M2 macrophages induced by prostaglandin E2 and IL-6 from cervical carcinoma are switched to activated M1 macrophages by CD4+ Th1 cells

Monocytes attracted by tumor-induced chronic inflammation differentiate to APCs, the type of which depends on cues in the local tumor milieu. In this work, we studied the influence of human cervical cancer cells on monocyte differentiation and showed that the majority of cancer cells either hampered monocyte to dendritic cell differentiation or skewed their differentiation toward M2-like macrophages. Blocking studies revealed that M2 differentiation was caused by tumor-produced PGE(2) and IL-6. TGF-β, IL-10, VEGF, and macrophage colony-stimulating factor did not play a role. Notably, these CD14(+)CD163(+) M2 macrophages were also detected in situ. Activation of cancer cell-induced M2-like macrophages by several TLR-agonists revealed that compared with dendritic cells, these M2 macrophages displayed a tolerogenic phenotype reflected by a lower expression of costimulatory molecules, an altered balance in IL-12p70 and IL-10 production, and a poor capacity to stimulate T cell proliferation and IFN-γ production. Notably, upon cognate interaction with Th1 cells, these tumor-induced M2 macrophages could be switched to activated M1-like macrophages that expressed high levels of costimulatory molecules, produced high amounts of IL-12 and low amounts of IL-10, and acquired the lymphoid homing marker CCR7. The effects of the interaction between M2 macrophages and Th1 cells could partially be mimicked by activation of these APCs via CD40 in the presence of IFN-γ. Our data on the presence, induction, and plasticity of tumor-induced tolerogenic APCs in cervical cancer suggest that tumor-infiltrated Th1 cells can stimulate a tumor-rejecting environment by switching M2 macrophages to classical proinflammatory M1 macrophages.     

Role of the transient receptor potential vanilloid 5 (TRPV5) protein N terminus in channel activity, tetramerization, and trafficking

The epithelial Ca(2+) channel transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry site for active Ca(2+) reabsorption in the kidney. The TRPV5 channel is a member of the TRP family of cation channels, which are composed of four subunits together forming a central pore. Regulation of channel activity is tightly controlled by the intracellular N and C termini. The TRPV5 C terminus regulates channel activity by various mechanisms, but knowledge regarding the role of the N terminus remains scarce. To study the role of the N terminus in TRPV5 regulation, we generated different N-terminal deletion constructs. We found that deletion of the first 32 residues did not affect TRPV5-mediated (45)Ca(2+) uptake, whereas deletion up to residue 34 and 75 abolished channel function. Immunocytochemistry demonstrated that these mutant channels were retained in the endoplasmic reticulum and in contrast to wild-type TRPV5 did not reach the Golgi apparatus, explaining the lack of complex glycosylation of the mutants. A limited amount of mutant channels escaped the endoplasmic reticulum and reached the plasma membrane, as shown by cell surface biotinylation. These channels did not internalize, explaining the reduced but significant amount of these mutant channels at the plasma membrane. Wild-type TRPV5 channels, despite significant plasma membrane internalization, showed higher plasma membrane levels compared with the mutant channels. The assembly into tetramers was not affected by the N-terminal deletions. Thus, the N-terminal residues 34-75 are critical in the formation of a functional TRPV5 channel because the deletion mutants were present at the plasma membrane as tetramers, but lacked channel activity.     

In Vivo Occupancy of Mitochondrial Single-Stranded DNA Binding Protein Supports the Strand Displacement Mode of DNA Replication

Mitochondrial DNA (mtDNA) encodes for proteins required for oxidative phosphorylation, and mutations affecting the genome have been linked to a number of diseases as well as the natural ageing process in mammals. Human mtDNA is replicated by a molecular machinery that is distinct from the nuclear replisome, but there is still no consensus on the exact mode of mtDNA replication. We here demonstrate that the mitochondrial single-stranded DNA binding protein (mtSSB) directs origin specific initiation of mtDNA replication. MtSSB covers the parental heavy strand, which is displaced during mtDNA replication. MtSSB blocks primer synthesis on the displaced strand and restricts initiation of light-strand mtDNA synthesis to the specific origin of light-strand DNA synthesis (OriL). The in vivo occupancy profile of mtSSB displays a distinct pattern, with the highest levels of mtSSB close to the mitochondrial control region and with a gradual decline towards OriL. The pattern correlates with the replication products expected for the strand displacement mode of mtDNA synthesis, lending strong in vivo support for this debated model for mitochondrial DNA replication.     

