Pan-lysyl oxidase inhibition disrupts fibroinflammatory tumor stroma, rendering cholangiocarcinoma susceptible to chemotherapy.

BACKGROUND: Cholangiocarcinoma (CCA) features highly desmoplastic stroma that promotes structural and functional resistance to therapy. Lysyl oxidases (LOX, LOXL1-4) catalyze collagen cross-linking, thereby increasing stromal rigidity and facilitating therapeutic resistance. Here, we evaluate the role of lysyl oxidases in stromal desmoplasia and the effects of pan-lysyl oxidase (pan-LOX) inhibition in CCA. METHODS: Resected CCA and normal liver specimens were analyzed from archival tissues. Spontaneous and orthotopic murine models of intrahepatic CCA (iCCA) were used to assess the impact of the pan-LOX inhibitor PXS-5505 in treatment and correlative studies. The functional role of pan-LOX inhibition was interrogated through in vivo and ex vivo assays. RESULTS: All 5 lysyl oxidases are upregulated in CCA and reduced lysyl oxidase expression is correlated with an improved prognosis in resected patients with CCA. Spontaneous and orthotopic murine models of intrahepatic cholangiocarcinoma upregulate all 5 lysyl oxidase isoforms. Pan-LOX inhibition reversed mechanical compression of tumor vasculature, resulting in improved chemotherapeutic penetrance and cytotoxic efficacy. The combination of chemotherapy with pan-LOX inhibition increased damage-associated molecular pattern release, which was associated with improved antitumor T-cell responses. Pan-LOX inhibition downregulated macrophage invasive signatures in vitro, rendering tumor-associated macrophages more susceptible to chemotherapy. Mice bearing orthotopic and spontaneously occurring intrahepatic cholangiocarcinoma tumors exhibited delayed tumor growth and improved survival following a combination of pan-LOX inhibition with chemotherapy. CONCLUSIONS: CCA upregulates all 5 lysyl oxidase isoforms, and pan-LOX inhibition reverses tumor-induced mechanical forces associated with chemotherapy resistance to improve chemotherapeutic efficacy and reprogram antitumor immune responses. Thus, combination therapy with pan-LOX inhibition represents an innovative therapeutic strategy in CCA.

Sclerotherapy of the Post renal Transplant Lymphoceles: A Meta-Analysis.

PURPOSE: This study evaluated the effectiveness of sclerotherapy in treating lymphoceles after kidney transplantation, focusing on factors such as recurrence rates and procedural success. MATERIALS AND METHODS: Retrospective studies using sclerotherapy as the only form of treatment for postrenal transplant lymphoceles were included. All studies used percutaneous transcatheter sclerotherapy as treatment, and the success rate of the intervention was recorded. Sixty-one references were obtained by manually searching the MEDLINE (n = 20), Embase (n = 41), and Cochrane Library databases (n = 0) for retrospective research studies that included the keywords "sclerotherapy post renal transplant lymphoceles." After removing 3 duplicates, 50 of the remaining articles were excluded after the screening, and the remaining studies were extracted for demographic data and our primary outcome of the success rate of sclerotherapy. RESULTS: A descriptive analysis of the outcomes and complication rates associated with sclerotherapy interventions for lymphoceles is provided. A high degree of variation across the different studies was observed. According to the Kruskal-Wallis test, there was no correlation between the sclerosant used and the sclerotherapy complication rate (P = .472) or the success rate (P = .591). There was also no correlation between the gender of the patient and the success rate; however, there was a significant difference in the complication rate by gender (P < .005). CONCLUSIONS: In conclusion, different sclerosant products have been used for therapy with no consensus on the most efficacious product because the success rate has been variable. In addition, the gender of the patient may influence the complication rates associated with sclerotherapy for lymphoceles in patients post-kidney transplant.

Comparable and Complimentary Modalities for Treatment of Small-Sized HCC: Surgical Resection, Radiofrequency Ablation, and Microwave Ablation.

BACKGROUND: Over the past decade, there has been continual improvement in both ablative and surgical technologies for the treatment of hepatocellular carcinoma (HCC). The efficacy of ablative therapy compared to surgical resection for HCC has not been thoroughly evaluated using multiple large-scale randomized controlled trials. By international consensus, if a patient is eligible, surgery is the primary curative treatment option, as it is believed to confer superior oncologic control. OBJECTIVE: to determine the efficacies of percutaneous ablative therapies and surgical resection (SR) in the treatment of HCC. Data sources, study appraisal, and synthesis methods: A meta-analysis using 5 online databases dating back to 1989 with more than 31,000 patients analyzing patient and tumor characteristics, median follow-up, overall survival, and complication rate was performed. RESULTS: Ablative therapies are suitable alternatives to surgical resection in terms of survival and complication rates for comparable patient populations. For the entire length of the study from 1989-2019, radiofrequency ablation (RFA) produced the highest 5-year survival rates (59.6%), followed by microwave ablation (MWA) (50.7%) and surgical resection (SR) (49.9%). In the most recent era from 2006 to 2019, surgical resection has produced the highest 5-year survival rate of 72.8%, followed by RFA at 61.7% and MWA at 50.6%. Conclusions and key findings: Depending on the disease state and comorbidities of the patient, one modality may offer superior overall survival rates over the other available techniques. Interventional ablative methods and surgical resection should be used in conjunction for the successful treatment of small-sized HCC.

