heard that even after Bill Gates was quite wealthy, it was still moderately easy to get a date with him. However, he would present a list of test questions that would tend to select for intelligence that would need to be completed if a woman wanted a second date. I see those actions on Gates' part as an attempt at self-protection.
@Redfox Wow


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Homemade Tether Car
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Homemade Tether Car
Well, it is officially done now with the exception of a test run. And that will be done in the spring of 2019. I got the plans from a member on a Facebook group. I cut it out and painted it with white for a basecoat followed by a simple neon blue and neon green paint job using craft paints. I followed up with sealing with Minwax Polyurethane in a rattle can for a fuel proof finish. It is nothing fancy, but I had wanted one for a while and with the mention of an upcoming contest got my butt in gear. SO here it is. Looking forward to trying it out.
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Re: Homemade Tether Car
Good job, hope it's a good runner.
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Re: Homemade Tether Car
Thank you. Fingers are crossed as I had not yet run this engine since rebuilding and polishing it up. Even if it goes round and round at a snails pace, I'll still be happy.
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Pretty similar to a kit that Blackhawk Models used to sell. Is it balsa or pine? Heavier might be better once it gets up some momentum.
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I do not honestly know what kind of wood it is. It was just a scrap piece I had laying around that was the right size. Sorry I could not be of help.
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Detection of autoreactive T cells using MHC II tetramers is difficult because of the low affinity of their TCR. We have generated a class II tetramer using the IAs class II molecule combined with an autoantigenic peptide from myelin proteolipid protein (PLP; PLP139–151) and used it to analyze myelin PLP139–151-reactive T cells. Using monomers and multimerized complexes labeled with PE, we confirmed the specificity of the reagent by bioassay and flow cytometry. The IAs tetramers stimulated and stained the PLP139–151-specific 5B6 TCR transgenic T cells and a polyclonal cell line specific for PLP139–151, but not a control T cell line specific for PLP178–191. We used this reagent to optimize conditions to detect low affinity autoreactive T cells. We found that high pH (∼8.0) and neuraminidase treatment enhances the staining capacity of PLP139–151 tetramer without compromising specificity. Furthermore, we found that induction of calcium fluxing by tetramers in T cells may be used as a sensitive measure to detect autoreactive T cells with a low affinity. Taken together, the data show that the tetrameric reagent binds and stimulates PLP139–151-reactive T cells with specificity. This tetrameric reagent will be useful in studying the evolution of PLP139–151-specific repertoire in naive mice and its expansion during the autoimmune disease experimental autoimmune encephalomyelitis.
Major histocompatibility complex molecule-peptide tetramer technology has been used to detect Ag-specific T cells during an immune response in vivo. Although there has been much success in generating class I tetramers, specific class II tetramers have been rather more difficult to generate. (1, 2, 3, 4, 5). Work with class II tetramers already suggests that the detection of autoreactive cells is more difficult than the detection of foreign Ag-specific cells, because of the smaller size and lower affinity of the autoreactive T cell repertoire in the peripheral immune compartment (6, 7, 8, 9). Furthermore, some of the peptides that are relevant in experimental models of autoimmunity bind with low affinity to their MHC class II molecules, as shown for IAg7 or IAu (10, 11, 12).
We and others have identified proteolipid protein (PLP)3139–151 and PLP178–191 as the major immunogenic and encephalitogenic epitopes that induce murine experimental autoimmune encephalomyelitis in the SJL mouse strain (13, 14, 15, 16). Although these epitopes of PLP bind MHC II IAs with similar affinities, the immune response to PLP139–151 is always dominant (16). Recently, based on limiting dilution analysis, we reported that naive SJL mice have a high frequency (1/20,000) of PLP139–151-reactive CD4 T cells circulating in the periphery (17). It is very clear that limiting dilution analysis does not give a true estimate of Ag-reactive cells in the repertoire, and to be able to determine more precisely the frequency of PLP139–151-reactive cells in the naive peripheral repertoire and to monitor changes in the frequency of these cells during the development of an autoimmune disease, we generated PLP139–151/IAs tetramers.
In this study, we report generation of PLP139–151/IAs tetramers, which stain PLP139–151-reactive cells with specificity. We have defined optimum conditions for staining using MHC II tetramers specific for a self-Ag. Several factors influence the staining with class II IAs multimers, including the pH of the staining medium and neuraminidase (NA) treatment during the staining reaction. We show that analysis by flow cytometry identifies only a fraction of autoantigen-specific T cells, which are able to interact with specific peptide-MHC complexes as judged by studying the ability of these cells to flux calcium.
Materials and Methods
Female SJL mice (4–8 wk of age) were obtained from The Jackson Laboratory (Bar Harbor, ME). They were housed in specific pathogen-free conditions at the animal facility of the Harvard Institutes of Medicine. The mice were kept in accordance with the guidelines set out in the animal protocols of Harvard Medical School. The 5B6 TCR transgenic (Tg) mice specific for myelin PLP139–151 have been described previously (18) and were also housed at the same facility.
Peptides, immunization protocols, and generation of short-term polyclonal T cell lines and 5B6 TCR Tg T cells
The PLP peptides, PLP139–151 (HSLGKWLGHPDKF), PLP178–191 (NTWTTCQSIAFPSK), and Theiler's murine encephalomyelitis virus (TMEV) VP270–86 (WTTSQEAFSHIRIPLP) were synthesized using F-moc chemistry (QCB BioSource International, Hopkinton, MA). All peptides were HPLC purified. The identity of peptides was confirmed by mass spectroscopy, and they were dissolved in sterile water before use. For immunizations, 100 μg of each peptide was administered emulsified in CFA in multiple sites s.c. Ten days later, the regional lymph nodes were pooled, and single-cell suspensions were obtained after lysing the erythrocytes using 1× ammonium chloride potassium buffer (BioWhittaker, Walkersville, MD). The lymph node cells (LNC) were restimulated in 24-well plates at a density of 5 × 106 cells/ml in complete medium supplemented with 20 μg of the corresponding peptides and APC as described (16). The complete medium consisted of DMEM, 10% FCS, 2 mM l-glutamine, 10 mM HEPES, 2 mM mercaptoethanol, penicillin (1000 U/ml), and streptomycin (1 μg/ml) (BioWhittaker). After 2 days in culture, complete medium containing IL-2 (5 μM; AB Biotechnologies, Columbia, MD) was added into the cultures. These cultures were then restimulated with the peptide (20 μg) and APC every 2 wk, two to three times. The 5B6 Tg T cells specific for PLP139–151 are described elsewhere (18). A T cell line from the LNC of naive 5B6 Tg T cells was generated by repeated stimulation with PLP139–151 as above.
