The Role of Alpha 6 Integrin in Prostate Cancer Migration and Bone Pain in a Novel Xenograft Model

Of the estimated 565,650 people in the U.S. who will die of cancer in 2008, almost all will have metastasis. Breast, prostate, kidney, thyroid and lung cancers metastasize to the bone. Tumor cells reside within the bone using integrin type cell adhesion receptors and elicit incapacitating bone pain and fractures. In particular, metastatic human prostate tumors express and cleave the integrin A6, a receptor for extracellular matrix components of the bone, i.e., laminin 332 and laminin 511. More than 50% of all prostate cancer patients develop severe bone pain during their remaining lifetime. One major goal is to prevent or delay cancer induced bone pain. We used a novel xenograft mouse model to directly determine if bone pain could be prevented by blocking the known cleavage of the A6 integrin adhesion receptor. Human tumor cells expressing either the wildtype or mutated A6 integrin were placed within the living bone matrix and 21 days later, integrin expression was confirmed by RT-PCR, radiographs were collected and behavioral measurements of spontaneous and evoked pain performed. All animals independent of integrin status had indistinguishable tumor burden and developed bone loss 21 days after surgery. A comparison of animals containing the wild type or mutated integrin revealed that tumor cells expressing the mutated integrin resulted in a dramatic decrease in bone loss, unicortical or bicortical fractures and a decrease in the ability of tumor cells to reach the epiphyseal plate of the bone. Further, tumor cells within the bone expressing the integrin mutation prevented cancer induced spontaneous flinching, tactile allodynia, and movement evoked pain. Preventing A6 integrin cleavage on the prostate tumor cell surface decreased the migration of tumor cells within the bone and the onset and degree of bone pain and fractures. These results suggest that strategies for blocking the cleavage of the adhesion receptors on the tumor cell surface can significantly prevent cancer induced bone pain and slow disease progression within the bone. Since integrin cleavage is mediated by Urokinase-type Plasminogen Activator (uPA), further work is warranted to test the efficacy of uPA inhibitors for prevention or delay of cancer induced bone pain

The Mycobacterium tuberculosis Phagosome Is a HLA-I Processing Competent Organelle

Mycobacterium tuberculosis (Mtb) resides in a long-lived phagosomal compartment that resists maturation. The manner by which Mtb antigens are processed and presented on MHC Class I molecules is poorly understood. Using human dendritic cells and IFN-γ release by CD8+ T cell clones, we examined the processing and presentation pathway for two Mtb–derived antigens, each presented by a distinct HLA-I allele (HLA-Ia versus HLA-Ib). Presentation of both antigens is blocked by the retrotranslocation inhibitor exotoxin A. Inhibitor studies demonstrate that, after reaching the cytosol, both antigens require proteasomal degradation and TAP transport, but differ in the requirement for ER–golgi egress and new protein synthesis. Specifically, presentation by HLA-B8 but not HLA-E requires newly synthesized HLA-I and transport through the ER–golgi. Phenotypic analysis of the Mtb phagosome by flow organellometry revealed the presence of Class I and loading accessory molecules, including TAP and PDI. Furthermore, loaded HLA-I:peptide complexes are present within the Mtb phagosome, with a pronounced bias towards HLA-E:peptide complexes. In addition, protein analysis also reveals that HLA-E is enriched within the Mtb phagosome compared to HLA-A2. Together, these data suggest that the phagosome, through acquisition of ER–localized machinery and as a site of HLA-I loading, plays a vital role in the presentation of Mtb–derived antigens, similar to that described for presentation of latex bead-associated antigens. This is, to our knowledge, the first description of this presentation pathway for an intracellular pathogen. Moreover, these data suggest that HLA-E may play a unique role in the presentation of phagosomal antigens

Association between plasma phospholipid saturated fatty acids and metabolic markers of lipid, hepatic, inflammation and glycaemic pathways in eight European countries: a cross-sectional analysis in the EPIC-InterAct study.

