High Density Lipoprotein Stimulated Migration of Macrophages Depends on the Scavenger Receptor Class B,Type I,PDZK1 and Akt1 and Is Blocked by Sphingosine 1 Phosphate Receptor Antagonists

HDL carries biologically active lipids such as sphingosine-1-phosphate (S1P) and stimulates a variety of cell signaling pathways in diverse cell types, which may contribute to its ability to protect against atherosclerosis. HDL and sphingosine-1-phosphate receptor agonists, FTY720 and SEW2871 triggered macrophage migration. HDL-, but not FTY720-stimulated migration was inhibited by an antibody against the HDL receptor, SR-BI, and an inhibitor of SR-BI mediated lipid transfer. HDL and FTY720-stimulated migration was also inhibited in macrophages lacking either SR-BI or PDZK1, an adaptor protein that binds to SR-BI''s C-terminal cytoplasmic tail. Migration in response to HDL and S1P receptor agonists was inhibited by treatment of macrophages with sphingosine-1-phosphate receptor type 1 (S1PR1) antagonists and by pertussis toxin. S1PR1 activates signaling pathways including PI3K-Akt, PKC, p38 MAPK, ERK1/2 and Rho kinases. Using selective inhibitors or macrophages from gene targeted mice, we demonstrated the involvement of each of these pathways in HDL-dependent macrophage migration. These data suggest that HDL stimulates the migration of macrophages in a manner that requires the activities of the HDL receptor SR-BI as well as S1PR1 activity.     

Results of the Implementation of a Pilot Model for the Bidirectional Screening and Joint Management of Patients with Pulmonary Tuberculosis and Diabetes Mellitus in Mexico

Background

Recently, the World Health Organisation and the International Union Against Tuberculosis and Lung Disease published a Collaborative Framework for the Care and Control of Tuberculosis (TB) and Diabetes (DM) (CFTB/DM) proposing bidirectional screening and joint management.

Objective

To evaluate the feasibility and effectiveness of the CFTB/DM in Mexico. Design. Prospective observational cohort. Setting. 15 primary care units in 5 states in Mexico. Participants: Patients aged ≥20 years diagnosed with DM or pulmonary TB who sought care at participating clinics. Intervention: The WHO/Union CFTB/DM was adapted and implemented according to official Mexican guidelines. We recruited participants from July 2012 to April 2013 and followed up until March 2014. Bidirectional screening was performed. Patients diagnosed with TB and DM were invited to receive TB treatment under joint management. Main outcome measures. Diagnoses of TB among DM, of DM among TB, and treatment outcomes among patients with DM and TB.

Results

Of 783 DM patients, 11 (1.4%) were unaware of their TB. Of 361 TB patients, 16 (4.4%) were unaware of their DM. 95 TB/DM patients accepted to be treated under joint management, of whom 85 (89.5%) successfully completed treatment. Multiple linear regression analysis with change in HbA1c and random capillary glucose as dependent variables revealed significant decrease with time (regression coefficients (β)  = −0.660, (95% confidence interval (CI), −0.96 to −0.35); and β = −1.889 (95% CI, −2.77 to −1.01, respectively)) adjusting by sex, age and having been treated for a previous TB episode. Patients treated under joint management were more likely to experience treatment success than patients treated under routine DM and TB programs as compared to historical (adjusted OR (aOR), 2.8, 95%CI 1.28–6.13) and same period (aOR 2.37, 95% CI 1.13–4.96) comparison groups.

Conclusions

Joint management of TB and DM is feasible and appears to improve clinical outcomes.     

Number and Distribution of Mouse Retinal Cone Photoreceptors: Differences between an Albino (Swiss) and a Pigmented (C57/BL6) Strain

We purpose here to analyze and compare the population and topography of cone photoreceptors in two mouse strains using automated routines, and to design a method of retinal sampling for their accurate manual quantification. In whole-mounted retinas from pigmented C57/BL6 and albino Swiss mice, the longwave-sensitive (L) and the shortwave-sensitive (S) opsins were immunodetected to analyze the population of each cone type. In another group of retinas both opsins were detected with the same fluorophore to quantify all cones. In a third set of retinas, L-opsin and Brn3a were immunodetected to determine whether L-opsin⁺cones and retinal ganglion cells (RGCs) have a parallel distribution. Cones and RGCs were automatically quantified and their topography illustrated with isodensity maps. Our results show that pigmented mice have a significantly higher number of total cones (all-cones) and of L-opsin⁺cones than albinos which, in turn, have a higher population of S-opsin⁺cones. In pigmented animals 40% of cones are dual (cones that express both opsins), 34% genuine-L (cones that only express the L-opsin), and 26% genuine-S (cones that only express the S-opsin). In albinos, 23% of cones are genuine-S and the proportion of dual cones increases to 76% at the expense of genuine-L cones. In both strains, L-opsin⁺cones are denser in the central than peripheral retina, and all-cones density increases dorso-ventrally. In pigmented animals S-opsin⁺cones are scarce in the dorsal retina and very numerous in the ventral retina, being densest in its nasal aspect. In albinos, S-opsin⁺cones are abundant in the dorsal retina, although their highest densities are also ventral. Based on the densities of each cone population, we propose a sampling method to manually quantify and infer their total population. In conclusion, these data provide the basis to study cone degeneration and its prevention in pathologic conditions.     

