extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Medicine

text:
Immunization of Chlamydia pneumoniae (Cpn)-Infected Apobtm2SgyLdlrtm1Her/J Mice with a Combined Peptide of Cpn Significantly Reduces Atherosclerotic Lesion Development

Objective To investigate the antigenic effect of a peptide containing two epitopes of Chlamydia pneumoniae (Cpn) on atherosclerotic lesion formation in mice infected with Cpn. Materials and Methods Six-week-old Apobtm2SgyLdlrtm1Her/J mice were immunized using a repetitive immunization multiple-sites strategy with KLH-conjugated peptides derived from the major outer membrane protein and the putative outer membrane protein 5 of Cpn. Mice were fed a high-fat diet and infected with Cpn twice during the 10-week diet period. Lesions were evaluated histologically; local and systemic immune responses were analyzed by immunohistochemistry of aorta samples and cytokine measurements in plasma samples and splenocyte supernatants. Results Mice immunized with the combined Cpn peptide showed a greater reduction in lesion size compared to mice immunized with either epitope alone [54.7% vs 39.8% or 41.72%] and was also associated with a significant decrease in lesion area in descending aortas compared with those in controls (88.9% for combined Cpn peptide, 81.9% for MOMP peptide and 75.7% for Omp5, respectively). This effect was associated with a shift in the cellular composition of plaques towards decreased inflammatory cell and increased regulatory T-cell content. Additionally, the effect was also connected with decreased secretion of proinflammatory cytokines and increased production of anti-inflammatory cytokines demonstrated in plasma and in supernatant on stimulated spleen cells. Conclusions Atherosclerotic lesion formation may be promoted by Cpn infection in the presence of a high-fat diet, and reduced by immunization with the combined Cpn peptide. The combined peptide has more potential than either epitope alone in reducing atherosclerotic lesion development through Treg expansion.

Introduction
Chlamydia pneumoniae (Cpn) [1] is an important human pathogen that causes atypical pneumonia and is associated with various chronic inflammatory diseases such as atherosclerosis, a major cause of cardiovascular disease and death in the Western world [2][3][4][5][6]. Although the epidemiological importance of Chlamydia infection in atherosclerosis is not well defined, the potential role of Cpn in coronary atherosclerosis may be related more to acceleration of the disease or to the systemic effects of persistent infection than to sudden initiation of infarction by acute infection [7]. However, the theoretical role of Cpn in acceleration of atherosclerosis is still controversial [8][9][10]. Although an association between Cpn infection and coronary atherosclerosis has been reported, the association is less clear for the effect of peptide antigen derived from Cpn on the formation of atherosclerotic lesion. In addition, an epitope of the major outer membrane protein (MOMP) of Cpn (AA 67-74: GDYVFDRI) and the putative outer membrane protein 5 (Omp5) of Cpn (AA 284-292: QAVANGGAI) share high homology, with two sequence locations of ApoB protein (http://web.expasy.org/sim/). ApoB protein plays a crucial role in atherosclerosis as immunization with some peptides derived from ApoB protein reduce atherosclerotic lesion in several mouse models. Indeed, this molecular mimicry (share high homology) was recently demonstrated in our laboratory in which an epitope containing both sequences of AA 67-74 (GDYVFDRI) and AA 284-292 (QAVANGGAI) has an effect on atherosclerotic lesion reduction in a protein scaffold in non-infected mice with Chlamydophia [11]. In this study, we investigate the effect of a linear peptide containing these two putative epitopes derived from MOMP and Omp5 of Cpn on atherosclerotic lesion formation in Cpn-infected Apob tm2Sgy Ldlr tm1Her /J mice.

Materials and Methods
The immunizing peptides derived from MOMP (AA 67-74: GDYVFDRI, designated as MOMP peptide) and Omp5 (AA 283-291: QAVANGGAI, designated as Omp5 peptide), and a combined peptide containing the MOMP and the Omp5 peptides (designated as a combined Cpn peptide) coupled by a polyglycine [(Gly)5] linker, were used in this study in a Keyhole limpet hemocyanin (KLH)-conjugated form. All of the peptides used in the study, including ApoB peptide and human HSP60 (hHSP60) peptide, were synthesized by Severn biotech Ltd (Worcestershire, UK).

Animal Experiments
The experiments were approved by the Animal Welfare Committee of the University of Szeged and conform to the Directive 2010/63/EU of the European Parliament.
Apob tm2Sgy Ldlr tm1Her /J mice (these mice produce ApoB100 only, and are LDL receptor deficiency).were used in our study in a total of 5 groups (3 sample and 2 control groups). Each group included 6 mice (5-6-week-old males; similar body weight, 32.2662.12 g [measured at the end of the experiment]) and the experiment was repeated. Mice were immunized with KLHconjugated peptides mixed with Alum adjuvant subcutaneously according to a repetitive immunization multiple sites strategy (RIMMS) as described earlier [11,12]. For infection, mice were inoculated intranasally with 2610 6 inclusion forming units (IFU) of Cpn (CV-6, cardiovascular strain) [13] in 25 mL of phosphate buffered saline (PBS) at week 4 and at week 8. This dose was chosen based on survival and symptoms observed in mice after infection with different infection doses (Table S1). CV-6 strain of Cpn was propagated in HEp-2 cells and partially purified as described earlier [14]. The mice were sacrificed at the end of week 12 (a high-fat diet was started at week 2 and continued for 10 weeks). For detection of Cpn-specific DNA, polymerase chain reaction (PCR) was performed as described by Tong and Sillis [15]. The MOMP of Cpn was chosen as a target for amplification in a nested PCR. All primers were synthesized in Life Technologies Ltd (Paisley, UK). The external primers (Table S2) amplified a 333 base-pair product (first-stage PCR) from the genomic DNA purified from lung homogenates of tested mice in both infected and non-infected (negative control) groups. The internal primers amplified a 207 base-pair product (second-stage PCR) using the first-stage PCR product as a template. To confirm if the mice were infected, further detection of Cpn-specific IgG in the mouse sera by indirect immunofluorescence was carried out. A HEp-2 cell (ATCC) monolayer grown on 13-mm coverslips in 24-well plates was infected with Cpn (CWL029) at a multiplicity of infection (m.o.i.) of 1. At 48 h of infection, cells were fixed with ice-cold acetone (10 min, 220uC). PBS-rehydrated cells were stained with Cpn MOMP-specific mouse monoclonal antibody (DAKO, Budapest, Hungary) and FITC-labelled anti-mouse IgG secondary antibody (SIGMA, Dorset, UK). Coverslips were examined under an ultraviolet-microscope, and evenly distributed bright fluorescing inclusions were visible. Mouse sera diluted in PBS were used as the primary antibody for staining the similarly treated monolayers and the same FITC-conjugated secondary antibody was applied to detect serum IgG binding to inclusions. Reciprocal of serum dilution producing clearly discernable inclusions was determined as Cpn-specific titre of the mouse serum. The repetitive immunization multiple-sites strategy (RIMMS) was adopted [9,10] and mice were sacrificed at the end of week 12 (a high-fat diet was started at week 2 and continued for 10 weeks).

Tissue Preparation and Antibody Response Measurements
Twelve weeks after the first immunization, tissues were harvested and mounted in Optimal Cutting Temperature (OCT) for immunohistochemical analyses and in paraffin for lesion measurement. Atherosclerosis in aortic roots was examined by Image-Pro Plus TM software, version 4.0 ((Media Cybernetics, Bethesda, MD, USA). Peptide-specific antibody titers were measured by ELISA following the manufacturer's instructions. To assess whether immunization with the combined Cpn peptide led to changes in lipoprotein profiles, the cholesterol, triglyceride, HDL-and LDL-cholesterol profiles of the mice were analyzed using a pooled plasma sample from each group after being fed a high-fat diet for 10 weeks.

Immunohistochemical Analyses, Morphometric Analyses and Quantitative Measurements of Atherosclerosis
OCT-embedded samples were used for immunohistochemical analyses. Sections of paraffin-embedded tissues were stained with hematoxylin and eosin (HE) and elastin/van Gieson (Sigma) for histological examination and were evaluated using an Olympus U-ULH Optical microscope (Olympus Optical Co. Ltd, Tokyo, Japan).
Both the atherosclerotic lesions in the aortic sinus and the lesions in the descending aortas were measured. The descending aortas were evaluated for the extent of atherosclerosis.

Measurement of Cytokines
Plasma levels of interleukin (IL)-10, transforming growth factor (TGF)-b, tumor necrosis factor (TNF)-a and interferon (IFN)-c were measured by ELISA following the manufacturer's instructions (R&D systems, Abingdon, UK). IL-10 and TNF-a levels in the lesions were quantified by immunohistochemical analyses (rat anti-mouse TNF-a and IL-10 purchased from BioLegend, CA, USA). Levels of concanavalin-A (Con A)-induced IL-10, TGF-b, TNF-a and IFN-c in splenocyte cultures were also measured.

