TLR-dependent phagosome tubulation in dendritic cells promotes phagosome cross-talk to optimize MHC-II antigen presentation

Dendritic cells (DCs) phagocytose large particles like bacteria at sites of infection and progressively degrade them within maturing phagosomes. Phagosomes in DCs are also signaling platforms for pattern recognition receptors, such as Toll-like receptors (TLRs), and sites for assembly of cargo-derived peptides with major histocompatibility complex class II (MHC-II) molecules. Although TLR signaling from phagosomes stimulates presentation of phagocytosed antigens, the mechanisms underlying this enhancement and the cell surface delivery of MHC-II–peptide complexes from phagosomes are not known. We show that in DCs, maturing phagosomes extend numerous long tubules several hours after phagocytosis. Tubule formation requires an intact microtubule and actin cytoskeleton and MyD88-dependent phagosomal TLR signaling, but not phagolysosome formation or extensive proteolysis. In contrast to the tubules that emerge from endolysosomes after uptake of soluble ligands and TLR stimulation, the late-onset phagosomal tubules are not essential for delivery of phagosome-derived MHC-II–peptide complexes to the plasma membrane. Rather, tubulation promotes MHC-II presentation by enabling maximal cargo transfer among phagosomes that bear a TLR signature. Our data show that phagosomal tubules in DCs are functionally distinct from those that emerge from lysosomes and are unique adaptations of the phagocytic machinery that facilitate cargo exchange and antigen presentation among TLR-signaling phagosomes.Professional phagocytes take up large particles, such as bacteria, by phagocytosis and submit them to an increasingly harsh environment during phagosome maturation (1). Phagocytes concomitantly alert the immune system that an invader is present via signaling programs initiated by pattern recognition receptors, such as Toll-like receptors (TLRs) (2). Conventional dendritic cells (DCs) also alter and optimize phagosome maturation and TLR-signaling programs to preserve bacterial antigens for loading onto MHC class I and class II (MHC-II) molecules and optimize cytokine secretion to stimulate and direct T-cell responses to the invading agent (3, 4). DC presentation of soluble antigen is facilitated by TLR-driven tubulation of lysosomes that harbor MHC-II–peptide complexes and by consequent fusion of tubulovesicular structures with the plasma membrane (5–7); however, little is known about the mechanism by which signaling pathways influence the formation or presentation of phagosome-derived MHC-II–peptide complexes, key processes in the adaptive immunity to bacterial pathogens.TLRs respond to microbial ligands at the plasma membrane and in intracellular stores (8). TLR stimulation at the plasma membrane, endosomes, or phagosomes elicits distinct signaling pathways via two sets of adaptors, TIRAP (or MAL)-MyD88 and TRAM-TRIF (8, 9), which induce proinflammatory cytokine secretion and other downstream responses. TLRs such as TLR2 and TLR4 are recruited to macrophage and DC phagosomes at least partly from an intracellular pool (10–13), and signal autonomously from phagosomes independent of plasma membrane TLRs (11, 14, 15). Autonomous phagosomal signaling from TLRs or Fcγ receptors enhances the degradation of phagocytosed proteins and assembly of MHC-II with their derived peptides (14–16). Phagosomal TLR signaling has been proposed to also promote the reorganization of phagosome-derived MHC-II-enriched compartments (MIICs) to favor the delivery of MHC-II–peptide complexes to the plasma membrane (17), analogous to TLR-stimulated formation of tubules from MIICs/lysosomes (18–20) that fuse with the plasma membrane (7) and extend toward the immunologic synapse with T cells (5). Tubules emerge from phagosomes in macrophages shortly after phagocytosis and likely function in membrane recycling during early phagosome maturation stages (21–23), but tubules at later stages that might facilitate the presentation of phagosome-derived MHC-II–peptide complexes have not been reported previously. Moreover, a role for TLR signaling in formation of phagosome-derived tubules has not been established.Herein we show that in DCs, maturing phagosomes undergo extensive tubulation up to several hours after phagocytosis, and that tubulation requires TLR and MyD88 signaling and an intact actin and microtubule cytoskeleton. Unlike lysosome tubulation, phagosome tubulation is not essential for MHC-II–peptide transport to the cell surface. Rather, it contributes to content exchange among phagosomes that carry a TLR signature, and thereby enhances presentation of phagocytosed antigens from potential pathogens.     

Genetics of feline hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is characterized by an abnormal increase in myocardial mass that affects cardiac structure and function. HCM is the most common inherited cardiovascular disease in humans (0.2%) and the most common cardiovascular disease in cats (14.7%). Feline HCM phenotype is very similar to the phenotype found in humans, but the time frame for the development of the disease is significantly shorter. Similar therapeutic agents are used in its treatment and it has the same complications, such as heart failure, thromboembolism and sudden cardiac death. In contrast to humans, in whom thousands of genetic variants have been identified, genetic studies in cats have been limited to fragment analysis of two sarcomeric genes identifying two variants in MYBPC3 and one in MYH7. Two of these variants have also been associated with human disease. The high prevalence of the reported variants in non-affected cats hinders the assumption of their pathogenicity in heterozygotes. An in-depth review of the literature about genetic studies on feline HCM in comparison with the same disease in humans is presented here. The close similarity in the phenotype and genotype between cats and humans makes the cat an excellent model for the pathophysiological study of the disease and future therapeutic agents.     