MHC II IAs constructs and generation of soluble MHC molecules and IAs multimeric complexes
The cDNA constructs were made by PCR using plasmids encoding IAs α- and β-chains. The cDNAs encoding the signal peptides and extracellular domains of the IAs α- and β-chains were amplified first, and the sequence for PLP139–151 peptide (5′-CATTCTTTGGGAAAATGGCTAGGACATCCCGACAAGTTT-3′) was then introduced in the IAs β sequence by overlapping PCR. The amino acid sequences of the amino-terminal region of the IAs β-chain/PLP139–151 peptide/linker sequences were as follows: MALQIPSLLLSAAVVVLMVLSSPGTEG (IAs β leader); GDSGT (linker); HSLGKWLGHPDKF (PLP139–151); and GSGSGS (linker). The IAs β carboxyl-terminal residue linked to the 7-aa linker is "K." The sequences corresponding to Fos and Jun dimerization domains were amplified by PCR from IAg7 constructs (19). They were attached to the 3′-end of IAs α and IAs β extracellular domains, respectively, through a 7-aa linker (Val-Asp-(Gly)5) that contained a SalI restriction site. The amino acid sequences for Fos and Jun linkers were the following: Fos, VDGGGGG (linker at the amino-terminal end)-Fos sequence-SAGGG (linker at carboxyl-terminal end); and Jun, VDGGGGG (linker at the amino-terminal end)-Jun sequence-no linker at the carboxyl-terminal end. The identities of these constructs were confirmed by sequencing. The IAsα cassette containing the Fos domain was cloned into the BamHI site of pAcAB3 vector (BD PharMingen, San Diego, CA), while the IAsβ cassette containing the peptide and Jun dimerization sequence was introduced into the BglII site. The Bir A site (LGGIFEAMKMELRD) for biotinylation was incorporated into the 3′-end distal to the Fos sequence in the α-chain. To generate IAs-TMEV70–86, the sequence for PLP139–151 peptide was excised by using KpnI and BamHI enzymes and replaced with a nucleotide sequence encoding TMEV70–86 (5′-TGGACCACCAGCCAGGAAGCGTTCAGCCATATCCGCATCCCGCTGCCG-3′).
The soluble MHC molecules were expressed in a baculovirus expression system by infecting Sf9 insect cells (BD PharMingen). The expression of MHC α- and β-chains was confirmed by SDS-PAGE and Western blot analysis using rabbit polyclonal antisera for Fos and Jun (1:500). To identify the leucine zippers Fos and Jun, we raised antisera against them in rabbits using peptides coding for Fos and Jun zippers, which were kindly provided by Dr. P. Kim (Massachusetts Institutes of Technology, Cambridge, MA). Large-scale production of soluble MHC molecules was accomplished by using spinner flasks connected with spargers (Bellco Biotechnology, Vineland, NJ). The Sf9 cells at a density of 1 × 106 cells/ml were infected using a high-titer supernatant of the rIAs baculovirus (1 × 108 PFU/ml). The cells were grown in serum-free insect cell medium (BD BaculoGold Max-XP; BD PharMingen) for 3–5 days. The MHC proteins in the baculovirus-infected supernatants were concentrated using a Prep/Scale-TFF 1 ft2 cartridge (30k; Millipore, Bedford, MA). The proteins were then purified on an affinity column prepared using MKS4 mAb (American Type Culture Collection, Manassas, VA), and the proteins were concentrated to >5 mg/ml using Centricon-plus 20 columns (Millipore). The IAs recombinant protein yield was ∼1 mg/L.
Biotinylation of MHC monomers was conducted at 30°C overnight using biotin protein ligase enzyme at an optimized concentration of 25 μg/10 nmol of substrate according to the manufacturer's recommendations (Avidity, Denver, CO). The unincorporated biotin was removed by extensive dialysis with 1× PBS (pH 7.5). The multimeric complexes were then generated using streptavidin (SA)-PE (Molecular Probes, Eugene, OR) at different ratios between biotinylated protein and SA-PE ranging from 2:1 to 16:1. Native gels (6% acrylamide) using unconjugated SA confirmed the extent of biotinylation. As a negative control, a tetrameric reagent was also derived for a foreign Ag, TMEV VP270–86 that also binds IAs (20, 21).
T cell proliferative response
Soluble IAs monomers, IAs-PLP139–151 or IAs-TMEV70–86, were coated to 96-well plates overnight in bicarbonate buffer (pH 9.0). The plates were washed twice with 1× PBS, and freshly activated polyclonal T cells specific for PLP139–151, PLP178–191, and TMEV70–86, or 5B6 Tg T cells were added at a density of 2 × 105 cells/well. After 48 h of culture, the cells were pulsed with 1 μCi of [3H]thymidine, and 16 h later, the plates were harvested using a Wallac (Gaithersburg, MD) liquid scintillation counter. The proliferative response was measured as cpm and expressed as mean cpm in triplicate wells.
MHC II IAs tetramer staining based on flow cytometric analysis
Three to 6 days following the activation of polyclonal T cell lines or 5B6 Tg T cells, the cell suspensions were enriched for T cells by Ficoll-Hypaque density gradient centrifugation. The cells were incubated with IAs multimers in DMEM supplemented with IL-2 (5 μM) at 37°C for 3 h at a concentration of 30 μg/ml. The cells were then washed once with 4 ml of buffer containing 1× PBS, 2% FCS, and sodium azide (0.1%). This was followed by staining with anti-CD4 Ab (clone RM4.5, CD4-APC) and 7-AAD (BD PharMingen). After incubating at room temperature (RT) for 20 min, the cells were washed, as above, and acquired using a FACSort (BD Biosciences, San Jose, CA). These conditions were used for all of the tetramer staining reactions, unless otherwise indicated. To define the optimum conditions for tetramer staining, the following parameters were studied: concentration of tetramers, duration of staining, temperature requirement (4–37°C), amount of FCS (0–10%), pH of the staining medium (DMEM containing IL-2) at the initiation of culture (5.0–9.0), and the effect of anti-TCRcβ (clone H57597; BD PharMingen) and anti-CD3 (clone 145-2C11; BD PharMingen). For all tetramer staining reactions, we did not purify the multimeric complexes, because there was no difference between staining with the unpurified or the purified material obtained from gel filtration columns (Superose 12; Pharmacia, Piscataway, NJ) (data not shown). The number of tetramer-PE (PLP139–151 or TMEV70–86)-positive cells was then determined in live (7-AAD-negative), CD4-positive populations.