Fluorescence Activated Cell Sorting Analysis
Spleen cells from mice immunized with either Cpn peptides or KLH (control) were used for T regular (Treg) cells measurement using a Treg detection kit (Miltenyi Biotec, Surrey, UK). Cells were analyzed with a Beckman-Coulter FC-500 Analyzer (Bachman Coulter, High Wycombe, UK).

Statistical Analyses
Data are reported as mean6standard error of the mean (6SEM), unless otherwise indicated. Figures were plotted using graph-pad Prism 5.01 and Sigma plot 9.0. For atherosclerotic lesion size, data were compared and intergroup differences were conducted using one-way ANOVA for multiple comparisons and post hoc bonferroni test. Other data were analyzed using Student's t-test (2-tailed analyses). Non-parametric distributions were analysized using Mann-Whitney U test for pairwise comparisons and the Kruskal-Wallis test for multiple comparisons. Differences between groups were considered significant at P values below 0.05.

Detection of Cpn Infection
The MOMP of Cpn was chosen as a target for amplification in nested PCR DNA from infected samples only, and produced a band of the expected size after the first PCR amplification. Similarly, in the second amplification, a positive band of the correct size was obtained only from using the first PCR product as a template originally from infected and non-infected mice ( Figure 1). Cpn DNA was detected from the lung homogenates by PCR as shown in Figure 1a, and the primers used for PCR as shown in Table S1. In addition, a genus specific epitope within Chlamydia lipopolysaccharde (LPS) was detected from the lesion sites of aortic sinus in Cpn-infected mice by Chlamydia LPS antibody (MCA 2718, AbD serotec) compared that of mice without Cpn infection (control), suggesting that Cpn bacteria were captured at the lesion site when mice were infected with Cpn bacteria as shown in Figure 1b.

Peptide-Specific Immunoglobulin G in Sera of Immunized Mice
A peptide-induced specific antibody response was observed when the MOMP peptide, the Omp5 peptide and the combined Cpn peptide were used as ELISA antigens (Figures 2A-D), when compared with a KLH control, which produced little immune response ( Figures 2C and D). The antibody immune response at week 12 was slightly lower than that at week 2 ( Figure 2A-D) apart from combined Cpn peptide-induced antibody at week 12. High antibody concentrations against either the Omp5 peptide or the MOMP peptide at week 2 were detected in immunized mice despite a relatively lower immune response in the MOMPpeptide-immunized mice than in the Omp5-peptide-immunized mice at week 12.

Peptide-Specific Immunoglobulin Subtypes G1 and G2a in Antiserum of Immunized Mice
A peptide-induced specific IgG1 response was observed, when compared with a KLH control ( Figure 2E). There was little or no IgG2a immune response ( Figure 2F) in pooled antiserum. Immunization with a KLH-conjugated ApoB and hHSP60 peptides was described earlier [11]. Sera from these experiments and sera of Cpn peptide-immunized mice were used for testing cross-reaction of peptide-specific antibodies. ApoB and Cpn peptides, individually, induced high levels of peptide-specific IgG in mouse sera two weeks after the first immunization compared to the control group immunized with KLH alone. Certain level of cross-reaction was observed between ApoB peptide and Cpn peptide antisera ( Figure 2G), as well as between Cpn peptide and ApoB peptide antisera ( Figure 2H) [16].

Effect of Treatment With Cpn Peptides on Plasma Lipid Levels
Slightly lower concentrations of cholesterol and LDL were observed in the infected control mice than in the non-infected controls (Table 1). In agreement with previous report by Blessing et al, there was no significant difference observed between the infected and non-infected mice [17]. In addition, there was little change in either triglyceride or HDL concentration observed in non-infected and infected control mice as well as in mice infected after immunization with the combined Cpn peptide. These levels were remained the same as those in infected mice after immunization with the combined Cpn peptide.

Reduction of Atherosclerotic Lesion Size in the Aortic Sinus
Representative sections from the aortic sinuses of mice are shown in Figure 3A. The lesion size in infected control mice was 5% larger than that in non-infected controls (32% vs. 27%), but it did not show significant. In contrast, the lesion size in mice immunized with the combined Cpn peptide was smaller than that in either the MOMP peptide-or the Omp5-peptide-immunized mice (14% vs. 19% and 14% vs. 18%, respectively) ( Figure 3B). The former pair of peptide antigens showed significant difference (P = 0.045), but this was not in case of the later pairs (P = 0.05). No significant difference in lesion size between mice immunized with either the MOMP peptide or the Omp5 peptide alone was observed.
Reduction in lesion size, expressed as a percentage of the lesion size in the aortic sinus of the control mice, was 54.7% following immunization with the combined Cpn peptide versus 39.8% with the MOMP peptide and 41.7% with the Omp5 peptide ( Figure 3C). In addition, the lesion reduction in mice immunized with the combined Cpn peptide was significantly greater (P = 0.046) than that in MOMP peptide-immunized mice ( Figure 3C).
In addition, we assessed oil red O (ORO) lipid levels in atherosclerotic lesions in the aortic sinus. Representative sections from the aortic sinuses of mice are shown in Figure S1A. The lipid level at the lesion sites in infected control mice was approximately 7-fold higher than in the MOMP-peptide-immunized mice (32.5% versus 4.6%, respectively), 6-fold higher than in the Omp5peptide-immunized mice (32,5% versus 5.2%, respectively) and 9fold higher than in the combined Cpn peptide-immunized mice (32.5% versus 3.6%, respectively) ( Figure S1B). Lipid level in mice immunized with the combined peptide was significantly lower than that in either the MOMP-peptide-immunized mice (4.6% versus 3.6%, P = 0.028) or the Omp5-peptide-immunized mice (5.2% versus 3.6%, P = 0.001), respectively. Furthermore, we examined the impact of treatment with Cpn peptides on the collagen in these lesions. The reduction of atherosclerosis in mice treated with these peptides was associated with an increased collagen content: 4-fold for the combined Cpnpeptide-immunized mice versus control mice (24.461.2% versus 5.960.5%; P,0.001), 3-fold for the MOMP peptide-immunized mice (19.461.3% versus 5.960.5%; P,0.001), and 3-fold for the Omp5 peptide-immunized mice (18.961.5% versus 5.960.5%; P,0.00) ( Figures S2A and B), respectively. Mice immunized with the combined peptides showed a significant collagen increase compared to mice immunized with either the MOMP-or the Omp5-peptide alone (P = 0.007 and P = 0.004, respectively).

Reduction of Atherosclerotic Lesion Size in Descending Aortas
Lesion size in the descending aortas of infected control mice was greater than that in non-infected control mice (32.0% vs. 17.0%, P,0.01) ( Figure 4A). Lesion size in the descending aortas in mice immunized with the combined peptide was smaller than that in either the MOMP-peptide-immunized (3.4% vs. 5.8%) or the Omp5-peptide-immunized (3.4% vs. 7.8%) mice respectively ( Figure 4B). There was, however, no significant difference in lesion size between mice immunized with either the MOMP peptide or the Omp5 peptide, or between mice immunized with either the MOMP peptide or the combined Cpn peptide; a significant difference (P,0.01) was found between mice immunized with the Omp5 peptide versus the combined Cpn peptide. Reduction in lesion size, expressed as a percentage of lesion size in descending aortas, was 88.9%, 81.9% and 75.7% from mice immunized with the combined Cpn peptide, the MOMP peptide and the Omp5 peptide, respectively, showing a similar trend to that observed in aorta sinus ( Figure 4C). The calculated percentage reduction in lesion size showed a significant difference (P = 0.006) between mice immunized with the Omp5 peptide versus the combined Cpn peptide ( Figure 4C).

Amount of Inflammatory Cells in The Atherosclerotic Lesions
The percentage of macrophage-occupied area in the lesions in mice immunized with the combined peptide was 7.8%, significantly lower than that in mice immunized with the Omp5 peptide (12.2%, P = 0.016), but not with the MOMP peptide (12.3%, P = 0.084) alone. In contrast, mice immunized with the KLH showed a 34.1% occupation ( Figures 5A and B).
The proportion of anti-CD11c + -stained lesion area was 5.260.4% in mice immunized with the combined Cpn peptide, lower than that with either the MOMP peptide (8.360.5%, P = 0.006) or the Omp5 peptide (8.460.5%, P = 0.002) alone, and the control group (32.962.8%, P,0.001) ( Figure 5C). In addition, the proportion of anti-CD11c + -stained lesion area co-localized with CD68 + was 71.264.5% for the combined peptide, 77.769.5% for the MOMP peptide and 74.667.0% for the Omp5 peptide, compared with 95.760.8% for the controls ( Figure 5D).
Forkhead box P3 (Foxp3) is a transcriptional regulator of CD4 + CD25 + regulatory T cells [18]. Thus, we chose to investigate whether the compromised suppressor function of Tregs observed in mice immunized with these peptides was associated with Foxp3 up-regulation. We found that the CD4 + -occupied area in the lesion was significantly reduced in mice immunized with these peptides (P,.0.001) compared with that of control mice immunized with the KLH only (33086268 mm 2 versus 10,60761435 mm 2 for the MOMP peptide, 27796525 mm 2 versus 10,60761435 mm 2 for the Omp5 and 17616359 mm 2 versus 10,60761435 mm 2 for the combined Cpn peptide, respectively) (Figures 5E and F). In addition, the CD4 + -occupied area in lesions in mice immunized with the combined Cpn peptide developed significantly smaller occupied areas in lesions than those of mice immunized with the MOMP peptide (P = 0.006) (Figures 5E and F). In contrast, the proportion of Foxp3 + area colocalized with CD4 + area in mice immunized with these peptides was increased compared with that in control mice immunized with the KLH alone, showing 21.4% (P = 0.001) for the combined Cpn peptide, 8.4% (P = 0.006) for the MOMP peptide, and 10.0% (P,0.001) for the Omp5 peptide, versus 1.1% for the control (Figures 5E and G). In addition, a statistically significant difference (P,0.05) was observed between the combined Cpn peptide and either the MOMP peptide or the Omp5 peptide ( Figures 5E and G).
Consistently, the flow cytometric analysis of spleen cells showed significantly increased expression of Foxp3 + in mice immunized with these peptides compared with that of the KLH control mice (P#0.001, Figures 5H and I). Similar to the observation in the lesions, higher expression of Foxp3 + was found in mice immunized with the combined Cpn peptide than those of mice immunized with either the MOMP peptide (P = 0.006) or the Omp5 peptide (P = 0.003).