Anaglyph of Retinal Stem Cells And Developing Axons: Selective Volume Enhancement In Microscopy Images

Retinal stem cell culture has become a powerful research tool, but it requires reliable methods to obtain high‐quality images of living and fixed cells. This study describes a procedure for using phase contrast microscopy to obtain three‐dimensional (3‐D) images for the study of living cells by photographing a living cell in a culture dish from bottom to top, as well as a procedure to increase the quality of scanning electron micrographs and laser confocal images. The procedure may also be used to photograph clusters of neural stem cells, and retinal explants with vigorous axonal growth. In the case of scanning electron microscopy and laser confocal images, a Gaussian procedure is applied to the original images. The methodology allows for the creation of anaglyphs and video reconstructions, and provides high‐quality images for characterizing living cells or tissues, fixed cells or tissues, or organs observed with scanning electron and laser confocal microscopy. Its greatest advantage is that it is easy to obtain good results without expensive equipment. The procedure is fast, precise, simple, and offers a strategic tool for obtaining 3‐D reconstructions of cells and axons suitable for easily determining the orientation and polarity of a specimen. It also enables video reconstructions to be created, even of specimens parallel to the plastic base of a tissue culture dish, It is also helpful for studying the distribution and organization of living cells in a culture, as it provides the same powerful information as optical tomography, which most confocal microscopes cannot do on sterile living cells. Anat Rec, 297:770–780, 2014. © 2014 Wiley Periodicals, Inc.     

Conservative management of cholestasis with and without fever in acute biliary pancreatitis

The presence of cholestasis in both mild and severe forms of acute biliary pancreatitis(ABP)does not justify,of itself,early endoscopic retrograde cholangiography(ERC)or endoscopic sphincterotomy(ES).Clinical support treatment of acute pancreatitis for one to two weeks is usually accompanied by regression of pancreatic edema,of cholestasis and by stone migration to the duodenum in 60%-88%of cases.On the other hand,in cases with both cholestasis and fever,a condition usually characterized as ABP associated with cholangitis,early ES is normally indicated.However,in daily clinical practice,it is practically impossible to guarantee the coexistence of cholangitis and mild or severe acute pancreatitis.Pain,fever and cholestasis,as well as mental confusion and hypotension,may be attributed to inflammatory and necrotic events related to ABP. Under these circumstances,evaluation of the bile duct by endo-ultrasonography(EUS)or magnetic resonance cholangiography(MRC)before performing ERC and ES seems reasonable.Thus,it is necessary to assess the effects of the association between early and opportune access to the treatment of local and systemic inflammatory/infectious effects of ABP with cholestasis and fever, and to characterize the possible scenarios and the subsequent approaches to the common bile duct,directed by less invasive examinations such as MRC or EUS.     

Long‐Term Evaluation of Periodontal Parameters and Implant Outcomes in Periodontally Compromised Patients: A Systematic Review

Background : The aim of this systematic review is to evaluate the long‐term outcomes of patients with periodontitis submitted to periodontal therapy/maintenance and implant placement. Methods: Studies reporting clinical and/or long‐term implant outcomes from partially edentulous patients with periodontitis who were treated and followed periodontal maintenance for ≥5 years were considered eligible for the review. Screening of the articles, data extraction, and quality assessment were conducted independently and in duplicate. Results: Search of MEDLINE, EMBASE, and CENTRAL databases resulted in 959 papers, and of them 931 were excluded after title/abstract assessment. The full texts of 28 potentially eligible publications were screened, but only 10 studies met inclusion criteria. Most of the included studies (77.8%) presented a medium/high methodologic quality. The results demonstrated that patients with a diagnosis of periodontitis had satisfactory implant outcomes. Implant survival was high (92.1%) within studies reporting 10 years of follow‐up. Parameters related to probing depth, clinical attachment level, and bone loss around teeth increased the occurrence of peri‐implantitis and implant loss. Non‐attendance to periodontal maintenance and smoking habits were also associated with less favorable implant outcomes. Conclusions: This systematic review confirmed that implant therapy can be successfully used in patients with a diagnosis of periodontitis who underwent proper therapy and regular periodontal maintenance. Residual pockets, non‐attendance to the periodontal maintenance program, and smoking were considered to be negative factors for the long‐term implant outcomes.     

A Specific Angiographic View of Left Coronary Artery Bifurcation in the Left Main Percutaneous Coronary Intervention Era

Objectives

We propose a right lateral (90–120° RAO) with 30° cranial angiographic view to expose the bifurcation of the left main coronary artery (LMCA) when previously used routine projections were inefficient at clearly showing this region.

Background

Little has been published in the medical literature regarding angiographic projections dedicated to special anatomies.

Methods

A total of 84 patients were subjected to the proposed projections. A reproducibility study, conducted with the participation of 2 independent observers, judged the effectiveness of the proposed projection. The Prevalence and Bias Adjusted Kappa (PABAK) index, with a 95% confidence interval (CI), was used to demonstrate the intensity of intra/inter‐observer agreement.

Results

The proposed projection was efficient in 79% of the angiograms, with agreement of 0.76 (0.6–0.9; P ≤ 0.001). The origins and the proximal segments of: the anterior descending coronary artery were exposed in 89% of the angiograms, agreement of 0.86 (0.7–1.0; P ≤ 0.001); the circumflex artery were exposed in 83% of the angiograms, with agreement of 0.72 (0.5–1.0; P ≤ 0.001); and the intermediate branch, when present, were exposed in 89% of the angiograms, agreement of 0.79 (0.6–1.0; P ≤ 0.001).

Conclusion

The right lateral (90–120° RAO) with 30° cranial projection is effective, safe, and reproducible. In special situations where routine projections fail, this proposed projection can reveal important details of the anatomy of the bifurcation of the LMCA during conventional coronary angiography or be the working projection during coronary angioplasty. (J Interven Cardiol 2016;29:293–299)