BLP and MDP pre-treated RACs enhance the disease-inducing ability of IRBP-specific T cells.

<p>IRBP1–20-specific T cells from the draining lymph nodes and spleens of donor B6 mice immunized with IRBP1–20 were stimulated with IRBP1–20 and irradiated naïve splenic APCs or RACs from either wild-type B6 or NOD2 knockout mice that had been left untreated or had been pre-treated with BLP and/or MDP as described in Fig. 2, then the T cell blasts (5×10⁶ cells/mouse) were transferred to naïve mice (n = 6/group). Eyes from the recipient mice were collected for histological evaluation on day 18 post-transfer. Histopathological lesions in the eyes of one representative mouse in each group (A) and average disease scores of both eyes in 6 individual mice that received T cells (<i>B</i>) are shown. **<i>p<</i>0.01 compared with RAC treated with medium, BLP, or MDP alone using Mann-Whitney U test.</p

RACs activated by MDP plus BLP induce antigen-specific uveitogenic T cell proliferation and differentiation into Th1and Th17 cells.

<p>(A): MDP plus BLP-treated RACs are effective in presenting uveitogenic peptide, leading to the proliferation of IRBP1–20-specific T cells. T cells from in vivo IRBP1–20-primed B6 mice at day 12 were cultured with RACs in the presence of IRBP1–20 and proliferation was measured. Prior to co-culture with T cells, cultured RACs were treated for 24 h with BLP and/or MDP, washed, and treated with MMC. (B & C): MDP plus BLP-pre-treated RACs induce IRBP-specific T cells to produce pro-inflammatory cytokines. The experimental paradigm was as in (A), but, after 48 h, cytokines in the supernatants were measured by ELISA. The values are the mean ± SEM for three individual experiments. *<i>p<</i>0.05, **<i>p<</i>0.01 compared with RAC treated with medium, BLP or MDP alone in one-way ANOVA.</p

BLP plus MDP induces RACs to express costimulatory molecules.

<p>(A) RACs were incubated for 48 h in, from left to right, medium alone or medium containing BLP or MDP or both, then were stained with monoclonal Abs against CD80, CD40, or ICOSL, and analyzed by flow cytometry. Negative control samples were stained with an isotype-matched control IgG Ab (empty filled). Numbers in each histogram represent mean fluorescence intensity values. One of three reproducible experiments is shown. (B) The antigen-specific uveitogenic T cell expansion induced by MDP plus BLP-pre-treated RACs is inhibited by anti-ICOSL and anti-CD80 Abs. T cells from in vivo IRBP1-20-primed B6 mice at day 12 were cultured with MDP plus BLP-pre-treated RACs and IRBP1-20 in the absence or presence of 10 μg/ml of anti-CD80, CD40, and ICOSL Abs either alone or in combination or their rat IgG2a, κ isotype control, then proliferation by the responder T cells were determined. C: Cytokine production of the antigen-specific uveitogenic T cells induced by MDP plus BLP-pre-treated RACs is inhibited by anti-ICOSL and CD80 Abs. The experimental paradigm was as in (B), but, after 48 h, cytokines in culture supernatants were measured by ELISA. The values are the mean ± SEM for three individual experiments. *<i>p<</i>0.05, **<i>p<</i>0.01 compared with control in one-way ANOVA.</p

Medical Costs Associated with Diabetes Complications in Medicare Beneficiaries Aged 65 Years or Older with Type 2 Diabetes

   Objective: To estimate medical costs associated with 17 major diabetes-related complications and treatment procedures among Medicare beneficiaries aged ≥65 years with type 2 diabetes. Methods: Data were from the 2006–2017 100% Medicare claims database among beneficiaries enrolled in fee-for-service plans. Records with type 2 diabetes and complications were identified using International Classification of Diseases codes, Ninth Revision and Tenth Revision, and diagnosis-related group codes. The index year was the year when a person was first identified with diabetes with an inpatient claim, or an outpatient claim plus another inpatient/outpatient claim in the 2 years following the first claim in Medicare. Included individuals were followed from index years until death, discontinuation of plan coverage, or December 31, 2017. Fixed-effect regression was used to estimate the cost in years when the complication event occurred and in subsequent years. The total cost for each complication was calculated for 2017 by multiplying the complication prevalence by the cost estimate. All costs were standardized to 2017 U.S. dollars. Results: Our study included 10,982,900 persons with type 2 diabetes. Follow-up ranged from 3 to 10 years. The three costliest complications were kidney failure treated by transplantation (occurring year $79,045; subsequent years$17,303), kidney failure treated by dialysis ($54,394;$38,670), and lower-extremity amputation ($38,982;$8,084). Congestive heart failure accounted for the largest share (18%) of total complication cost.  Conclusions: Costs associated with diabetes complications were substantial. Our cost estimates provide essential information needed for conducting economic evaluation of treatment/programs to prevent/delay diabetes complications in Medicare beneficiaries.  </p

The synergistic effect of BLP and MDP on TNF-α production is cell-type specific.