Expression of Anti-Inflammatory Cytokines and Pro-Inflammatory Cytokines
To further characterize the effect of immunization with the combined Cpn peptide, we analyzed IL-10 expression in aortic  lesions of mice immunized with these peptides ( Figure 6A). The proportion of CD4 + cells expressing IL-10 was significantly higher in mice immunized with the combined Cpn peptide compared with control mice (.2-fold; 9.0% versus 3.6%, P = 0.024). Mice immunized with either the MOMP peptide or the Omp5 peptide showed similar proportions of IL-10 (6.0% and 5.6%, respectively) and showed significantly higher proportions in lesion sites compared with that in controls (P,0.05). Mice immunized with the combined Cpn peptide had a greater IL-10-positive area compared with either the MOMP peptide or the Omp5 peptide alone; but this increased level did not show statistical significance ( Figure 6B). Immunohistochemical analyses of TNF-a showed significantly smaller TNF-a-occupied areas in lesions of mice immunized with the combined Cpn peptide compared with controls (6.0% for the combined peptide vs. 9.2% for the MOMP peptide, 9.0% for the Omp5 peptide and 22.4% for the controls). The difference was statistically significant for the MOMP peptide (P,0.05), but not for the Omp5 peptide when compared with the combined Cpn peptide (Figures 6C and D).

Level of Atheroprotective and Atherogenic Cytokines in Plasma and in Supernatants of Stimulated Splenocytes
Plasma levels of atheroprotective cytokine IL-10 (P,0.05) and TGF-b (P,0.01) were significantly increased in mice immunized with the combined Cpn peptide compared with either the MOMP peptide or the Omp5 peptide ( Figures 7A and B). The levels of these two cytokines were also significantly increased in mice immunized with either the MOMP peptide (P,0.05) or the Omp5 peptide (P,0.05) (Figures 7A and B) versus controls. Plasma levels of TNF-a were significant reduced by immunization with the combined peptide compared with either the MOMP (P,0.05) or the Omp5 peptide ( Figure 7C); all peptides produced a reduction in TNF-a secretion versus controls. A similar trend was obtained for these peptides in respect of plasma levels of IFN-c ( Figure 7D). Although no statistically significant difference was found between the combined Cpn peptide and the MOMP peptide, a significant difference in IFN-c levels was found in mice immunized with the combined Cpn peptide compared with the Omp5 peptide (P,0.05) ( Figure 7D).
In addition, supernatants of the splenocytes from mice immunized with these peptides individually showed significantly increased secretion of IL-10 ( Figure 7E) and TGF-b ( Figure 7F), stimulated with 10 or 100 mg/mL of Con A (P,0.001). The incremental secretion of IL-10 and TGF-b in the combined Cpn peptide-immunized mice was significantly higher than either the MOMP or the Omp5 peptide-immunized mice (P,0.05). Significantly decreased secretion of TNF-a ( Figure 7G) and IFNc ( Figure 7H) was found in the supernatants of splenocytes in mice immunized with these peptide antigens. A significantly greater decrease in the secretion of TNF-a was observed in mice immunized with the combined Cpn peptide versus immunized with either the MOMP peptide or the Omp5 peptide alone ( Figure 7G; P,0.05) stimulated with 100 mg/mL of ConA (P,0.05). A significantly greater decrease in secretion of IFN-c was detected in the combined Cpn peptide-immunized mice versus those in either the MOMP-peptide or the Omp5 peptideimmunized mice ( Figure 7H, P,0.05), stimulated with 10 or 100 mg/mL of ConA (P,0.05).
Furthermore, IL-10 concentration was significantly higher in the supernatant of spleen cells from the combined Cpn peptideimmunized mice stimulated by the same antigen peptide than that after stimulation by PBS (P,0.05, Figure 7I). In contrast, IFN-c was significantly lower in supernatant of spleen cells from the combined Cpn peptide-immunized mice stimulated by the same antigen peptide than that of mice stimulated by PBS (P,0.05, Figure 7J). In the similar experimental conditions, supernatant of spleen cells from the naive mice failed to show these differences.

Evaluation of Antigen-Induced Specific Treg Function and Specific Cellular Immune Response
To assess whether functional Treg cells were induced by immunization, we co-cultured antigen-specific Treg cells with CD4 + effector T-cells (CD4 + CD25 -T-cells). Proliferation of effector T-cells from control mice immunized with KLH in response to stimulation with KLH at 1 mM did not show suppression in a dose-dependent manner in the presence of Treg cells from KLH-immunized mice (Figures. 8A, B and F). In contrast, Inhibition of effector T-cell proliferation was achieved from sampling mice immunized with either the MOMP peptide ( Figures 8A, C and F  as well as the combined peptide ( Figures 8A, E and F), when cocultured CD4 + CD25 2 effector T cells with CD4 + CD25 + Treg cells isolated from these mice in response to stimulation with related antigen. The differences were significant when adding Tregs to the effector cells at the ratios between 4:1,16:1 (P,0.05,,0.001) compared with that without the addition of Tregs. Additionally, the differences were significant when compared different concentration of added Treg in sampling mice at the ration between 4:1,16:1 of Treg and T effector cells (P,0.05,,0.001).