NA treatment
22). The cells were then washed in 1× PBS and used for flow cytometric analysis. The effect of NA on lymphocytes was analyzed by forward-scatter (FSC) vs side-scatter (SSC) profiles, and the expression of surface markers, namely CD3 (clone 145 2C11), CD4 (clone RM4.5), CD25 (clone 7D4), and CD69 (clone H1.2F3) and their corresponding isotype controls (all FITC-conjugated; BD PharMingen). The results were then compared between NA-treated and NA-untreated cell populations. Before tetramer staining, the polyclonal T cells, which were either rested or freshly activated with peptides and APC for 5 days, were obtained by Ficoll-Hypaque density gradient centrifugation and used for NA treatment. The tetramer staining was conducted on NA-treated cells, as described above, with or without anti-TCRcβ (clone H57597) or anti-CD28 (clone 37.51) (BD PharMingen).7 cells/ml were used for NA treatment at a concentration of 0.7 U/ml (NA type X from Clostridium perfringens; Sigma-Aldrich, St. Louis, MO) in serum-free DMEM at 37°C for 1 h (
Measurement of calcium fluxing
Flow cytometric analysis.
The T cells enriched from SJL mice immunized with PLP139–151 were restimulated twice in vitro using the PLP139–151 peptide and APC. Five days after the second stimulation, the T blasts were obtained by Ficoll-Hypaque density gradient centrifugation. The cells at a density of 106–107 cells/ml were loaded with the membrane-permeable form (penta-aceto-oxymethyl (AM) ester) of Indo-1 (2 μg/ml) for 1 h at 37°C in DMEM. Cellular esterases cleave the AM moiety, resulting in the trapping of Indo-1 in the cell (23). After washing in 1× PBS, the cells were resuspended in DMEM to a density of 3 × 106 cells/ml and stored in the dark for 15 min at RT until analysis. The release of intracellular Ca2+ was determined using a FACSVantage SE (BD Biosciences). The cytometer was optimized using ionomycin (2 μg/ml; Sigma-Aldrich) as a positive control such that the Indo-1 emission ratio of 405 over 490 nm was maximized, and the ratio was plotted against time. To study the tetramer-induced response, the cells were acquired for ∼1 min to obtain the baseline response, and the IAs tetramers were added into the cell suspensions in 1 ml at a concentration of 30 μg/ml. The response was monitored for ∼9 min. The sample tubes containing the cells and reagents were kept at 37°C during acquisition of the data.
Confocal microscopy.
The PLP139–151-specific T cell lines were used for measuring the Ca2+ fluxing by confocal microscopy using Fluo-4 which excites at 488 nm and emits at 516 nm. Cells at a density of 1 × 107/ml were loaded with AM esters of Fluo-4 (2 μg/ml), and the mixture was incubated at 37°C for 1 h in DMEM (23). Following washing in 1× PBS, the cells were rested at RT for 15 min in the dark. The adequacy of cell loading was tested by treating a sample with ionomycin (2 μg/ml), and the cell suspension (20 μl) was loaded onto 0.25 mm, express-lane slides (Plus Gold; Grace Bio-Labs, Bend, OR). The fluorescence intensity of the responding cells was measured using a pseudocolor plot in an LSM 510 laser-scanning inverted microscope equipped with argon ion lasers (Zeiss, Heidelberg, Germany). To determine the response for IAs tetramers, the PLP139–151 and TMEV70–86 tetramers were added at a concentration of 30 μg/ml, and the response was measured immediately based on the intensity of pseudocolor (green). The responses to the above stimuli were measured by counting both the number and the intensity of each responding cell by using IPLab software (Sanalytics, Fairfax, VA). The percentage of PLP139–151 tetramer-induced Ca2+ fluxing in the responding cells was calculated as follows: (number of cells responding to PLP139–151 tetramers − number of cells responding to TMEV tetramers)/(number of cells responding to ionomycin − number of cells responding to TMEV tetramers) × 100.
Generation of IAs multimers
The IAs monomers could be successfully expressed in a baculovirus expression system. Soluble IAs proteins affinity purified on MKS4 columns were resolved by SDS-PAGE, and staining with Coomassie blue revealed two major bands of the expected sizes. These were ∼40 and ∼28 kDa, respectively, and represented the IAs α- and β-chains. This suggests that the MHC proteins obtained from the MKS4 Ab column were pure (Fig. 1⇓A). The identity of the bands was confirmed by Western blotting using the polyclonal anti-Fos and anti-Jun rabbit antisera, because IAs α- and β-chains were linked to Fos and Jun leucine zipper domains, respectively (Fig. 1⇓B). The presence of PLP139–151 or TMEV70–86 peptide in the soluble IAs proteins was confirmed by N-terminal amino acid sequencing (Molecular Biology Core Facility, Dana-Farber Cancer Institute). The formation of IAs multimeric complexes was verified on a native nonreducing gel using unconjugated SA. To form tetramers, the biotinylated MHC proteins and SA were mixed at ratios ranging from 2:1 to 16:1. The IAs multimers included di-, tri-, and tetrameric complexes, and only ∼50–60% of the IAs monomers was biotinylated (data not shown). Consistently, the ratios of 4:1 to 8:1 generated the most multimeric complexes. It has been suggested that the use of the term "tetramer" is a misnomer, given the fact that higher-order complexes are present when PE-labeled SA is used for multimerization (3). Hence, in this article, the terms tetramers and multimers have been used interchangeably.
Specificity of IAs tetramers
We have provided two lines of evidence to show that the PLP139–151- or TMEV70–86-MHC proteins identify T cell populations specific for appropriate peptide-MHC complexes. First, the in vitro proliferation assay presented in Fig. 2⇓ confirms that the IAs monomers stimulate their corresponding polyclonal T cell lines but not lines specific for another autoantigen. The specificity of the PLP139–151-IAs molecule was further verified by stimulating Tg T cells obtained from a PLP139–151-specific SB6TCR Tg mouse, and as expected, activated the 5B6 Tg T cells. These cell lines showed no response to the control TMEV-IAs tetramers (Fig. 2⇓). As a control, we used polyclonal T cells specific for PLP178–191 which were not stimulated by either PLP139–151 or TMEV70–86 monomers confirming that the IAs monomers were highly specific (Fig. 2⇓). Second, using flow cytometry, the tetrameric reagents (PLP139–151 or TMEV70–86) were used to stain various T cell lines including a polyclonal PLP139–151-specific T cell line, T cells from the PLP139–151-specific 5B6 Tg mice, a TMEV VP270–86-specific T cell line, and a control polyclonal cell line specific for PLP178–191. As shown in Fig. 3⇓, TMEV70–86 tetramer stained the TMEV-specific T cell line and not any other cells, whereas PLP139–151-IAs tetramer stained the PLP139–151-specific polyclonal T cell line and the 5B6 TCR Tg T cells. Neither of the tetramers stained PLP178–191-specific T cells (Fig. 3⇓). The PLP139–151 tetramers stained only 46% of 5B6 Tg T cells. This may be due to a fraction of cells in the Tg T cells that express endogenous TCR α- and/or β-chains.