BACKGROUND: Accumulating evidence suggests that individual circulating saturated fatty acids (SFAs) are heterogeneous in their associations with cardio-metabolic diseases, but evidence about associations of SFAs with metabolic markers of different pathogenic pathways is limited. We aimed to examine the associations between plasma phospholipid SFAs and the metabolic markers of lipid, hepatic, glycaemic and inflammation pathways. METHODS: We measured nine individual plasma phospholipid SFAs and derived three SFA groups (odd-chain: C15:0 + C17:0, even-chain: C14:0 + C16:0 + C18:0, and very-long-chain: C20:0 + C22:0 + C23:0 + C24:0) in individuals from the subcohort of the European Prospective Investigation into Cancer and Nutrition (EPIC)-InterAct case-cohort study across eight European countries. Using linear regression in 15,919 subcohort members, adjusted for potential confounders and corrected for multiple testing, we examined cross-sectional associations of SFAs with 13 metabolic markers. Multiplicative interactions of the three SFA groups with pre-specified factors, including body mass index (BMI) and alcohol consumption, were tested. RESULTS: Higher levels of odd-chain SFA group were associated with lower levels of major lipids (total cholesterol (TC), triglycerides, apolipoprotein A-1 (ApoA1), apolipoprotein B (ApoB)) and hepatic markers (alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamyl transferase (GGT)). Higher even-chain SFA group levels were associated with higher levels of low-density lipoprotein cholesterol (LDL-C), TC/high-density lipoprotein cholesterol (HDL-C) ratio, triglycerides, ApoB, ApoB/A1 ratio, ALT, AST, GGT and CRP, and lower levels of HDL-C and ApoA1. Very-long-chain SFA group levels showed inverse associations with triglycerides, ApoA1 and GGT, and positive associations with TC, LDL-C, TC/HDL-C, ApoB and ApoB/A1. Associations were generally stronger at higher levels of BMI or alcohol consumption. CONCLUSIONS: Subtypes of SFAs are associated in a differential way with metabolic markers of lipid metabolism, liver function and chronic inflammation, suggesting that odd-chain SFAs are associated with lower metabolic risk and even-chain SFAs with adverse metabolic risk, whereas mixed findings were obtained for very-long-chain SFAs. The clinical and biochemical implications of these findings may vary by adiposity and alcohol intake

Understanding the effect of producers' attitudes, perceived norms, and perceived behavioral control on intentions to use antimicrobials prudently on New York dairy farms

Understanding farmers' behavior, motivations, and perceptions toward antimicrobial use can influence how veterinarians translate research into practice and guide effective ways of implementing protocols. A multidisciplinary team investigated behavioral tendencies of New York dairy farmers toward antimicrobial use by administering a survey modeled with the reasoned action approach. This approach is a framework from social psychology containing the constructs attitude, perceived norms, and perceived behavioral control, and is used in combination with structural equation modeling to determine what drives intentions. Multiple indicators and multiple causes (MIMIC) models were then used to determine the effects of beliefs on their underlying constructs. The objective of the study was to provide direct and indirect measures of the constructs using survey data to determine importance of and associations with intention to use antimicrobials prudently. The structural equation model indicated that perceived behavioral control explained intention. Thus, farmers who feel capable of prudent use expressed positive intentions. Attitude and perception of others also had influence to a lesser extent. MIMIC models showed that the most important attributes of instrumental attitude were increasing profitability, decreasing risk of residues, and increasing herd health. Contributing attributes of affective attitude were job satisfaction, decreasing resistance, and increasing milk production. For perceived norms, the attributes were opinions/approval of family and peers, veterinarians, and milk processors. Finally, for perceived behavioral control, attributes focused on saving money on labor and treatment, ability to fit into the daily routine, and effectiveness with veterinary guidance. In conclusion, the best approach for adoption of practices might be presentation of examples of successful strategies by other producers, particularly in peer groups. In addition, veterinarians should provide the tools and guidance needed to produce economic gain, reduction of risks associated with residues and resistance, and positive experiences when using the tactics. </p

Mtb proteins require retrotranslocation for presentation.

<p>(A,B) DC were treated with exoA or cycloheximide for one hour prior to infection with H37Rv-eGFP (A) or addition of CFP10 and CFP10_3–11 (B). DC were harvested, fixed, and assessed for their ability to stimulate T cell clones by IFN-γ ELISPOT as described. Each bar reflects the mean±SEM of at least three experiments. ND, not done. (C) DC were treated with exoA, exoA/PJ34, or BSA/PJ34 for one hour prior to infection with vaccinia virus expressing eGFP. After 16–18 hours, DC were harvested and GFP expression analyzed by flow cytometry. Data are representative of three experiments. (D,E) DC treated with exoA, BSA, exoA/PJ34, or BSA/PJ34 for one hour were subsequently infected with H37Rv-eGFP (D) or pulsed with antigen (E) overnight, harvested, fixed, and assessed for their ability to stimulate T cell clones by IFN-γ ELISPOT. Data are representative of two experiments. (F) DC were treated with exoA, exoA/PJ34, or BSA/PJ34 for one hour prior to infection with vaccinia virus expressing HIV p24. After 16–18 hours, DC were harvested, fixed and used to stimulate the HIV p24_306–316-specific CD8⁺ clone 16A7 in an IFN-y ELISPOT assay. Data are representative of two experiments.</p

HLA-I, loading machinery, and HLA-I:peptide complexes are present in highly pure Mtb phagosomes.