Discussion
In this study, we investigated the effect of the combined peptide derived from Cpn proteins of MOMP and Omp5 in modulating experimental atherosclerosis in Apob tm2Sgy Ldlr tm1Her /J mouse-a strain that develops high levels of atherosclerosis after being fed a high-fat diet [16]. The effect of immunization with the combined peptide was compared with that of the two peptides used singly. The two peptides were selected for coupling based on our hypothesis that they share high homology with ApoB protein which is associated with atherosclerosis.
In line with previous reports by Blessing et al [17] and Ezzahiri et al [19], our results showed that infection of mice with Cpnbacteria promotes the formation of atherosclerotic lesion. Unlike that reported by Blessing et al, our results did not show statistical significance on promoting the lesion in mice infected with Cpn bacteria compared with that of uninfected mice following feeding with a high-fat diet for 10 weeks. These results suggest that feeding with a high-fat diet for longer than 10 weeks may be necessary to form sufficient lesion area, as the data from Blessing et al have shown that the fast lesion increasing time seems between 10-16 weeks [17]. Additionally, other factors, such as different mouse or bacterial strains and different inoculating doses may also affect lesion formation. We report the novel finding that either the MOMP peptide or the Omp5 peptide significantly reduces atherosclerotic lesion when used as a peptide antigen in Apob tm2Sgy Ldlr tm1Her /J mice infected with Cpn bacteria. A different study was shown that a recombinant protein containing both of the MOMP peptide and the Omp5 peptide sequence significantly reduced atherosclerotic lesion in non-infected mice [11], indicating infection of mice with Cpn is an independent factor for functional study of these two peptides. The mechanism of this function may be due to molecular mimicry, as the combined Cpn peptides can cross-react with antiserum induced by human ApoB peptide (also called ApoB100 661-681 peptide). Although certain level of cross-reaction was observed between ApoB peptide and Cpn peptide antisera, strong cross-reaction (approx net 1.5 OD value) between Cpn peptide and ApoB peptide antisera was striking. In fact, autoantibodies to this ApoB peptide as well as to other ApoB100 peptides are present in human plasma and are associated with decreased cardiovascular risk. In addition, immunization with this peptide reduces atherosclerotic lesion in several knock-out mouse models including LDL receptor 2/2 / human ApoB-100 mice [20], ApoE 2/2 mice [21] and Apob tm2Sgy Ldlr tm1Her /J mice [16]. Vaccination with a modified Streptococcus pneumoniae (Spn) has been reported to decrease atherosclerotic lesion formation through molecular mimicry between Spn and oxidized low-density lipoprotein (oxLDL) [22], which has been demonstrated to play a role in the development of atherosclerotic lesions [23,24]. Immunization of oxLDL or apolipoprotein B (ApoB) peptide of oxLDL reduces atherosclerotic lesion formation [16,25] in either ApoE 2/2 or Apob tm2Sgy Ldlr tm1Her / J mouse models. However, molecular mimicry can occur in the absence of any true sequence homology between Cpn-derived peptide and ApoB peptide by using the computer-based software 'SIM-Alignment Tool' for protein or peptide sequences (http://web.expasy.org/tools/sim), in agreement with the report by Kohm et al [26]. In this case, it could explain why antigenic surfaces, rather than sequence homology, dictate molecular mimicry [27]. The data from our present study also demonstrate that reduction of the lesion, either in the aortic sinus or the descending aorta, in mice immunized with the combined Cpn peptide is significantly greater than with either the MOMP-or the Omp5peptide alone. In addition, the reduction in lipid level at lesion sitesin mice immunized with the combined Cpn peptide is significantly greater than that in mice immunized with either the MOMP or the Omp5 peptide alone. Furthermore, the reduction of atherosclerotic lesion in mice treated with these peptides was associated with an increase in collagen content. Collagen content in mice immunized with the combined Cpn peptide is significantly greater than that in mice immunized with either the MOMP-or the Omp5-peptide alone. These data suggest that the Cpn-derived combined peptide may have an additive effect.
Immunization Keyhole limpet hemocyanin (KLH) with the combined Cpn peptide was associated with intracellular responses that influence cellular infiltration into atherosclerotic lesions, as the levels of macrophages, activated CD4 + T cells and dendritic cells (markers of early lesion formation) were decreased compared with those in controls [28]. In addition, CD4 T-cells from the mice immunized with the combined Cpn peptide harbor 2-fold more Foxp3 + Treg cells compared with either the MOMP peptide or the Omp5 peptide and approximately 20-fold more compared with controls. As Th2-type IgG1, and not Th1-type IgG2a, anti-Cpn peptide antibody titers were observed, indicating an association of Cpn peptide treatment with Th2 response which was enhanced by Treg [29,30].
It appears that there is a shift toward decreased proinflammatory cytokine secretion and increased anti-inflammatory cytokine production in antigen-immunized mice, as the main biological function of IL-10 and TGF-b is to limit and terminate the inflammatory responses [31]. IL-10 has been reported to be a global suppressor of immune responses as well as an immunoregulator of the Th-2 cell response [31] while IL-17A (previously called IL-17) is a unique T helper lineage that regulates tissue inflammation [32]. Evidence was obtained from CD4 + expressing pro-atherogenic cytokine IL-17A [33,34], which was shown decreased IL-17A population in peptide antigen-immunized mice in this study.
The suppressive function of CD4 + CD25 + Foxp3 + natural Tregs appears to require only cell-cell contact or proximity in vitro, whereas the in vivo function of these cells is associated with secretion of IL-10 and TGF-b [11,35]. We demonstrated in vitro that the atheroprotective effect paralleled an induction of Treg suppression of antigen-specific effector T-cells, thus suggesting Tregs have an active role in the control of the atherosclerotic process. Treg cells are characterized by the expression of Foxp3, which has a crucial role in their suppression function [36]. Collectively, our study has provided evidence that the mechanism of lesion reduction by subcutaneous immunization with Cpn-derived hepta-or nona-peptide is duo to antigen-induced specific Treg expansion, which suppresses T effector cell proliferation along with increased atheroprotective cytokines and decreased proinflammatory cytokines. Notably, IL-17A that was thought to play a pro-atherogenic role in atherosclerosis [37,38] was down-regulated, however, the relevance of IL-17A to human atherosclerosis remains poorly defined.
In conclusion, we have provided proof of principle evidence that the combined Cpn peptide antigen has an additive effect as this peptide in most of experiments, if not all, showed statistically significant difference when compared to either the MOMP-or the Omp5-peptide antigen used singly (Table S3). It appears that modulation of atherosclerosis-related autoimmunity by antigen-specific activation of Tregs represents a novel approach for the development of bivalent vaccines against atherosclerosis.  Figure S3 Assessment of IL-17A expression level in splenocytes from Ldlr tm1Her Apob tm2Sgy J mice infected with Cpn bacteria and fed a high-fat diet after immunization with Cpn peptides vs controls infected with Cpn bacteria and fed a high-fat diet after immunization with KLH only. A. Representative flow cytometry plots for IL-17A expressing CD4+ population in spleen cells. Spleen cells from mice infected with Cpn bacteria and fed a high-fat diet after immunization with Cpn peptides and control mice infected with Cpn bacteria and fed a high-fat diet after immunization with KLH only were purified using a CD4 + purification kit (Miltenyi Biotec, Surrey, UK) according to manufacturer's protocols. B. Bar chart presentation of flow cytometry analysis. Data represent mean 6 SEM from 3 independent samples. (TIF)


Novel Oncology Therapeutics: Targeted Drug Delivery for Cancer

Despite the progress in techniques for cancer prevention, detection, and treatment, as well as for increasing the public awareness in recent years, this disease is projected to become the leading cause of death worldwide. Advancements in omics, analytical procedures, and high throughput screening in the last five years have led to the realization that human diseases and especially cancer are more complex than were originally conceived. Cancer is not a static entity that can be easily monitored and manipulated. It is characterized by a dynamic and time-dependent network of constantly altered molecular and cellular interactions between players in different pathways. This network is not invariable and rigid but is constantly reshaped and altered conforming to the pliable signaling processes/responses implicated. Its complexity is apparent by the fact that the disease state is not a disruption of a single node or specific nodes in the network organism but is organism-patient dependent, thus requiring personalized perspective approaches.