Optimum conditions for IAs tetramer staining
Because we want to use the tetramers to analyze the evolution of the autoimmune response to PLP139–151 in vivo, we optimized staining conditions to detect maximal numbers of autoreactive cells with specificity. The conditions we examined included the concentration of tetramer, duration of staining, temperature requirement, the presence of FCS, and the pH of the staining reaction (Fig. 4⇓). Optimization was conducted using T cell lines that contain a polyclonal population of Ag-specific CD4 T cells, conditions which will closely mimic those expected in normal SJL mice immunized for the induction of experimental autoimmune encephalomyelitis. As a negative control, the TMEV70–86 tetramer-PE was used and the results were compared between PLP139–151 and TMEV70–86 tetramers.
We tested the concentration of tetramer for staining from 15–120 μg/ml at 37°C for 3 h. It was found that the staining with tetramer increased proportional to the concentration of tetramer added in the staining reaction (Fig. 4⇑A). However, as the amount of tetramer increased, the nonspecific binding of the unrelated tetramer TMEV70–86 also increased. Based on this data, a concentration of 30 μg/ml was chosen for increased specificity and for economy of the reagent. The tetramer was tested by staining for various times (1.5–6 h), but a duration of 3 h appeared sufficient for maximum tetramer staining with the PLP139–151 tetramer (Fig. 4⇑B). Staining was also tested at various temperatures including RT and 4 and 37°C. Although the percentage of tetramer-positive T cells was greater when the staining was done at RT, 37°C was preferred, because the background staining for TMEV tetramer was negligible (Fig. 4⇑C). We tested the effect of the presence of FCS in the staining reaction. We noticed that FCS in the medium appeared to enhance nonspecific staining with the control TMEV tetramer. Although this nonspecific binding varied between experiments at different concentrations of FCS, the least binding with the control TMEV tetramers was obtained when no FCS was used in the medium (Fig. 4⇑D). We then tested the effect of pH on the staining reaction. We observed that the best tetramer staining was obtained when the tetramers were incubated with cells in DMEM (pH 8.0) with a minimal background for the control TMEV tetramer (Fig. 4⇑E).
Lastly, it has been suggested that coincubation of cells with anti-TCRcβ enhances tetramer staining (2). Hence, we compared the effects of two Ab, namely anti-CD3 (145 2C11) and anti-TCRcβ (H57597), on staining with the tetramer. Of these, while anti-CD3 Ab showed no effect on tetramer staining, anti-TCRcβ Ab marginally increased tetramer staining. This was observed with both PLP139–151-specific polyclonal and 5B6 Tg T cells (data not shown).
Effect of NA treatment on tetramer staining
NA has been used to enhance staining of CD8 cells by class I tetramers (24). We tested the effect of NA treatment on CD4 cells using the PLP139–151-specific T cell line. We noticed that addition of NA in the reaction mixtures resulted in change in FSC vs SSC profiles, when NA-treated and NA-untreated samples were compared. For example, the percentage of cells in gate 2 in NA-treated samples was increased by 2-fold to 11% as compared with 5% in NA-untreated sample, indicating that there is an increase in both size and granularity of the treated cells. This suggests that the NA treatment may have activated the cell populations (Fig. 5⇓A). Corresponding to this shift, the percentage of cells in gate 1 was reduced in NA-treated sample (74% in NA-treated vs 88% in NA-untreated cultures) (Fig. 5⇓A). To confirm whether the NA treatment activates T cells, the activation status of the cell populations was verified in both NA-treated and untreated populations by staining for the expression of the activation markers (CD25 and CD69). Although the differences in the expression levels of these markers were not statistically significant, the percentage of CD25- and CD69-positive T cells increased in NA-treated cells. Although the percentage of CD4- or CD3-positive T cells did not change significantly between NA-treated and untreated samples, a minor population of CD4high subset was evident in the NA-treated samples (data not shown).
We then determined the effect of NA on IAs-PLP139–151 tetramer staining. This was addressed in two ways, using a rested and activated polyclonal T cell line specific for PLP139–151. The tetramer staining was done first on rested cells, where the percentage of tetramer-positive T cells is generally low (Fig. 5⇑B). The cells were treated with NA, followed by staining with tetramers. Should the NA influence the tetramer staining, then the percentage of tetramer-positive T cells was expected to be greater in NA-treated samples. As predicted, the PLP139–151-positive T cells in the resting cell population was 0.22% compared to 2.15% following NA treatment (Fig. 5⇑B). Although the background staining for TMEV tetramer also increases, the percentage of TMEV70–86 tetramer-positive NA-treated cells was negligible (0.2%) in relation to the staining intensity obtained with PLP139–151 tetramer. We then determined the effect of NA on activated T cells, which were freshly stimulated, with PLP139–151 and APC. Three days following activation, the tetramer staining was done using PLP139–151 or TMEV70–86 tetramers with or without addition of anti-TCRcβ and anti-CD28. As shown in Fig. 6⇓, the NA pretreatment enhanced the tetramer staining for PLP139–151 by 2-fold in NA-treated cells (NA-untreated, 2.4%, vs NA-treated, 4.8%). It is also evident that in all the PLP139–151 tetramer-treated samples, the CD4 cells appeared to increase in size, presumably due to the tetramer-induced activation. The addition of anti-TCRcβ together with tetramers enhanced the tetramer binding regardless of tetramers (Fig. 6⇓). Nonetheless, the specificity of the tetramer staining was still maintained (PLP139–151 tetramer, NA treatment alone, 3.4%, vs NA-treatment plus anti-TCRcβ, 6.2%; TMEV70–86 tetramer, NA-treatment alone, 0.1%, vs NA-treatment plus anti-TCRcβ, 0.3%). The effect of anti-CD28 treatment on tetramer staining was not significant (Fig. 6⇓).
Tetramer preferentially stains CD4high T cells
The LNC from SJL mice immunized with PLP139–151 were restimulated twice in vitro every 2–3 wk using peptide and APC. In freshly activated T cells, as expected, there was the appearance of two live CD4 populations, namely CD4low and CD4high (Fig. 7⇓). In these subsets, we determined the frequency of PLP139–151-reactive cells in the CD25+ population compared with the frequency of TMEV70–86 tetramer-positive cells. The frequency of PLP139–151 tetramer-positive cells was higher in the CD25+CD4high population (8.6%) compared with that of the CD25+CD4low subset (0.5%). The background staining with TMEV70–86 tetramers was negligible, although also higher in the CD25+CD4high population (0.6%) compared with that of the CD25+CD4low population (0.04%) (Fig. 7⇓). These data are consistent with the expected enrichment of Ag-specific cells in the CD25high subset, as well as with recently reported data showing that activated cells have a CD4high phenotype (3, 25). However, the differences we observed in tetramer staining of rested and activated cell lines led us to investigate whether analyzing cells for a functional interaction with tetramer might be a more sensitive measure of their presence than flow cytometric staining alone.