Image-Based Grouping during Binocular Rivalry Is Dictated by Eye-Of-Origin

Prolonged viewing of dichoptically presented images with different content results in perceptual alternations known as binocular rivalry. This phenomenon is thought to be the result of competition at a local level, where local rivalry zones interact to give rise to a single, global dominant percept. Certain perceived combinations that result from this local competition are known to last longer than others, which is referred to as grouping during binocular rivalry. In recent years, the phenomenon has been suggested to be the result of competition at both eye- and image-based processing levels, although the exact contribution from each level remains elusive. Here we use a paradigm designed specifically to quantify the contribution of eye- and image-based processing to grouping during rivalry. In this paradigm we used sine-wave gratings as well as upright and inverted faces, with and without binocular disparity-based occlusion. These stimuli and conditions were used because they are known to result in processing at different stages throughout the visual processing hierarchy. Specifically, more complex images were included in order to maximize the potential contribution of image-based grouping. In spite of this, our results show that increasing image complexity did not lead to an increase in the contribution of image-based processing to grouping during rivalry. In fact, the results show that grouping was primarily affected by the eye-of-origin of the image parts, irrespective of stimulus type. We suggest that image content affects grouping during binocular rivalry at low-level processing stages, where it is intertwined with eye-of-origin information.     

The Spleen Responds to Intestinal Manipulation but Does Not Participate in the Inflammatory Response in a Mouse Model of Postoperative Ileus

Background

Postoperative ileus is characterized by a transient impairment of the gastrointestinal motility after abdominal surgery. The intestinal inflammation, triggered by handling of the intestine, is the main factor responsible for the prolonged dysmotility of the gastrointestinal tract. Secondary lymphoid organs of the intestine were identified as essential components in the dissemination of inflammation to the entire gastrointestinal tract also called field effect. The involvement of the spleen, however, remains unclear.

Aim

In this study, we investigated whether the spleen responds to manipulation of the intestine and participates in the intestinal inflammation underlying postoperative ileus.

Methods

Mice underwent Laparotomy (L) or Laparotomy followed by Intestinal Manipulation (IM). Twenty-four hours later, intestinal and colonic inflammation was assessed by QPCR and measurement of the intestinal transit was performed. Analysis of homeostatic chemokines in the spleen was performed by QPCR and splenic cell populations analysed by Flow Cytometry. Blockade of the egress of cells from the spleen was performed by administration of the Sphingosine-1-phosphate receptor 1 (S1P₁) agonist CYM-5442 10 h after L/IM.

Results

A significant decrease in splenic weight and cellularity was observed in IM mice 24 h post-surgery, a phenomenon associated with a decreased splenic expression level of the homeostatic chemokine CCL19. Splenic denervation restored the expression of CCL19 and partially prevented the reduction of splenocytes in IM mice. Treatment with CYM-5442 prevented the egress of splenocytes but did not ameliorate the intestinal inflammation underlying postoperative ileus.

Conclusions

Intestinal manipulation results in two distinct phenomena: local intestinal inflammation and a decrease in splenic cellularity. The splenic response relies on an alteration of cell trafficking in the spleen and is partially regulated by the splenic nerve. The spleen however does not participate in the intestinal inflammation during POI.     

Inhibition of complex I of the electron transport chain causes O2-{middle dot}-mediated mitochondrial outgrowth

Recent evidence indicates that oxidative stress is central to the pathogenesis of a wide variety of degenerative diseases, aging, and cancer. Oxidative stress occurs when the delicate balance between production and detoxification of reactive oxygen species is disturbed. Mammalian cells respond to this condition in several ways, among which is a change in mitochondrial morphology. In the present study, we have used rotenone, an inhibitor of complex I of the respiratory chain, which is thought to increase mitochondrial O₂^–· production, and mitoquinone (MitoQ), a mitochondria-targeted antioxidant, to investigate the relationship between mitochondrial O₂^–· production and morphology in human skin fibroblasts. Video-rate confocal microscopy of cells pulse loaded with the mitochondria-specific cation rhodamine 123, followed by automated analysis of mitochondrial morphology, revealed that chronic rotenone treatment (100 nM, 72 h) significantly increased mitochondrial length and branching without changing the number of mitochondria per cell. In addition, this treatment caused a twofold increase in lipid peroxidation as determined with C11-BODIPY^581/591. Finally, digital imaging microscopy of cells loaded with hydroethidine, which is oxidized by O₂^–· to yield fluorescent ethidium, revealed that chronic rotenone treatment caused a twofold increase in the rate of O₂^–· production. MitoQ (10 nM, 72 h) did not interfere with rotenone-induced ethidium formation but abolished rotenone-induced outgrowth and lipid peroxidation. These findings show that increased mitochondrial O₂^–· production as a consequence of, for instance, complex I inhibition leads to mitochondrial outgrowth and that MitoQ acts downstream of this O₂^–· to prevent alterations in mitochondrial morphology. rhodamine 123; video-rate confocal microscopy; superoxide; MitoQ     

Postural changes in capillary pressure in the hallux of healthy volunteers.