Despite the progress in techniques for cancer prevention, detection, and treatment, as well as for increasing the public awareness in recent years, this disease is projected to become the leading cause of death worldwide. Advancements in omics, analytical procedures, and high throughput screening in the last five years have led to the realization that human diseases and especially cancer are more complex than were originally conceived. Cancer is not a static entity that can be easily monitored and manipulated. It is characterized by a dynamic and time-dependent network of constantly altered molecular and cellular interactions between players in different pathways. This network is not invariable and rigid but is constantly reshaped and altered conforming to the pliable signaling processes/responses implicated. Its complexity is apparent by the fact that the disease state is not a disruption of a single node or specific nodes in the network organism but is organism-patient dependent, thus requiring personalized perspective approaches.
Numerous challenges hamper effective cancer treatment and development of effective drugs such as ineffective therapeutic drug concentration reaching the tumor site, lifethreatening side-effects caused by nonspecific tissue distribution of anticancer agents, and acquired resistance of the cancer cell upon chemotherapy that triggers cross-resistance to a wide range of different drugs.
Such multifactorial states require the development of very delicate approaches in the course of the drug discovery pipeline. The scientific roots of the drug development philosophy should be shifted from the traditional concept of the "magic bullet" drug (i.e., scalped for a single drug target) to the formulation of a navigated vehicle which could spatiotemporally deliver the drug in the correct location and the appropriate time. Thus, the term targeted drug delivery should give its place to navigated drug delivery since it is not only the cytotoxic drug that targets a specific cellular location but rather a vehicle that navigates the course of the loaded drug to the appropriate site of action. Such drug loaded and navigated vehicles in order to enhance the selective uptake of the cytotoxic agent by the tumor cells and spare the normal cells, should consist of a multidimensional architecture ( Figures  1 and 2). The major components of these vehicles are the transporting vehicle (i.e., lipid), the cytotoxic agent that is loaded, the "programmable" navigating/targeting agent (i.e., receptor specific ligand) that enables the appropriate delivery routes to avoid toxicity on healthy proliferating cells as also ineffective concentration of the cytotoxic agent : Navigated drug delivery: the drug delivery vehicle is equipped with a "programmable navigation system" that allows the transportation of the "blind" cytotoxic agent in the correct cellular location. In the absence of the drug delivery vehicle that is tagged with navigating delivery routes, toxicity is triggered on healthy proliferating cells against the anticancer agent, and ineffective therapeutic drug concentration reaches the tumor site.
to the tumor site (Figure 2), and the "stealth" nanocarriers (biocompatibility polymers, i.e., PEG) that enhance the short plasma half-life of the drug-loaded transporting vehicle.
In line with the challenges raised by the complexity of cancer, the aim of the present thematic issue is to provide an accumulation of innovative molecularly targeted cancer therapies and entrepreneurial methods of drug delivery in cancer (Table 1). It contains 12 papers embracing most aspects of cancer related to the exploration of active targeted nanoparticles for cancer treatment and diagnosis, including the exploitation of novel cancer drug targets, vaccine design against cancer, innovative methods for drug delivery-based on focused ultrasound or convection enhanced delivery, and mathematical modeling as an indispensable tool to analyze the transport processes and predict the outcome of anticancer treatment.
A constant developmental effort is being conducted for innovative nanoparticles to meet unmet needs in the transportation of imaging and therapeutic agents for cancer diagnosis and therapy. This happens since the nanoscale of these particles allows sculpting of diversity in their capabilities thus, enabling them to respond to a diverse array of functional requirements. Therefore, nanoparticles have been considered as appropriate vehicles to provide an ideal platform for personalized approaches to cancer diagnosis and therapy in cancer disease management. A. D. Miller presents Clinical attrition rates are a critical issue in drug development. In oncology, fourfold higher rates of attrition have been determined in respect of other indications. This clearly pinpoints the unmet necessity to steer for novel and traditionally unexplored drug targets to be employed in the drug discovery process towards the development of novel anticancer drugs. In this light, V. Buxhofer-Ausch et al. describe the capacity of members of the organic anion transporter family (OATP) to serve as tumor biomarkers and effective cancer drug targets. The importance of these drug targets is due to their implication on the uptake of clinically important drugs and hormones, thereby affecting drug disposition and cell penetration. An OATP-targeted therapy holds promises to combat cancer efficiently and with lesser side effects due to the tissue specific expression of different OATP members and specifically their differential expression in various cancer and normal tissues.
Enzymes of the sulfatase pathway could offer another appealing cancer drug target. S. Lena et al. provide a comprehensive review about the expression and function of enzymes of the sulfatase pathway, particularly of steroid sulfatase (STS), in breast, endometrial, ovarian, and colorectal cancer. Furthermore, it highlighted the applicability of STS inhibitors to function as enzyme-based cancer imaging agents applied in the biomedical imaging technique positron emission tomography for the diagnosis and therapy of estrogensensitive cancers.
Since cancer is considered not only as a genetic but also as an epigenetic disease and tumorigenesis involves multiple genetic and epigenetic alterations that contribute to the transformation of normal cells towards a malignant phenotype, epigenetics should also be the focus of discovering novel cancer drug targets. E. Hatzimichael and T. Crook reviewed important advances in the field of cancer epigenetics and specifically provide an overview of the clinical use of epigenetics as cancer biomarkers and current progress in the utilization of epigenetic drugs in solid and blood cancers.
Navigated delivery of cytotoxic agents to special sites or organs is a challenging issue that needs to be addressed in order to surpass systematic toxicity. These challenges become even more exigent for drug delivery in brain tumors where intrinsic difficulties are met due to the hurdles faced to cross the brain-tumor and blood-brain barriers. To establish a targeting vehicle for glioblastoma cells, T. Kasai et al. exploited the capability of chlorotoxin to selectively bind to matrix metalloproteinase-2 and other proteins on glioma cell surfaces once it was fused to human IgG-Fcs and displayed on the surface of bionanocapsules. This chlorotoxin loaded bionanocapsule showed specific affinity to the surface of glioma cells and internalized into the cytoplasmic space suggesting that is a promising drug delivery system for targeting glioblastoma.
In order to establish a target-based agent to be specifically delivered to osteosarcomas, K. Hayashi et al. focused on a lectin (Eucheuma serra agglutinin) both as a tumor-targeting agent and as an antitumor agent. Lectins are carbohydrate binding proteins that are highly specific for sugar moieties on the surface of tumor cells. The authors formulated a drug delivery system containing PEGylated Span 80 vesicles which immobilized the specific lectin. They noted that the specific system was not only selectively targeting osteosarcoma cells but was also cytotoxic to the targeted cells emphasizing the dual role adopted by lectins both as navigating and cytotoxic agents.
To achieve an effective approach for enhanced uptake of anticancer drug-loaded vehicles by tumor cells, M. W. Ndinguri et al. explored cell surface proteoglycan CD44 targeting as a way to selectively deliver therapeutic agents encapsulated inside colloidal delivery systems. CD44 has been recognized as a contributor to tumor chemoresistance and as a cancer cell and cancer stem cell biomarker due to its overexpression in cancer compared to normal cells (haematopoietic, epithelial, and neuronal cells). To target CD44, the authors used a triple-helical peptide sequence derived from type IV collagen that was incorporated in doxorubicin-loaded liposomes (composed of DSPG, DSPC, cholesterol, and DSPE-PEG-2000). The CD44-targeted liposomes were found effective in reducing tumor size, highlighting their potential to be used for selective chemotherapeutic treatment of CD44 overexpressing tumor cells.
The immune system has evolved to protect the body against microorganism invasion and thereby prevent diseases. Thus, cancer vaccines could boost the immune system to enable it to combat cancer more effectively. In vaccine development, a major aim is to induce strong, specific T cell responses. This is achieved by targeting antigens to cell surface molecules on dendritic cells (DCs) that efficiently stimulate T cell responses. The paper by V. Apostolopoulos et al. focuses on the most attractive cell surface receptors expressed on DCs to be used as targets for antigen delivery for cancer immunotherapy. The DC cell surface receptors M. Thanou and W. Gedroyc provided a comprehensive overview on the utilization of MRI-guided focused ultrasound (MRgFUS) as a new method of drug delivery. This methodology combines a high intensity focused ultrasound beam that can be used as an external stimulus to activate drug delivery in tissues and Magnetic Resonance Imaging system (MRI) which visualizes patient anatomy and controls the treatment by continuously monitoring the tissue effect. The advantages of being noninvasive as well as controlled and focus could establish this methodology as valuable tool in clinic to increase drug targeting and tissue specific drug delivery.
The transport of anticancer drugs and their consequence on tumor cells implicates an array of physical and biochemical processes. Since multiple steps are involved in the drug-transport, drug-release, and drug-uptake pipelines, mathematical models have become an indispensible tool to analyze the transport processes and predict the outcome of anticancer treatment. Mathematical modeling of cancer provides a tool to assist our realization on the interaction and drug-mediated perturbation of such complex processes, thus, fruitfully contributing to the optimization and refinement of drug delivery. W. Zhan and X. Y. Xu present the development of an improved mathematical model that was applied to an idealized geometry consisting of tumor and normal tissues. They predicted the efficacies of direct intravenous administration and thermosensitive liposome-mediated delivery and illustrated that thermo-sensitive liposome-mediated delivery provides a lower drug concentration in normal tissues than direct infusion of non-encapsulated drug as also a significantly higher peak intracellular concentration. These computational results furnish a projection on the effectiveness of two different treatments, within a mathematical framework, and set the basis to develop and corroborate optimized treatments with reduced risk of associated side effects as also Journal of Drug Delivery 5 effective tumor cell killing in a short time period of treatment.
Due to the complexity of the disease, efficient cancer therapy can emerge only upon interscience collaboration. This special issue aims to accumulate current knowledge on molecularly targeted cancer therapies and innovative methods of drug delivery in cancer. We believe that this accumulative knowledge will assist to accelerate progress developing more precise navigated drug delivery in cancer based on innovative tools.


s2fieldsofstudy: Biology Chemistry

text:
Dependence of Micelle Size and Shape on Detergent Alkyl Chain Length and Head Group

Micelle-forming detergents provide an amphipathic environment that can mimic lipid bilayers and are important tools for solubilizing membrane proteins for functional and structural investigations in vitro. However, the formation of a soluble protein-detergent complex (PDC) currently relies on empirical screening of detergents, and a stable and functional PDC is often not obtained. To provide a foundation for systematic comparisons between the properties of the detergent micelle and the resulting PDC, a comprehensive set of detergents commonly used for membrane protein studies are systematically investigated. Using small-angle X-ray scattering (SAXS), micelle shapes and sizes are determined for phosphocholines with 10, 12, and 14 alkyl carbons, glucosides with 8, 9, and 10 alkyl carbons, maltosides with 8, 10, and 12 alkyl carbons, and lysophosphatidyl glycerols with 14 and 16 alkyl carbons. The SAXS profiles are well described by two-component ellipsoid models, with an electron rich outer shell corresponding to the detergent head groups and a less electron dense hydrophobic core composed of the alkyl chains. The minor axis of the elliptical micelle core from these models is constrained by the length of the alkyl chain, and increases by 1.2–1.5 Å per carbon addition to the alkyl chain. The major elliptical axis also increases with chain length; however, the ellipticity remains approximately constant for each detergent series. In addition, the aggregation number of these detergents increases by ∼16 monomers per micelle for each alkyl carbon added. The data provide a comprehensive view of the determinants of micelle shape and size and provide a baseline for correlating micelle properties with protein-detergent interactions.