Calcium fluxing as a measurement of tetramer binding
We tested whether activating with tetramer could initiate Ca2+ fluxes in responding T cells and whether this could be used as a more sensitive measure of the frequency of detecting autoreactive T cells. This was examined by measuring intracellular Ca2+ using both flow cytometric analysis and confocal microscopy. To show that activation with tetramer-MHC could activate cells specifically, we used Indo-1 for flow cytometric analysis, which provides a ratiometric measure of Ca2+ fluxing (23). We observed that the response occurred in a PLP139–151-specific cell line in <1 min after the tetramer was added, and this response persisted for ∼5 min before returning to resting levels (Fig. 8⇓A). By comparison, the TMEV tetramer did not induce Ca2+ fluxing over the basal level in the PLP139–151-specific T cell line, indicating that the response was specific. This was shown clearly when the mean Indo-1 ratios of calcium-fluxed responses to PLP139–151 and TMEV70–86 tetramers were overlaid (Fig. 8⇓A).
To quantitate the frequency of responding cells, we used confocal microscopy with Fluo-4 (26) and a PLP139–151-specific cell line. As shown in Fig. 8⇑B, the PLP139–151 tetramer induced Ca2+ fluxing in a ∼3-fold greater number of cells than the TMEV-treated sample. These responses were compared with that of ionomycin (positive control), which is an ionophore, and it induces Ca2+ response in all the viable loaded cells. The response to ionomycin was taken as 100%, and this was used to normalize the responses to the IAs tetramers. We found that the PLP139–151 tetramer induced the release of intracellular Ca2+ in 36% of the total number of cells. In parallel by flow cytometry, only 4% of the cells were specifically stained by PLP139–151 tetramers. Thus, confocal evaluation allowed us to detect a higher frequency of Ag-specific T cells than did flow cytometric analysis. When the mean intensity of the responses was compared between different stimuli, it was relatively greater with ionomycin than IAs tetramers (data not shown). However, the mean intensities of the responses between PLP139–151 and TMEV70–86 tetramers were similar.
We have generated IAs multimeric complexes for both self (PLP139–151) and foreign (TMEV VP70–86) Ags that allow us to detect the PLP139–151-specific T cells. TMEV tetramers, besides being used as a negative control, will allow us to detect T cells specific for a foreign Ag and to compare this to the induction of self T cell responses. The development of MHC II tetramers has been difficult for many reasons, including the instability of soluble MHC II molecules particularly complexed with self-Ag, a lack of suitable Ab to purify the soluble MHC proteins, and the inadequate capacity of these reagents to stain naive or low-affinity self-reactive T cells. In our hands, we failed to express the soluble IAs monomers for PLP139–151, presumably due to the instability of the α- and/or β-chains of IAs, with the PLP139–151 peptide to form stable dimers in the absence of the transmembrane domains. We could overcome this difficulty by adding the Fos and Jun leucine zipper domains to the MHC II α- and β-chains, respectively. Use of these leucine zipper dimerization domains greatly facilitated assembly of the heterodimer, an approach that has previously been used for the expression of soluble IAg7, HLA-DQ8, and HLA-DR2 heterodimers (19, 27, 28).
Several lines of evidence suggest that the tetramers we have generated are specific for appropriate T cells in that they can activate relevant cells in a bioassay, stain the cells specifically, and induce Ca2+ fluxes in the specific T cells. Furthermore, development of a control tetramer for TMEV will also allow us to compare self- and foreign Ag-reactive repertoires. As reported by others (2, 3, 25, 29, 30), the tetramer staining in our system was best seen in activated cells corresponding to the CD25CD4high subset. The reason for this activation dependency for binding of MHC II tetramers to the TCR is not clear. Conceivably, activation results in reorientation and configuration of TCR and cell surface molecules leading to an increase in the avidity of TCR-MHC binding. Whether higher expression of CD4 on activated T cells further contributes to binding avidity is not clear yet. But in studies of non-autoantigen-specific tetramers, CD4 has not been shown to play a critical role in tetramer binding (1). In agreement with the data published on anti-TCRcβ (H57597), we also demonstrated an enhancing effect on tetramer staining, although in our case the enhancement was marginal (data not shown). Nonetheless, this effect could still be beneficial, especially when the rare low-avidity, autoreactive T cells in the naive repertoire are to be examined. It is not known how anti-TCRcβ influences tetramer staining. It is unlikely that the anti-TCRcβ-mediated effect can be ascribed to the activation phenomenon, because the clone 145 2C11, which is also an activating Ab, when used in parallel with tetramer, did not increase the staining; rather, it appeared to reduce the tetramer staining in both PLP139–151 and 5B6 Tg T cell systems (data not shown).
We evaluated several factors to optimize the conditions for tetramer staining: concentration (30 μg/ml), duration (minimum 3 h), incubation temperature (37°C), reaction medium containing IL-2 without FCS, and pH were all critical in getting maximal staining without increasing the background. Although similar conditions have been described for other MHC II tetramers with respect to concentration, duration, and the temperature requirements (1, 2, 3, 31, 32), we observed in our system that the FCS in the staining medium enhanced nonspecific binding even at low concentrations. To maintain healthy cells in the cultures, we routinely carry out the tetramer staining in DMEM supplemented with IL-2. Similarly, we report here that the pH of the medium appears to be critical for tetramer binding in that the IAs tetramer staining was better when the pH of the medium was relatively alkaline. We tested a wide pH range of the culture medium (pH 5.0–9.0), and the best staining was obtained when the pH was between 8.0 and 8.4 with negligible background staining with the control TMEV IAs tetramer and without compromising cell viability. We opted for pH 8.0 at which the viability of the cells was maximal as determined by 7-AAD staining. We speculate that the alkaline pH may facilitate better accessibility of T cells to the tetramers.
We used NA in our system to test whether it enhances the tetramer staining as demonstrated for the MHC I system (24). We found NA at 0.7 U/ml as optimum based on cell viability analysis using 7-AAD and tetramer staining (data not shown). NA has been used to enhance B cell function, and it acts by removing the sialic acid from the surface of the cells (22, 33). It is suggested that the removal of sialic acid results in a reduction in the net charge of cells in the interacting populations, in our case APC and T cells, which leads to enhanced cell-cell adhesiveness, which is critical for T cell stimulation (22). Our preliminary data suggest that NA treatment may partially activate T cells, and that may be the reason for increased tetramer binding. Our data would suggest that the NA might induce changes in the membrane fluidity and geometry of the surface molecules involved in TCR-MHC-peptide interaction. Although there was no significant increase in CD69 and CD25 expression, the change in size is suggestive of this phenomenon.