Comparative Study of 17-AAG and NVP-AUY922 in Pancreatic and Colorectal Cancer Cells: Are There Common Determinants of Sensitivity?

The use of heat shock protein 90 (Hsp90) inhibitors is an attractive antineoplastic therapy. We wanted to compare the effects of the benzoquinone 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin) and the novel isoxazole resorcinol–based Hsp90 inhibitor NVP-AUY922 in a panel of pancreatic and colorectal carcinoma cell lines and in colorectal primary cultures derived from tumors excised to patients. PANC-1, CFPAC-1, and Caco-2 cells were intrinsically resistant to 17-AAG but sensitive to NVP-AUY922. Other cellular models were sensitive to both inhibitors. Human epidermal growth factor receptor receptors and their downstream signaling pathways were downregulated in susceptible cellular models, and concurrently, Hsp70 was induced. Intrinsic resistance to 17-AAG did not correlate with expression of ATP-binding cassette transporters involved in multidrug resistance. Some 17-AAG-resistant, NVP-AUY922–sensitive cell lines lacked NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme and activity. However, colorectal LoVo cells still responded to both drugs in spite of having undetectable levels and activity of NQO1. Pharmacological and biologic inhibition of NQO1 did not confer resistance to 17-AAG in sensitive cell lines. Therefore, even though 17-AAG sensitivity is related to NQO1 protein levels and enzymatic activity, the absence of NQO1 does not necessarily convey resistance to 17-AAG in these cellular models. Moreover, NVP-AUY922 does not require NQO1 for its action and is a more potent inhibitor than 17-AAG in these cells. More importantly, we show in this report that NVP-AUY922 potentiates the inhibitory effects of chemotherapeutic agents, such as gemcitabine or oxaliplatin, and other drugs that are currently being evaluated in clinical trials as antitumor agents.     

The increased presence of repetitive motifs in the KDDR-plus recombinant protein,a kinesin-derived antigen from Leishmania infantum,improves the diagnostic performance of serological tests for human and canine visceral leishmaniasis

Visceral leishmaniasis (VL) is caused by protozoa belonging to the Leishmania donovani complex and is considered the most serious and fatal form among the different types of leishmaniasis, if not early diagnosed and treated. Among the measures of disease control stand out the management of infected dogs and the early diagnosis and appropriate treatment of human cases. Several antigens have been characterized for use in the VL diagnosis, among them are the recombinant kinesin-derived antigens from L. infantum, as rK39 and rKDDR. The main difference between these antigens is the size of the non-repetitive kinesin region and the number of repetitions of the 39 amino acid degenerate motif (6.5 and 8.5 repeats in rK39 and rKDDR, respectively). This repetitive region has a high antigenicity score. To evaluate the effect of increasing the number of repeats on diagnostic performance, we designed the rKDDR-plus antigen, containing 15.3 repeats of the 39 amino acid degenerate motif, besides the absence of the non-repetitive portion from L. infantum kinesin. Its performance was evaluated by enzyme-linked immunosorbent assay (ELISA) and rapid immunochromatographic test (ICT), and compared with the kinesin-derived antigens (rKDDR and rK39). In ELISA with human sera, all recombinant antigens had a sensitivity of 98%, whereas the specificity for rKDDR-plus, rKDDR and rK39 was 100%, 96% and 71%, respectively. When evaluated canine sera, the ELISA sensitivity was 97% for all antigens, and the specificity for rKDDR-plus, rKDDR and rK39 was 98%, 91% and 83%, respectively. Evaluation of the ICT/rKDDR-plus, using human sera, showed greater diagnostic sensitivity (90%) and specificity (100%), when compared to the IT LEISH (79% and 98%, respectively), which is based on the rK39 antigen. These results suggest that the increased presence of repetitive motifs in the rKDDR-plus protein improves the diagnostic performance of serological tests by increasing the specificity and accuracy of the diagnosis.     

Degradation of glyphosate and other pesticides by ligninolytic enzymes

The ability of pure manganese peroxidase (MnP), laccase, lignin peroxidase (LiP) and horseradish peroxidase (HRP) to degrade the widely used herbicide glyphosate and other pesticides was studied in separate in vitro assays with addition of different mediators. Complete degradation of glyphosate was obtained with MnP, MnSO₄ and Tween 80, with or without H₂O₂. In the presence of MnSO₄, with or without H₂O₂, MnP also transformed the herbicide, but to a lower rate. Laccase degraded glyphosate in the presence of (a) 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), (b) MnSO₄ and Tween 80 and (c) ABTS, MnSO₄ and Tween 80. The metabolite AMPA was detected in all cases where degradation of glyphosate occurred and was not degraded. The LiP was tested alone or with MnSO₄, Tween 80, veratryl alcohol or H₂O₂ and in the HRP assay the enzyme was added alone or with H₂O₂ in the reaction mixture. However, these enzymes did not degrade glyphosate. Further experiments using MnP together with MnSO₄ and Tween 80 showed that the enzyme was also able to degrade glyphosate in its commercial formulation Roundup^® Bio. The same enzyme mixture was tested for degradation of 22 other pesticides and degradation products present in a mixture and all the compounds were transformed, with degradation percentages ranging between 20 and 100%. Our results highlight the potential of ligninolytic enzymes to degrade pesticides. Moreover, they suggest that the formation of AMPA, the main metabolite of glyphosate degradation found in soils, can be a result of the activity of lignin-degrading enzymes.