Introduction
Detergents have played a significant role in advancing the understanding of membrane protein structure and function. Detergent monomers in solution self-assemble at and above the critical micelle concentration (cmc) to form micelles. Micelles adopt globular shapes (e.g., spheres, ellipsoids, and cylinders) of various sizes, determined by the detergent head group structure and alkyl chain length [1]. The hydrophilic head groups of the detergent comprise a solvent-exposed outer shell, while the hydrophobic alkyl chains are sequestered from water and form the micelle core. Thus, the cross-sectional composition of the micelle is structurally similar to the lipid bilayer with hydrocarbon chains bound by hydrophilic head groups.
Consequently, micelles are often employed as mimics of lipid bilayers to solubilize and stabilize integral membrane proteins for structural and functional in vitro studies [2][3][4][5]. Although nonmicellar amphiphilic systems, such as nanodiscs [6] and bicelles [7], have been used in membrane protein studies, so far detergents have demonstrated more successes in high-resolution structure determination of membrane proteins. Nonetheless, stable, functional protein-detergent complexes (PDC) are difficult to obtain because protein denaturation and aggregation often occur. Determining the optimal conditions that yield a properly folded membrane protein relies heavily on exhaustive screening of detergents [8][9][10][11]. This need for empirical detergent screening stems from a lack of understanding of the physical forces between the detergent micelle and membrane protein.
Micelle size, shape, and detergent concentration need to be considered in evaluating a PDC for structural and biochemical studies. For instance, recent evidence suggests that the micelle hydrophobic thickness needs to match that of the membrane protein to maintain proper fold and function [12][13][14] (similar to the hydrophobic match proposed for membrane proteins in bilayers [15][16][17][18][19]). Therefore, a systematic investigation of the size and shape determinants of pure micelles will provide a baseline and foundation for the further understanding of micelle-protein interactions.
In this study, the sizes and shapes of micelles formed by four classes of detergents, phosphocholines, maltosides, glucosides, and lysophosphatidyl glycerols (Table 1), are investigated using smallangle X-ray scattering (SAXS). The detergents were selected based on their prevalence in membrane protein structural biology. Approximately 40% of the ,115 membrane protein structures determined by NMR were prepared in dodecyl phosphocholine (FC12) micelles while nearly 40% of the ,1200 membrane protein structures determined by X-ray crystallography were in octyl glucoside (OG), decyl maltoside (DM), or dodecyl maltoside (DDM) micelles [20]. Lysophosphatidyl glycerols (LPGs) have also facilitated solution NMR investigations of membrane proteins [21]. For each detergent class, multiple alkyl chain lengths were studied to address structure similarities within the class as well as common trends among all classes.
To date, few experimental studies have systematically investigated the effects of detergent structure on micelle geometry. In addition, a limited number of detergents and alkyl chain lengths have been investigated and inconsistencies in buffer conditions and methodologies between studies complicate comparison of detergent micelle properties. This study experimentally correlates the monomer detergent structure with micelle physical properties so that micelle shapes and sizes can be more accurately predicted, in particular with a view towards assessing PDC structure and function.
The shapes and sizes of micelles formed by the eleven detergents (Table 1) were analyzed under the same experimental conditions. The model independent and dependent parameters were then analyzed to establish trends in micelle size and shape. With an increase in the alkyl chain by a carbon atom, the aggregation number increases by 1663 detergent monomers. The short axis of the micelles increases with a distance expected for an additional alkyl carbon, while ellipticity (the ratio of the minor and major axes) is maintained. These results establish ellipsoid micelle models that are consistent through a detergent series and are expected to provide predictive measures for other detergent micelle size and shapes.
Several concentrations up to 200 mM were prepared for each detergent in a final buffer consisting of 20 mM phosphate buffer, pH 6.2, 150 mM NaCl, and 10% v/v D 2 O (necessary for the NMR deuterium lock). Detergent monomers do not contribute significantly to the observed scattering [22], and all scattering profiles presented are at concentrations well above the cmc (Table 1). Detergent concentrations were verified using 1D 1 H-NMR and standards of known concentration.
Three molecular weight standards were used for the X-ray scattering experiments: hen egg white lysozyme in 40 mM acetate buffer, pH 3.8 with 150 mM NaCl, horse heart cytochrome c (Sigma) in 100 mM acetate buffer, pH 4.6 with 0.5 M guanidinium hydrochloride, and bovine serum albumin (Sigma) in 20 mM HEPES buffer, pH 7.8 with 50 mM NaCl. Five concentrations were measured for each protein standard (up to 10.6 mg/ mL lysozyme, 4.2 mg/mL cytochrome c, and 8.6 mg/mL albumin) to determine any concentration dependent effects on the scattering. The scattering profiles that did not demonstrate concentration dependent changes were used to calculate k, the proportionality constant in Equation 6 (see Methods S1) that is used to determine micelle aggregation numbers.

SAXS Data Collection
SAXS data were measured at the XOR/BESSRC undulator beam line 12-ID-B of the Advanced Photon Source (Argonne, IL), with a sample-to-detector distance of 2 m and a Pilatus 2 M detector. The data were collected at 25uC using a custom-made sample holder [23] and an X-ray energy of 12 keV (corresponding to a wavelength of l = 1 Å ). The useable range of momentum transfer q was 0.02,q,0.3 Å 21 (q = 4p sin(h)/l, where 2h is the scattering angle and l is the x-ray wavelength). Additional descriptions of the beamline setup and measurement are previously published [23][24][25].
For the protein molecular weight standard samples, five exposures of 0.1 s were collected, image corrected, and circularly averaged. For the detergent samples, at least five exposures of 0.5 s each were collected. The absence of radiation damage was confirmed by comparing subsequent exposures of the same sample with no significant changes in the SAXS profile detected (data not shown). The five resulting profiles for each condition were averaged to improve signal quality. Matched buffer profiles were collected using identical procedures and subtracted from the sample scattering for background correction.
Analysis of the SAXS profiles for each sample followed the procedures outlined in Lipfert, et al. [22] for determination of micelle size and shape parameters (Methods S1). One noted exception was the use of a nonlinear, least-square fitting routine implemented in Igor Pro (WaveMetrics) as part of the NCNR analysis toolkit [26] to fit the two component (core-shell) models to the full scattering profiles (e.g. sphere, oblate, and prolate; Fig. 1). Although designed to model fits to small-angle neutron scattering (SANS) data, this procedure was readily adapted to SAXS data by replacing scattering length densities (effective atomic scattering powers in SANS) with electron densities for the micelle core and shell components. Agreement between model-independent and similar model-derived values was used for additional validation of the modeling approach.

Results and Discussion
Eleven micelle-forming detergents, classified by head group structure, were chosen for this study: zwitterionic phosphocholines with 10-, 12-, and 14-carbon alkyl chains, nonionic glucosides with 8-, 9-, and 10-carbon alkyl chains, nonionic maltosides with 8-, 10-, and 12-carbon alkyl chains, and ionic lysophosphatidyl glycerols with 14-and 16-carbon alkyl chains (Table 1). At least one detergent studied from each of the four classes has demonstrated success in membrane protein structure determination. Micelle scattering data were previously published for some detergents from each class; however, to obtain meaningful trends in micelle size and shape with changes in chain length, additional detergents were studied. The concentration series of scattering profiles, Guinier plots, calculated aggregation numbers, and the best model fit to the lowest detergent concentration (to reduce contributions from interparticle interference) for the FC14, OM, DG, and LMPG are presented in Figure 2, while data showing agreement with the previously reported detergents [22] are presented in Figures S2, S3, S4, S5, S6, S7, and S8. The data, fits, and resulting shapes and sizes of each detergent are described in Results S1, where we also compare the results to the available literature. The physical parameters from model-independent (subscript expt is used) and dependent (subscript model is used) measurements for all the detergents are provided in Table 2 and Table S1.
In summary, the micelles were modeled using a two-component ellipsoid ( Fig. 1) for which the ellipsoidal dimensions (a, b. and t), and the radius of gyration (Rg model ) and aggregation number (N model ) were determined (Table 2). In addition, model independent parameters (Rg expt , N expt ,and L expt ) were measured or calculated directly from the scattering profile or from Guinier analysis of the low q data ( Table 2). For further description of the physical parameters and determination of each, see Methods S1. Micelle geometries (sphere, oblate/prolate ellipsoid) for each detergent were determined by comparing the fits for each shape to the experimental SAXS profile (Fig. S9) and considering any physical constraints imposed on the model. In addition, a cylindrical model was applied to the DG scattering profile (Fig. S10), but did not provide a more reasonable fit (see Results S1). The phosphocholine micelle models share a common 2.7-3.0 Å uniform shell thickness and a prolate ellipsoid geometry. The maltoside micelle models share a common 5.4-5.8 Å uniform shell thickness and an oblate ellipsoid geometry. The glucoside micelle models share a common 2.9-3.5 Å uniform shell thickness and an oblate ellipsoid geometry. Finally, the LPG micelle models share a common 5.3-6.1 Å uniform shell thickness and an oblate ellipsoid geometry. All detergent micelle models have a minor core axis approximating the length of a detergent monomer with almost fully extended alkyl chains. The four detergent types with multiple alkyl chain lengths provided a foundation for a systematic investigation of the trends in micelle size and shape with varied detergent properties.