In this study, we have demonstrated that the tetramers induce Ca2+ fluxing in an Ag-specific manner which can be detected both by flow cytometry and confocal microscopy. The occurrence of Ca2+ flux following tetramer binding indicates engagement of the TCR by the tetramer, suggesting that the binding of IAs tetramers to the Ag-specific T cells is functional. Given the fact that the binding of MHC II multimers to the autoreactive T cells is poor, the measurement of Ca2+ fluxing may provide a more sensitive measure to identify autoreactive T cells. It is likely that the MHC II tetramers interact with most of the Ag-specific TCRs and induce Ca2+ fluxing even when conventional flow cytometry may not detect these cells. Therefore, it appears from our data that, in a rested polyclonal mixed T cell line, raised against the autoantigen PLP139–151, there is a population which can respond to PLP139–151 tetramer as measured by Ca2+ flux but that is not labeled with the tetramer as detected by conventional flow cytometry. Thus, enumerating and sorting the tetramer-positive cells based on Ca2+ flux may provide an additional tool to the traditional FACS-based staining of Ag-specific cells. The Ca2+ flux together with confocal microscopy will allow phenotypic characterization of responding T cells. In contrast, flow cytometric analysis based on Ca2+ flux will allow one to sort the Ag-specific T cells in mixed cell subsets much more rapidly.
In summary, we have defined an MHC II tetramer system that allows us to detect autoreactive T cells using PLP139–151 as a model self-Ag. In this study, we show that, apart from dose, incubation time, and temperature, the pH of the medium and the presence of FCS are critical for tetramer binding. Though the differences obtained with these changes in the protocol may be marginal, these incremental changes may be especially important when staining the rare Ag-specific T cells in the naive repertoire or in detecting low-avidity autoreactive T cells. We found that the NA treatment enhances the tetramer staining for PLP139–151 substantially, without loss of specificity. Furthermore, we show that the IAs tetramer induces Ca2+ fluxes in an Ag-specific manner and that cells not detected by flow cytometry can flux Ca2+ in response to tetramer binding. Hence, these strategies could be adapted for the detection of rare Ag-specific low-avidity autoreactive T cells in vivo for detecting the evolution of the autoreactive repertoire.
We thank Robert McGilp for his technical assistance in flow cytometric analysis of calcium fluxing.
↵1 J.R. is a recipient of Advanced Postdoctoral Fellowship by the National Multiple Sclerosis Society.
↵2 Address correspondence and reprint requests to Dr. Vijay K. Kuchroo, Room 706, Harvard Institutes of Medicine, Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115. E-mail address: vkuchroo{at}rics.bwh.harvard.edu
↵3 Abbreviations used in this paper: PLP, proteolipid protein; NA, neuraminidase; Tg, transgenic; TMEV, Theiler's murine encephalomyelitis virus; LNC, lymph node cell; SA, streptavidin; AM, penta-aceto-oxymethyl; RT, room temperature; FSC, forward scatter; SSC, side scatter.
- Received September 24, 2002.
- Accepted November 12, 2002.
- Copyright © 2003 by The American Association of Immunologists


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GP leaders to vote on support for fully salaried GP profession
The GPC could negotiate a new contract with Government under which GPs give up their independent contractor status and all go salaried, LMCs have suggested.
A motion to be presented by Oxfordshire LMC at the emergency LMC Conference at the end of the month suggests that because 'many younger GPs currently prefer to be salaried rather than partners', the GPC should look at 'future contractual models' which 'explore all options including movement away from independent contractor status'.
Although the Oxfordshire LMC motion is the only one scheduled for debate, several LMCs suggested similar motions to the agenda committee.
This included Mid Mersey LMC, which proposed a motion saying 'that the days of independent contractors delivering safe and sustainable general practice are coming to an end and calls upon the GPC to actively support the development of a salaried GP service'.
LMCs have voted on the topic before but the motion to move to a fully salaried service has always been voted down.
A Pulse survey of over 600 GPs last autumn found that support for a fully salaried model of general practice is growing, with 26% now in favour. However just over half, 54%, remained opposed.
The motion follows similar calls by former RCGP chair Professor Clare Gerada and NHS England in London, while NHS Hull CCG has also looked into plans for developing a fully salaried model - which it later discarded,
GPs in Scotland are already moving 'as close as possible' to a salaried service as part of the devolved health economy's new contract model.
The motion in full
OXFORDSHIRE: That conference believes that many younger GPs currently prefer to be salaried rather than partners, and that in looking at future contractual models, the GPC Executive Team should explore all options including movement away from independent contractor status.
Read the agenda in full here
Readers' comments (57)
I think most GPs are just in complete denial that the game has changed. Patients no longer care about 'the doctor patient relationship', they only care about what they want and only act how they do to get it. They are only out to get what they want and have no respect for us, even the ones that say or pretend they do- why else would they demand home visits when they are perfectly able to leave the house? Why else demand appointments immediately for trivial nonsense? And these are the ones that pretend to respect us.
If they respected us they wouldn't do these things.
as others have said, continuity of care is just another word for dependency. You get continuity of care by actually reviewing notes and not by simply knowing the patient for 30 years. Yes it's easier to say no to a drug seeker if you 'know them', but then it's not really that hard if you just say no and stick to it and they will leave.
The BMA Salaried GP scale is £55412 to £83617, model GP contract. However the consultant scale is £75249 to £101451 with a top up excellence award of between £2957 and £75,796. This to me means an actual pay scale of between £55412 and £177247. Please correct me if this is wrong. If you look at press releases consultant pay can be much higher than this.
Why can't a Salaried GP force have the same consultant pay scales?
To the last few comments- Some of you have touched on the crucial issue which is the notes. "Continuity" best suits those doctors who keep such poor medical records that only they really (if they can remember) know what is going on.
I have worked with these doctors. They are often well loved by their patients but keep inadequate notes, take weeks to deal with admin and seem to almost enjoy the chaos which they create by their selfish ways of working. Perhaps it boosts their ego and makes them feel important as they know people just have to work around them and make allowances.
Anyway if we are (and we are!) going to have 8-8, 7 day working, continuity will go out of the window. We will see increasing cross-cover and hub working. Only by sharing the record can this be achieved and I am already watching the panic in my locality of many of these rubbish doctors who refuse to share the record and are desperately trying to use patient confidentiality as a reason to protect themselves from the scrutiny of colleagues. It will be interesting to see how federated colleagues deal with the situation where they find substandard care. Let's see candour and transparency at work!
Having read the above comments - I for one can't wait for the day the current massive need for locums comes to an end. And before anyone bleats- I know that will be along time coming with current mantra of it's all about "supply and demand, dictated by market forces" blah-dee-blah-dee-blah.
It would seem that locum work is the 'dependency' that many GPs are being fed is the way to a utopian existence. In fact in our present predicament it is just the outcome of dysfunctional system. If we all decided to 'do it solely for the money' and forget about the silly bits like 'admin, meetings, pathology results' why not train to be a banker, and be a first aider at weekends. I would have said paramedic or A+E doc if 'whizz bang wallop, in and out' is what medicine should be for you, till I remembered that silly bits like 'admin, meetings, pathology results' is part of their responsibilities too!!
To some continuity of care has a purpose, to others clearly not. Each to their own, but it seems that we are in danger of throwing the baby out with bath water if attitudes persist that this fundamental aspect of GP'ing is diminished in the quest to 'earn a few more bucks'.