<p>RACs, RPE cells, or bone marrow derived DC were incubated with increasing concentrations of MDP in the presence or absence of BLP (0.1 μg/ml) for 12 h, then TNF-α in the culture supernatants was measured by ELISA. The results are the mean ± SEM for three independent experiments, each in duplicate. *<i>p</i><0.05, **<i>p</i><0.01 compared with cells treated with medium or MDP alone at different doses in two-way ANOVA with Fisher LSD test.</p

BLP and MDP synergistically enhance cytokine production by RACs.

<p>RACs were incubated with medium, MDP (1 μg/ml), BLP (0.1 μg/ml), or MDP plus BLP for 12 h, then the supernatants were collected for measurement of TNF-α (A) and IL-6 (B) by ELISA. The results are the mean ± SEM for three independent experiments, each in duplicate. **p<0.01 compared with RAC treated with medium, BLP or MDP alone in one-way ANOVA.</p

Characterization of RAC cultures.

<p>RACs from B6 mice were prepared as described in Materials and Methods. Before use, RAC were single-color stained with mAbs against GFAP (red), vimentin (green) and CRALBP (green) followed by immunofluorescence microscopy evaluation (A, 40 magnifications). The RACs were also two-color stained with mAbs against GFAP (green) and S-100 (red), GFAP and vimentin (red) or GFAP and GS (red) (B, 20 magnifications).</p

Medical Costs Associated with Diabetes Complications in Medicare Beneficiaries Aged 65 Years or Older with Type 1 Diabetes

   Aims: To estimate medical costs associated with 17 diabetes complications and treatment procedures among Medicare beneficiaries ≥65 years old with type 1 diabetes. Methods: Using the 2006–2017 100% Medicare claims database for beneficiaries enrolled in fee-for-service plans and Part D, we estimated the annual cost of 17 diabetes complications and treatment procedures. Type 1 diabetes and its complications and procedures were identified using ICD 9/10 codes, procedure codes, and diagnosis-related group codes. Individuals with type 1 diabetes were followed from the year when their diabetes was initially identified in Medicare (2006–2015) until death, discontinuing plan coverage, or December 31, 2017. Fixed-effect regression was used to estimate costs in the complication occurrence years and subsequent years. The cost-proportion of a complication was equal to the total cost of the complication, calculated by multiplying prevalence by the per person cost, divided by the total cost for all complications. All costs were standardized to 2017 US dollars. Results: Our study included 114,879 persons with type 1 diabetes with lengths of follow-up from 3 to 10 years. The costliest complications per person were kidney failure treated by transplantation (occurrence year $77,809; subsequent years$13,556), kidney failure treated by dialysis ($56,469;$41,429), and neuropathy treated by lower-extremity amputation ($40,698;$7,380). Sixteen percent of the total medical cost for diabetes complications was for treating congestive heart failure.  Conclusions: Costs of diabetes complications were large and varied by complications. Our results can assist in cost-effectiveness analysis of treatments and interventions for preventing or delaying diabetes complications in Medicare beneficiaries aged 65 years or older with type 1 diabetes.</p

Total Syntheses of (+)-Alopecuridine, (+)-Sieboldine A, and (−)-Lycojapodine A

(+)-Alopecuridine, (+)-sieboldine A, and (−)-lycojapodine A, three structurally unique and related lycopodium alkaloids, have been synthesized in enantiomeric forms through an efficient strategy. The main synthetic approach for (+)-alopecuridine features a semipinacol rearrangement of hydroxyl epoxide to construct the spiro 6,9-azacarbocycles with an all-carbon quaternary center and a late-stage SmI₂-mediated intramolecular coupling to form the 5-membered ring. Subsequently, the biomimetic synthesis of (+)-sieboldine A and (−)-lycojapodine A was accomplished successfully through two different bioinspired oxidations after a wide search for the oxidation methods. As a result, (+)-sieboldine A was derived from (+)-alopecuridine through an N-oxidation/nitrone formation process and (−)-lycojapodine A through an interesting cyclic hemiketal formation/oxidative diol cleavage pathway. These results confirmed the biogenetic relationship among the three alkaloids

Comparison of the standardization algorithms on the cylinder head model.

<p>(A) original model; (B) standardization result by Laplace; (C) standardization result by Taubin; (D) standardization result by Vollmer; (E) standardization result by Chen; (F) standardization result by our method.</p