Capillary circulation is delicately regulated by microvascular constriction mechanisms, thereby controlling capillary perfusion and transmural pressure. The influence of posture on capillary flow has been investigated in both diseased and healthy people. However, its influence on capillary pressure has rarely been investigated. We measured capillary pressures in the supine and sitting positions in the hallux of healthy volunteers. The capillaries in the eponychium of the hallux were punctured by using a micropipette connected to a micropressure system (900A, WPI). Also, peripheral arterial and venous pressures were measured in both positions. The rise in systolic capillary pressure from supine to sitting position (32 mmHg; from 39 to 71 mmHg, respectively) was significantly (P < 0.001) smaller than the rise in systolic arterial toe pressure (57 mmHg, from 87 to 144 mmHg, respectively) and venous pressure (41 mmHg, from 26 mmHg to 67 mmHg, respectively). This study shows that the postural rise in precapillary arteriolar pressure is not completely transmitted to the capillaries, probably because of activation of peripheral vasoconstriction mechanisms.     

Distinct uptake mechanisms but similar intracellular processing of two different toll-like receptor ligand-peptide conjugates in dendritic cells

Covalent conjugation of Toll-like receptor ligands (TLR-L) to synthetic antigenic peptides strongly improves antigen presentation in vitro and T lymphocyte priming in vivo. These molecularly well defined TLR-L-peptide conjugates, constitute an attractive vaccination modality, sharing the peptide antigen and a defined adjuvant in one single molecule. We have analyzed the intracellular trafficking and processing of two TLR-L conjugates in dendritic cells (DCs). Long synthetic peptides containing an ovalbumin cytotoxic T-cell epitope were chemically conjugated to two different TLR-Ls the TLR2 ligand, Pam(3)CysSK(4) (Pam) or the TLR9 ligand CpG. Rapid and enhanced uptake of both types of TLR-L-conjugated peptide occurred in DCs. Moreover, TLR-L conjugation greatly enhanced antigen presentation, a process that was dependent on endosomal acidification, proteasomal cleavage, and TAP translocation. The uptake of the CpG approximately conjugate was independent of endosomally-expressed TLR9 as reported previously. Unexpectedly, we found that Pam approximately conjugated peptides were likewise internalized independently of the expression of cell surface-expressed TLR2. Further characterization of the uptake mechanisms revealed that TLR2-L employed a different uptake route than TLR9-L. Inhibition of clathrin- or caveolin-dependent endocytosis greatly reduced uptake and antigen presentation of the Pam-conjugate. In contrast, internalization and antigen presentation of CpG approximately conjugates was independent of clathrin-coated pits but partly dependent on caveolae formation. Importantly, in contrast to the TLR-independent uptake of the conjugates, TLR expression and downstream TLR signaling was required for dendritic cell maturation and for priming of na?ve CD8(+) T-cells. Together, our data show that targeting to two distinct TLRs requires distinct uptake mechanism but follows similar trafficking and intracellular processing pathways leading to optimal antigen presentation and T-cell priming.     

Characterization of antigen-specific immune responses induced by canarypox virus vaccines

Avipoxvirus-based vectors, such as recombinant canarypox virus ALVAC, are studied extensively as delivery vehicles for vaccines against cancer and infectious diseases. Effective use of such vaccines is expected to benefit from proper understanding of the interaction between these viral vectors and the host immune system. We performed preclinical vaccination experiments in a murine tumor model to analyze the immunogenic properties of an ALVAC-based vaccine against carcinoembryonic Ag (ALVAC-CEA), a tumor-associated autoantigen commonly overexpressed in colorectal cancers. The protective CEA-specific immunity induced by this vaccine consisted of CD4(+) T cell responses with a mixed Th1/Th2 cytokine profile that were accompanied by potent humoral responses, but not by CEA-specific CD8(+) CTL immunity. In contrast, protective immunity induced by a CEA-specific DNA vaccine (DNA-CEA) consisted of Th1 and CTL responses. Modification of the ALVAC-CEA vaccine through coinjection of DNA-CEA, admixture with CpG oligodeoxynucleotides, or supplementation with additional transgenes encoding a triad of costimulatory molecules (TRICOM) did not result in induction of CEA-specific CTL responses. Even though these results suggested that ALVAC does not elicit Ag-specific CTLs, immunization with ALVAC vaccines against other Ags efficiently induced CTL responses. Our data show that the capacity of ALVAC vaccines to elicit CTL immunity against transgene-encoded Ags critically depends on the presence of highly immunogenic CTL epitopes in these Ags. This consideration needs to be taken into account with respect to the design and evaluation of vaccination strategies that use ALVAC-based vaccine.