Anonymous | GP Partner17 Jan 2016 12:23pm
I am one of those mercenary locums who you might think does not want to do the extra stuff. I do not consider these items of work to be silly at all. Quite the opposite in fact. I object the complete lack of recognition of the massive workload all these bits represent and the fact that partners are not properly remunerated for them. If someone wants to pay me to do their repeat script reviews or sort out their admin then I'm good to go. The trouble is that nobody in power or leadership seems to place appropriate value on this work which they trivialise.
I am genuinely sorry if, as a partner you feel got at by people like me, it is not my intention. It is just that these extra tasks are almost exclusively associated with the drudgery that partnership entails.
No we don't want to get rid of them at all. We need a few people who wish to fight to the death. Who else would be prepared to be the last man standing holding the obsolete 25 year lease and paying the staff redundancies?
Just watch, the next 3-4 years will be a competition to see which partners can jump ship the quickest. Its like a game of pass the parcel with the prize being bankruptcy.
Your turn...
Ditch the contract comrades!


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Medlock & West Realty
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According to our records, this business is located at 812 Main St. in Van Buren (in Crawford County), Arkansas 72956, the location GPS coordinates are: 35.436606 (latitude), -94.351968 (longitude). Medlock & West Realty is categorized under Relocation Services (SIC code 6531). Known organization it/internet support is Alice Medlock. Current estimates show that the unit has a sales volume of $100,000 and staff of approximately 2 people. You can contact the company by phone at (479) 474-7474.
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Business Wire
Henry Schein Chairman and CEO Stanley M. Bergman Honored for Advancing Diversity and Inclusion in Dentistry
Mr. Bergman Recognized During President's Luncheon at Greater New York Dental Meeting
MELVILLE, N.Y.–(BUSINESS WIRE)–Henry Schein, Inc. (Nasdaq: HSIC) today announced that Stanley M. Bergman, the Company's Chairman of the Board and Chief Executive Officer, was honored at the President's Luncheon during this week's 2019 Greater New York Dental Meeting (GNYDM) for his leadership in advancing diversity and inclusion in dentistry. Mr. Bergman was presented with a Presidential Citation by Dr. Chad Gehani, President of the American Dental Association (ADA), and a special recognition award by Dr. Lauro Medrano, GNYDM's General Chairman.
"I am honored to accept these awards on behalf of Team Schein and the countless colleagues in the dental industry who have been strong allies in fostering an environment in which diversity and inclusion are woven into the fabric of the dental profession and our industry," Mr. Bergman said. "As a result, we have seen the profession expand to more fully reflect the ever-growing diversity of our country, to strengthen the practitioner-patient relationship, and to improve public health. I speak for all of Team Schein when I say thank you to the ADA and the Greater New York Dental Meeting for this honor."
To demonstrate its commitment to diversity and inclusion, Henry Schein's Women's Leadership Network joined several industry partners to host the Women Dentists Leadership Conference during the GNYDM. Featuring women leaders from dentistry and other key stakeholders, the program aimed to empower and mentor current and future women dentists to utilize their talents and reach their full potential.
"It is fitting to present Stanley Bergman with this award at the Greater New York Dental Meeting, in a city where diversity is valued and cultural differences are celebrated," said Dr. Medrano. "Congratulations to Stan and all of Henry Schein. With your leadership we have spent years opening doors and expanding opportunities to ensure that our industry reflects the populations and communities we serve."
Henry Schein has helped drive this conversation for more than two decades. For example, the Company was one of four founding corporate sponsors of the ADA's Diversity in Leadership Institute, which was created to foster leadership training and a corporate mentoring network within the dental industry in order to improve the business management skills of dentists from racial, ethnic, or gender backgrounds that have been underrepresented in such roles. Henry Schein also provides financial and logistical support to many outreach missions within the U.S. and abroad.
"Stanley Bergman and Henry Schein have been a driving force in expanding opportunities in dentistry for years, and our industry is stronger for it," said Dr. Gehani. "Stan has long been an invaluable partner in our work to make diversity and inclusion a cornerstone of the American Dental Association, and we congratulate him and Team Schein on this much-deserved award."
About Henry Schein, Inc.
Henry Schein, Inc. (Nasdaq: HSIC) is a solutions company for health care professionals powered by a network of people and technology. With approximately 19,000 Team Schein Members worldwide, the Company's network of trusted advisors provides more than 1 million customers globally with more than 300 valued solutions that improve operational success and clinical outcomes. Our Business, Clinical, Technology, and Supply Chain solutions help office-based dental and medical practitioners work more efficiently so they can provide quality care more effectively. These solutions also support dental laboratories, government and institutional health care clinics, as well as other alternate care sites.
Henry Schein operates through a centralized and automated distribution network, with a selection of more than 120,000 branded products and Henry Schein private-brand products in stock, as well as more than 180,000 additional products available as special-order items.
A FORTUNE 500 Company and a member of the S&P 500® and the Nasdaq 100® indexes, Henry Schein is headquartered in Melville, N.Y., and has operations or affiliates in 32 countries. The Company's sales from continuing operations reached $9.4 billion in 2018, and have grown at a compound annual rate of approximately 13 percent since Henry Schein became a public company in 1995.
For more information, visit Henry Schein at www.henryschein.com, Facebook.com/HenrySchein, and @HenrySchein on Twitter.
Ann Marie Gothard
Vice President, Corporate Media Relations
(631) 390-8169
Business Wire
Shortages of Low-Skill, Middle-Skill, and High-Skill Workers Causing Revenue Declines and Other Headaches for Employers, TrueBlue's Latest Study Finds
TACOMA, Wash.–(BUSINESS WIRE)–While there has been a lot of discourse around the shortage of high-skill workers in the U.S., a new study by staffing giant TrueBlue shows a significant percentage of employers are also struggling with deficits in low-skill and middle-skill workers – and dealing with a host of business challenges as a result.
According to TrueBlue's nationwide survey, which included nearly 1,500 managers (HR, operational, and business), skills shortages are widening across skills categories:
- 32% of managers can't find workers to fill low-skill positions (generally classified as those that may or may not require a high school diploma and require little to no experience)
- 46% can't find workers for middle-skill jobs (typically require some experience and continuing education such as college courses, an apprenticeship or certification, but don't necessarily require a four-year college degree)
- 35% can't find workers for high-skill jobs (typically require a four-year degree or higher and specialized experience)
"Low unemployment coupled with globalization, accelerated technology advancement, and evolving work models are creating talent deficits across all skill levels within organizations," said Patrick Beharelle, CEO of TrueBlue. "The skills supply is not keeping up with demand, which is fueling a greater intensity in an already competitive labor market and adversely impacting productivity, service quality, and revenue growth for businesses."