<p>(A) Phagosomes were isolated by percoll gradient or magnetic purification and prepared for electron microscopy as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000374#s4" target="_blank">Materials and Methods</a>. (B) Magnetic bead-isolated phagosomes were analyzed by flow cytometry to assess HLA-II-PE (plasma membrane) and H37Rv-eGFP fluorescence as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000374#s4" target="_blank">Materials and Methods</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000374#ppat-1000374-g006" target="_blank">Figure 6</a>. The events shown represent a small proportion of the population of phagosomes isolated and the experiment is representative of three experiments. (C) DC were pulsed with magnetically-labeled H37Rv-eGFP for 20 minutes, washed, and incubated for 18 hr. After magnetic separation of Mtb phagosomes, flow organellometry was performed as described previously. Data are representative of three experiments. (D) Magnetically-isolated Mtb phagosomes were freeze-thawed and tested for their ability to stimulate D160 1-23 CD8⁺ T cell clones in the absence of additional APC. IFN-γ production was measured using ELISPOT. Data are representative of two experiments.</p

The Mtb phagosome contains HLA-I loading accessory molecules.

<p>(A) DC were pulsed with H37Rv-eGFP for 20 minutes, washed, and incubated for 40 minutes. Phagosomal fractions were prepared as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000374#ppat-1000374-g003" target="_blank">Figure 3</a> and stained with the indicated antibodies (top panel). Intact DC were fixed, permeabilized, and stained with the indicated antibodies (bottom panel). Data are representative of three experiments. (B) Quantitative analysis of Mtb phagosomes over time. The percent positive number represents Overton cumulative histogram subtraction of the isotype control from the indicated stain. Each bar represents the mean±SEM from three experiments per timepoint.</p

The Mtb phagosome retains characteristics of an early endosome.

<p>(A) Representative figure showing organelle distribution after percoll separation of homogenate from Mtb–infected DC. The plasma membrane was labeled with a PE-conjugated antibody to HLA-II prior to homogenization and fluorescence was detected by fluorometry. For detection of ER, fractions were assessed for the presence of TAP1 and PDI by western blot. An enzymatic assay for β-hexosaminidase was used for detection of lysosomes. Finally, fractions were examined for the presence of H37Rv-eGFP by flow cytometry and quantified using a reference latex bead population. For flow cytometric analysis of Mtb phagosomes, the final 2 ml (fractions 23–28) of the gradient were pelleted, fixed, permeabilized, and stained with antibodies of interest. (B) Magnetic bead and Mtb phagosomes were gated based on FSC/SSC (not shown) and then on LAMP-1/HLA-I (beads) or LAMP-1/GFP (Mtb). Arrows indicate the gated population. Analysis of phagosome maturation on one hour LAMP-1^lo/−/HLA-I⁺ magnetic bead phagosomes (top panel), one hour LAMP-1⁺/HLA-I^lo/− magnetic bead phagosomes (second panel), overnight LAMP-1⁺ magnetic bead phagosomes (third panel), and overnight Mtb phagosomes (bottom panel). Plots include isotype staining (shaded histograms) as well as staining with the indicated antibody (red lines). The amplified HLA-I signal on the HLA-I-FITC gated events is due to the use of primary and secondary antibody combination, with which we routinely see up to a log shift in signal over conjugated primary. (C,D) Quantitative analysis of phagosomes over time. The percent positive number represents Overton cumulative histogram subtraction of the isotype control from the indicated stain. Each bar represents the mean±SEM of three experiments per timepoint.</p

Mtb phagosomes contain minimal contamination.

<p>(A) HLA-A2⁻ or HLA-A2⁺ DC were infected with H37Rv-eGFP for 20 minutes, washed, and incubated for an additional 40 minutes. HLA-A2⁻ DC were mixed with uninfected HLA-A2⁺ DC, homogenized, and homogenate separated using 27% percoll as described or pelleted without percoll separation. Phagosomes were stained with an antibody to HLA-A2. Shaded histograms represent isotype staining. Data are representative of three experiments. (B) HLA-A2⁻ or HLA-A2⁺ LCL were fixed, permeabilized, and stained with an antibody to HLA-A2. (C,D) Mtb phagosomes (C) or intact DC (D) were analyzed for the presence of cis- and trans-golgi markers GM130 and golgin-97, respectively. Data in C are representative of three experiments each after a 40 minute or overnight chase. Data in D are representative of two experiments.</p