Comparison of the Two-component Ellipsoid Models and Model-free Derived Parameters
In order to validate the core-shell ellipsoid micelle model fits to the experimental data, comparisons were made between parameters that could be determined both from the geometric model and directly from the scattering profile (model-independent). The agreement between the model-independent radius of gyration, aggregation number, and dominant head group to head group length and that of the models strongly support the proposed ellipsoid models.
Radius of gyration (Rg). The radii of gyration calculated from the geometric model (Rg model ) are near or within the reported error from the Guinier analysis of the experimental data (Rg expt ) for all the detergents except the glucosides and FC14 (Table 2 and Fig. 3). However, evaluation of the values derived from the Guinier analysis must be taken with care as the scattering profile can be altered by attractive and/or repulsive effects between micelles (particularly at high concentrations), complicating accurate calculation of I(0) and Rg. In the case of FC14 and the glucosides, where there is some disagreement between the two Rg values, deviation from linearity in the low q region of the Guinier and Guinier fits (black lines). For DG, the Guinier region deviated from linearity at low q (denoted by *) as described in the results, and prevented accurate extrapolation to zero-scattering angle. Thus, aggregation numbers for DG could not be determined from the I(0) method, and the plot (panel D, see below) is not presented. An increase in scattering signal with increasing concentration is generally observed, except for LMPG at high concentration and low q (denoted with **) where interparticle effects described in the results become more apparent at higher concentrations. (C) Two-component ellipsoid fit (black solid line) and scattering intensity recorded at low detergent concentration (same color scheme as part A). The residuals of the fit are shown in the upper inset. Fit parameters are presented in Table 2. (D) Apparent aggregation numbers N expt (squares, same color scheme as in part A) obtained from the extrapolated forward scattering intensity and eq 7 described in Methods S1. The point at 0 mM (black) corresponds to the estimate obtained by linearly extrapolating the measured profiles for [c-cmc] #100 mM to zero micelle concentration (i.e. cmc). Errors are obtained from repeat fits using measurements from three molecular weight standards. Additional descriptions of micelle measurements and calculations can be found in Methods S1. doi:10.1371/journal.pone.0062488.g002 plots indicates micelle interactions that lead to an over-estimation of the size of the micelle.
Aggregation number (N). Aggregation numbers calculated from the hydrophobic core volume of the micelle model (N model ) were consistently larger (by about 10 monomers per micelle) than the aggregation numbers determined from the extrapolated forward scattering intensity (N expt ) ( Table 2 and Fig. 4A). This discrepancy between the aggregation numbers may be attributed to the additional separation between monomers, or monomer dynamics, in the micelle that is not accounted for in the direct volume calculation; that is, closest packing is assumed in the calculation. Although the geometric model parameters showed negligible concentration dependence (data not shown), a concentration dependence was observed in N expt (Fig. 2D) and Rg expt (data not shown), which was more pronounced in the charged micelles. The aggregation number from the forward scattering represents the value extrapolated back to the cmc concentration (zero micelle concentration) to provide an estimate free of concentrationdependent effects. The agreement of the experimentally determined aggregation numbers with the values reported in the literature further support the validity of the models. However, it should be noted that while the aggregation numbers determined for OM are consistent with the linear trends observed for the dependence of aggregation number on alkyl chain length (discussed below), these values do not agree with the available data from Affymetrix, Inc. (Table 1). Aggregation numbers from the forward scattering intensity are not reported for the glucoside detergents in this study (due to unreliable extrapolations of scattered intensity to zero scattering angle), and uncertainties in the wide range of published values provide only a qualitative comparison.
Dominant head group to head group length (L). The dominant head group to head group length describes the most frequently occurring distance between head groups separated by a pair of alkyl chains across the micelle. The 2 nd peak in the scattering profile provides a model-free assessment of the most frequently observed length scale, correlating to this distance L [13,22]. The length between opposing head groups from the model (L model ) are mostly within error or slightly shorter (1-2 Å ) than the corresponding distance determined from the position of the 2 nd peak in the SAXS profile (L expt ), except for FC10 and FC12 which are 1-2 Å longer ( Table 2 and Fig. 5A). A comparison between the two measured dominant head group to head group lengths was also useful in assessing the micelle model ellipsoid shape (oblate vs. prolate). Some fits to the scattering profiles did not agree with the structure of the detergent monomer and the observed L expt ; thus, they were not considered accurate models. Additionally, the dominant head group to head group length (L expt ) is measured at intermediate scattering angles and is not susceptible to the inter-particle interference observed at low q.  For FC14, an average Rg expt from the lower concentration data (#150 mM) was used because deviation from linearity in the Guinier region was observed; b The Guinier regions for the glucosides are mostly nonlinear in the range that q*Rg,1.3, however estimations were made from lower concentration data where possible. In addition, the rise in intensity as q 0 precluded the determination of forward scattering intensities and thus N expt could not be determined. Parameters were obtained from optimal core-shell ellipsoid model fits to the experimental SAXS data at given total concentrations of detergent in solution. doi:10.1371/journal.pone.0062488.t002

Head Group -Head Group Distances Increase with Alkyl Chain Length
With the data from multiple detergents systematically varying in chain length, the dependence of micelle physical parameters on chain length and head group could be investigated. The dominant head group -head group distances as determined from the position of the second maximum in the scattering profile (L expt ) are plotted as a function of alkyl chain length in Figure 5B. In each class of detergents studied, the dominant distance between head groups across the micelle increases by 2.5-3.0 Å with the addition of each carbon to the alkyl chain (note that the distance across the micelle contains two opposing alkyl chains). The short axial dimension of the fitted ellipsoid models similarly increases by 2.5-3.0 Å with each added alkyl carbon, while the thickness of the outer shell in the two-component is independent on alkyl chain length, as expected ( Table 2). The short ellipsoid axis dimension increases by ,2.5 Å for every two carbons added to the alkyl chain for the phosphocholines, ,2.4 Å for every two carbons added to the alkyl chain for the maltosides, ,1.5 Å for each carbon added to the alkyl chain for the glucosides, and ,2.5 Å for the two carbon increase in the LPG tail length ( Table 2). The average increase in alkyl chain length per added carbon unit for all micelles in this study is approximately 1.3160.13 Å (length of short axis (a or b) divided by the number of carbons), which is in good agreement with the additional length expected by the addition of each carbon, ,1.27 Å from Tanford's formula [27] for the maximum extension of the alkyl chain (l c = 1.5+1.265 n c ).

Aggregation Numbers Increase with Carbon Chain Length
The aggregation numbers calculated from the model for each detergent are plotted as a function of alkyl chain length in   Table 2. The average increase from all detergent micelles in this study indicates that micelle aggregation numbers increase linearly by approximately 1663 monomers per micelle with the addition of each carbon atom to the alkyl chain. This steady increase in aggregation number highlights the significant contributions to the increased hydrophobic interactions between adjacent monomers made by the addition to alkyl chain length.
Tanford predicted the maximal micelle aggregation numbers based on geometric packing of the alkyl chains in the hydrocarbon core for spherical and ellipsoidal micelles over a given range of ellipticities and chain lengths [28]. The detergent monomers in this case are assumed to have a maximum alkyl chain extension, which will result in overestimation of the aggregation number [29]. The best-fit model parameters for the phosphocholine data indicate a prolate ellipsoid geometry with a/b ,1.5, which corresponds to a predicted increase of ,14 monomers per micelle with each increased carbon in the alkyl chain, which is in agreement with the observed 12-15 monomers ( Table 2 and Fig. 5B). The oblate maltosides (glucosides were excluded for reasons discussed above) have a relative a/b of ,0.57 corresponding to a predicted increase of ,28 monomers per micelle with each increased carbon in the alkyl chain [28] and in reasonable agreement with the observed 20-25 monomers. The oblate geometry with a relative a/b of ,0.7 from the two LPG models corresponds to a predicted approximate increase of 20-25 monomers per micelle [28], which agrees well with the increase of 25 monomers per micelle determined in this study. In addition to aggregation number and core thickness, other measurements, such as radii of gyration, vary linearly with the number of carbons in the alkyl chain (1.5560.85 Å per carbon added; Table 2).
Using the geometric packing approach of Tanford, the calculated values for the aggregation numbers (e.g. 60, 83, and 110 for n c = 10, 12, and 14, respectively, for phosphocholine; 47, 58, 70, and 97 for n c = 8, 9, 10, and 12, respectively, for maltosides; and 105 for n c = 14 and 134 for n c = 16 for LPG) are also in good agreement with values determined in this study ( Table 2), given that the theoretical model likely presents an overestimation due to the assumed maximum extension of the alkyl chain. A comparison of the alkyl chain length predicted from the maximum extended chain length and determined from the micelle model indicates that the alkyl chains of the model are 80-90% of the maximum extension, consistent with a flexible yet closely-packed hydrocarbon core [27].

Aggregation Number Decreases with Steric Bulk or Electrostatic Repulsion
In addition to chain length, a major determinant of micelle aggregation number is the size and charge of the head group. The aggregation numbers of the nonionic series depend on the head group identity as can be observed by comparing the aggregation numbers for a single chain length (Fig. 4). Two previously reported data sets for (N-alkylamino)-1-deoxylactitols [30] and sucrose esters [31] are included in Figure 4B, to provide additional comparisons of head group effects. The general trend observed is that with an increase in steric bulk (nearest to the alkyl chain; Fig.  S11) the aggregation number decreases. The charged LPGs have even smaller aggregation numbers compared to that extrapolated for the polar sugar head groups. Phosphocholine prolate micelles cannot be directly compared based on head group to the oblate micelles because for the same chain length and aspect ratio, prolate micelles have a smaller aggregation number (larger surface area per head group) than the oblate micelle regardless of the repulsion that is likely to exist between the head groups [28].