Impact of Talent Shortages on Businesses
The top three business challenges managers are experiencing due to prolonged job vacancies within their organizations include:
- Quality – More than a third of managers (35%) reported that extended job vacancies have caused lower product or service quality.
- Turnover – 25% have seen higher employee turnover.
- Revenue – 23% said their companies experienced a decline in revenue.
To address talent shortages and minimize associated business impact, 2 in 5 companies (41 percent) reported that they plan to raise compensation for entry-level workers and nearly half (46 percent) plan to train and hire the long-term unemployed in the coming year.
Survey Methodology
This SurveyMonkey survey was conducted online in the U.S. by TrueBlue between September 23 and October 15, 2019. It included 1,499 managers (HR, operations and general). The survey was across regions, industries, and company sizes.
About TrueBlue
TrueBlue (NYSE: TBI) is a global leader in specialized workforce solutions that help clients achieve business growth and improve productivity. In 2018, the company connected approximately 730,000 people with work. TrueBlue's PeopleReady segment offers on-demand industrial staffing services, PeopleManagement offers contingent and productivity-based, on-site industrial staffing and driver staffing services, and PeopleScout offers recruitment process outsourcing (RPO) and managed service provider (MSP) solutions to a wide variety of industries. Learn more at www.trueblue.com.
Jennifer Grasz
Vice President, Corporate Communications
(312) 840-6327
Business Wire
Law Firm of Estey & Bomberger Reports: Uber Says Nearly 6,000 Rapes, Sexual Assaults Occurred in Two-year Period
SAN DIEGO–(BUSINESS WIRE)–The law firm of Estey & Bomberger reported today that Uber's long-awaited sexual assault report was released Dec. 5, with the ride-hailing company admitting that 5,981* passengers and drivers were raped or sexually assaulted between 2017-2018.
"I applaud Uber for releasing the data that acknowledges there is a problem with sexual assaults occurring in rideshare. While we believe these assaults were preventable, Uber's report represents a tremendous step for ride-hailing safety," said Estey & Bomberger attorney Mike Bomberger. "I think there are many positive measures Uber is taking. However, Uber still has an obligation to help the victims who have been raped and assaulted and facing a lifetime of emotional pain. They will need ongoing therapy."
Estey & Bomberger represents more than 100 ride-hailing sexual assault victims.
"It's important to remember when reading this report that only one in three women report their sexual assault," Bomberger said. "Therefore, the number of women who have been sexually assaulted is certainly much higher than reported here."
Bomberger reiterated his call for all ride-hailing trips to be digitally recorded.
"We're pleased that Uber is now testing cameras in Texas. That's the real solution to this problem – if drivers know they're being recorded they won't rape and assault," Bomberger said.
Estey & Bomberger is asking Lyft and Uber sexual assault victims, along with former employees of the ride-sharing firms, to contact its office by calling 866-964-1708 or emailing email@example.com.
*statistic courtesy NPR "Uber Received Nearly 6,000 U.S. Sexual Assault Claims in Past 2 Years," Dec. 5, 2019.
for Estey & Bomberger
Ed Vasquez, 408-420-6558
Business Wire
Best's Market Segment Report: AM Best Maintains Global Reinsurance Market Outlook at Stable
OLDWICK, N.J.–(BUSINESS WIRE)–AM Best has maintained a market segment outlook of stable on the global reinsurance industry for 2020, citing a stabilized pricing environment — albeit at levels below long-term adequacy — the continuing alignment between traditional and third-party capital and ongoing stability in the global life reinsurance segment.
A new Best's Market Segment Report, titled, "Market Segment Outlook: Global Reinsurance," states that although rates in the non-life reinsurance market have improved modestly, pricing has not kept adequate pace with the changing risk dynamics, as illustrated by loss development from events such as hurricanes Irma and Maria and Typhoon Jebi, and potential losses from more-recent events (e.g., Hurricane Dorian). Property catastrophe pricing still is being driven by the availability of third-party capital; however, the increasing interdependence between traditional capacity and third-party capital through joint ventures, retrocession and direct ownership should serve to more closely align return objectives for the market overall. Third-party capital also represents a benefit in the form of stabilized earnings of rated balance sheets, due to tail risk being assumed by this capital.
Overall market conditions are improving, but AM Best remains concerned about insufficient rate adequacy relating to certain U.S. casualty lines, a steady decline in the benefit of favorable reserve releases and the pervasive low interest rate environment. The collective effect of these factors requires underwriting discipline, and failure to react to these pressures could adversely affect the segment.
The report outlines other factors that are driving the stable market segment outlook, including:
- AM Best believes alternative third-party capital will hold the line on future return expectations following the recent heavy catastrophe loss years;
- A decline in capital consumption and earnings volatility, due in part to the increased utilization of third-party capital in retrocessionaire programs;
- Greater emphasis on underwriting discipline due to pressure on interest rates and potential slower economic growth globally;
- Improving pricing momentum driven by higher loss costs, coupled with lower loss reserve redundancies;
- Increased demand for non-life reinsurance due to primary companies' recent loss experience, as well as new risk transfer opportunities and mergers and acquisitions;
- Stable operating performance among life reinsurers, which continue to maintain defensible market positions and offer services beyond risk transfer that create hurdles for new entrants.
To access the full copy of the overall global reinsurance briefing, please visit http://www3.ambest.com/bestweek/purchase.asp?record_code=292334.
Separate briefings on the non-life and life reinsurance segments can be viewed at:
- Global non-life: http://www3.ambest.com/bestweek/purchase.asp?record_code=292333.
- Global life: http://www3.ambest.com/bestweek/purchase.asp?record_code=292320.
To view a video with AM Best Associate Director Scott Mangan about the global reinsurance market segment outlook, please visit http://www.ambest.com/v.asp?v=globalreoutlook1219.
AM Best is a global credit rating agency, news publisher and data provider specializing in the insurance industry. The company does business in more than 100 countries. Headquartered in Oldwick, NJ, AM Best has offices in cities around the world, including London, Amsterdam, Dubai, Hong Kong, Singapore and Mexico City. For more information, visit www.ambest.com.
Copyright © 2019 by A.M. Best Company, Inc. and/or its affiliates.
Robert DeRose
Senior Director
+1 908 439 2200, ext. 5435
Greg Carter
Managing Director
+44 20 7397 0288
Michael Porcelli, FSA
+1 908 439 2200, ext. 5548
Christopher Sharkey
Manager, Public Relations
+1 908 439 2200, ext. 5159
Jim Peavy
Director, Public Relations
+1 908 439 2200, ext. 5644
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If you want your card to provide a greater level of safety, you require to verify the security features of the printer to see if it would be good sufficient for your company. Because security is a significant concern, you may want to think about a printer with a password guarded operation. This indicates not everyone who may have access to the printer would be in a position to print ID playing cards. This built in security feature would make sure that only those who know the password would be able to operate the printer. You would not want just anyone to print an HID proximity card to gain access to limited areas.
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