Micelle Shape and Ellipticity Depend on Head Group Packing
Micelle shapes have been debated in the literature [28,[31][32][33][34][35]; however, the overwhelming evidence indicates that for single chain detergents the head group properties dictate the ellipticity (aspect ratio; a/b) of the micelle and that prolate and oblate micelles are observed [22,28,30,31,36]. Beyond this simplified treatment of the head group interactions, Iyer and Blankschtein [37] and Dupuy et al. [30] propose models for non-ionic surfactants that predict ellipticity and shape based on the packing and interactions between head groups (and solvent) that are independent of chain length. Their model also predicts that oblate micelles are preferred for small nonionic detergents, but that as the head group size increases or electrostatic repulsion occurs prolate micelles may be the preferred. The oblate ellipticity is predicted to approach spherical (aspect ratio of 1) as the head group increases in size or has repulsive electrostatic interactions (larger surface area). Indeed, this trend is observed in the ellipticities determined for the glucosides, maltosides, and LPGs (Table 2); the electrostatic repulsions between negatively charged head groups in LPG micelles gives rise to a more spherical micelle geometry compared to the uncharged glucosides and maltosides. The zwitterionic phosphocholines form prolate micelles indicating that the head group has significant steric and electrostatic repulsion and has a high surface area. This effect may be exaggerated due to counterion interactions at the surface with the prediction that ellipticity of the phosphocholine prolate micelle would be dependent on ionic strength. Also, as predicted by the Iyer and Blankschtein model, the micelle ellipticity does not change with an increase in chain length and aggregation number (Table 2).

Conclusions
The detergents investigated form micellar aggregates, which resemble core-shell ellipsoids, with near maximally extended alkyl chains comprising the core, and a compact shell formed by the detergent head groups which separates the hydrophobic core from the surrounding aqueous environment. Elliptical geometries provided the best fits to the micelle scattering data with dimensions that were consistent with the physical properties of the detergent monomer. The size of the micelles increased linearly with an increase in alkyl chain length; approximately 1663 monomers per micelle with the addition of each carbon atom. The increase in size was accommodated by an increase in the longer axis; however only to the length that maintained the ratio of the axes. These results provide a better understanding of the principles of detergent self-assembly, which will allow predictions of other micelle properties based on these principles. In addition, these results provide a foundation of physical properties important to the understanding of mixed detergent and PDC systems.

Supporting Information
Supplemental figures and tables are provided detailing the chemical structures of detergents, in addition to the SAXS profiles, Guinier plots, and calculated geometric parameters for each detergent micelle. Supplemental texts (Methods S1 and Results S1) describing additional details of the SAXS methodology and theory followed by a discussion of the results pertaining to each class of detergent micelle are also included as supporting information.  Table 2. (TIF) Figure S4 Scattering data, Guinier analysis, and two-shell ellipsoid fit for DM. (A) SAXS profiles (I(q)) of DM at total detergent concentrations of 15 (blue), 36 (yellow), 58 (red), 80 (purple), 99 (green), 152 (brown), and 204 (cyan) mM. (B) Guinier plot (ln(I) as a function of q 2 ) of the very low angle data (same color code as part A) and Guinier fits (black lines). An increase in scattering signal with increasing concentration is observed. (C) Apparent aggregation numbers N obtained from the extrapolated forward scattering intensity and eq 7 (squares, same color code as in part A). The point at 0 mM (black) corresponds to the estimate obtained by linearly extrapolating the measured profiles for [ccmc] #100 mM to zero micelle concentration (i.e. cmc). Errors are obtained from repeat fits using measurements from three molecular weight standards. (D) Two-component ellipsoid fit (black solid line) and scattering intensity recorded at a detergent concentration of 15 mM (blue, as before). The residuals of the fit are shown in the upper inset. Fit parameters are presented in Table 2. (TIF) Figure S5 Scattering data, Guinier analysis, and two-shell ellipsoid fit for DDM. (A) SAXS profiles (I(q)) of DDM at total detergent concentrations of 17 (blue), 32 (yellow), 57 (red), 75 (purple), 94 (green), 145 (brown), and 194 (cyan) mM. (B) Guinier plot (ln(I) as a function of q 2 ) of the very low angle data (same color code as part A) and Guinier fits (black lines). An increase in scattering signal with increasing concentration is observed. (C) Apparent aggregation numbers N obtained from the extrapolated forward scattering intensity and eq 7 (squares, same color code as in part A). The point at 0 mM (black) corresponds to the estimate obtained by linearly extrapolating the measured profiles for [ccmc] #100 mM to zero micelle concentration (i.e. cmc). Errors are obtained from repeat fits using measurements from three molecular weight standards. (D) Two-component ellipsoid fit (black solid line) and scattering intensity recorded at a detergent concentration of 17 mM (blue, as before). The residuals of the fit are shown in the upper inset. Fit parameters are presented in Table 2. (TIF) Figure S6 Scattering data, Guinier analysis, and two-shell ellipsoid fit for OG. (A) SAXS profiles (I(q)) of OG at total detergent concentrations of 50 (red), 75 (purple), 100 (green), and 150 (brown) mM. (B) Guinier plot (ln(I) as a function of q 2 ) of the very low angle data (same color code as part A). Aggregation numbers were unable to be determined from the I(0) method, and the plot is not presented. (C) Two-component ellipsoid fit (black solid line) and scattering intensity recorded at detergent concentration of 50 mM (red, as before). The residuals of the fit are shown in the upper inset. Fit parameters are presented in Table 2. (TIF) Figure S7 Scattering data, Guinier analysis, and two-shell ellipsoid fit for NG. (A) SAXS profiles (I(q)) of NG at total detergent concentrations of 25 (blue), 50 (red), 100 (green), and 200 (cyan) mM. (B) Guinier plot (ln(I) as a function of q 2 ) of the very low angle data (same color code as part A). Aggregation numbers were unable to be determined from the I(0) method, and the plot is not presented. (C) Two-component ellipsoid fit (black solid line) and scattering intensity recorded at detergent concentration of 25 mM (blue, as before). The residuals of the fit are shown in the upper inset. Fit parameters are presented in Table 2. (TIF) Figure S8 Scattering data, Guinier analysis, and two-shell ellipsoid fit for LPPG. (A) SAXS profiles (I(q)) of LPPG at total detergent concentrations of 5 (blue), 10 (yellow), 25 (red), 50 (purple), 100 (green), 150 (brown), and 200 (cyan) mM. (B) Guinier plot (ln(I) as a function of q 2 ) of the very low angle data (same color code as part A) and Guinier fits (black lines). An increase in scattering signal with increasing concentration is observed. Note that the Guinier region at high concentrations is highly distorted by interparticle repulsions between charged micelles. As this data was not collected with the same molecular weight standards, aggregation numbers determined from the forward scattering are not shown. (C) Two-component ellipsoid fit (black solid line) and scattering intensity recorded at detergent concentration of 5 mM (blue, as before). The residuals of the fit are shown in the upper inset. Fit parameters are presented in Table 2.

Supporting Information
(TIF) Figure S9 Best core-shell model fits using different geometries (oblate/prolate ellipsoid and sphere) to micelle scattering profiles for multiple alkyl chain lengths of each detergent class. (A) SAXS profiles (I(q)) of phosphocholine detergents (FC10: blue, FC12: green, and FC14: red) at total detergent monomer concentrations of 59, 77, and 97 mM (corresponding to ,1 mM of micelle), respectively, with the best model fits for each geometry shown as black lines. Residuals are shown above for each alkyl chain length using the same color coding. Similar comparisons are shown for: (B) maltoside detergents (OM: blue, DM: green, and DDM: red) at total monomer concentrations of 56, 80, and 94 mM, respectively, (C) glucoside detergents (OG: blue, NG: green, and DG: red) at total monomer concentrations of 50 mM, and (D) lysophosphatidyl glycerol detergents (LMPG: blue and LPPG: red) at total monomer concentrations of 16 and 25 mM, respectively. (TIF) Figure S10 Comparison of sphere (red), oblate ellipse (blue), and cylinder (cyan) geometrical model fits to OG scattering data. The sphere model contains a core radius of 17.1 Å with a shell thickness of 3.6 Å . The ellipse model is oblate and contains a core with semi-axes of 21.4 Å and 10.5 Å with a shell thickness of 3.1 Å . The cylinder model has a core radius of 12.3 Å with a shell thickness of 2.9 Å , and total length of 92.9 Å . Core, shell, and solvent contrasts were consistent between the models. (TIF) Figure S11 Head groups of each of the nonionic detergents compared in Figure 4. The atom attached to the alkyl chain is indicated by an arrow. The head groups are in order of increasing steric bulk from left to right (glucose,maltose,lactitol < sucrose ester). (TIF) Methods S1 Supporting information for methods of determining micelle size and shape from small angle X-ray scattering data. (DOCX) Results S1 Supporting information and results of micelle physical properties with comparison to relevant studies. (DOCX)