Understanding Solid-State Photochemical Energy Storage in Polymers with Azobenzene Side Groups.

Solar thermal fuel (STF) materials store energy through light-induced changes in the structures of photoactive molecular groups, and the stored energy is released as heat when the system undergoes reconversion to the ground-state structure. Solid-state STF devices could be useful for a range of applications; however, the light-induced structural changes required for energy storage are often limited or prevented by dense molecular packing in condensed phases. Recently, polymers have been proposed as effective solid-state STF platforms, as they can offer the bulk properties of solid materials while retaining the molecular-level free volume and/or mobility to enable local structural changes in photoresponsive groups. Light-induced energy storage and macroscopic heat release have been demonstrated for polymers with photoisomerizable azobenzene side groups. However, the mechanism of energy storage and the link between the polymer structure, energy density and storage duration has not yet been explored in detail. In this work, we present a systematic study of methacrylate- and acrylate-based polymers with azobenzene side groups to establish the mechanism of energy storage and release and the factors affecting energy density and reconversion kinetics. For polymers with directly attached azobenzene side groups, the energy storage properties are in line with previous work on similar systems, and the photoisomerization and reconversion properties of the azobenzene side groups mirror those of molecular azobenzene. However, the inclusion of an alkyl linker between the azobenzene side group and the backbone significantly increases the photoswitching efficiency, giving almost quantitative conversion to the Z isomeric state. The presence of the alkyl linker also reduces the glass transition temperature and leads to faster spontaneous thermal reconversion to the E isomeric form, but in all cases, half-lives of more than 4 days are observed in the solid state, which provides scope for applications requiring daily energy storage-release cycles. The maximum gravimetric energy density observed is 143 J g-1, which represents an increase of up to 44% compared to polymers with directly attached azobenzene moieties.

Outcomes after Child Physical Abuse and Association with Community-Level Social Determinants of Health.

BACKGROUND: Child physical abuse is a significant cause of pediatric injury and death. Previous studies have described disparities in outcomes for physically abused children according to insurance status. We hypothesized that children treated for physical abuse would be more likely to live in neighborhoods with increased socioeconomic deprivation. STUDY DESIGN: We performed a retrospective review of children who were admitted with suspected physical abuse from 2011 to 2021. Home addresses at the time of admission were used to assign an Area Deprivation Index (ADI) of the neighborhood. Clinicopathologic and outcome variables were compared between children from neighborhoods in the top 10th and bottom 90th national neighborhood ADI percentile. Univariate and multivariate logistic models were constructed. RESULTS: One hundred eighty-four children were included for analysis. Children from the top 10th (more impoverished) ADI percentile presented with more severe injuries, had higher area injury scores in the abdomen and extremities, and required admission to the intensive care unit more often, compared with children from the bottom 90th ADI percentile (all p Values <0.05). Children from high ADI neighborhoods were more likely to be discharged to a different caretaker than children from low ADI neighborhoods (71% caretaker change vs 49% caretaker change, p = 0.005). Univariate and multivariate logistic regression demonstrated statistically significant association between the ADI score and the need for caretaker change at the time of discharge (p = 0.004). CONCLUSIONS: Community-level social determinants of health are closely associated with child physical abuse. Child abuse reduction strategies might consider increased support for families with fewer resources and social support systems.

Social Determinants of Health are Associated With Postoperative Outcomes in Children With Complicated Appendicitis.

INTRODUCTION: Socioeconomic disadvantage has been associated with increased complicated appendicitis rates. Our purpose was to analyze the complex interactions between social determinants of health (SDOH) and postoperative outcomes in pediatric appendicitis. MATERIALS AND METHODS: Children who underwent appendectomy at our institution (1/2015-12/2020) were retrospectively reviewed. We used home addresses to determine composite measures of neighborhood/area-level socioeconomic advantage (Area Deprivation Index [ADI] and Social Deprivation Index [SDI]), and other area-level indicators. We created a novel, composite outcome score computed as a weighted average of eight outcome measures. Feature selection and exploratory factor analysis were used to create a multivariate model predictive of outcomes. RESULTS: Of 1117 children with appendicitis, 20.59% had complicated (perforated) appendicitis. Factor analysis identified two multivariate latent factors; Factor 1 contained SDI, ADI, and % unemployed in the population, and Factor 2 contained % Hispanic and % foreign-born in the population. Low Factor 2 scores (communities with more Hispanic/foreign-born residents) were associated with increased length of stay, more frequent postoperative percutaneous drainage, and increased postoperative imaging. CONCLUSIONS: Interactions between SDOH and pediatric surgical care go beyond the individual patient and suggest that vulnerable populations are exposed to contextual conditions that may impact outcomes. Specifically, neighborhood-level factors, including the prevalence of Hispanic ethnicity and foreign-born individuals, are associated with outcomes in pediatric patients with complicated appendicitis. Reducing disparities in complicated appendicitis outcomes may involve addressing neighborhood-level SDOH through strategic reallocation of healthcare resources and developing targeted interventions to improve access to pediatric surgical care in underserved communities.

Inferior Survival Is Associated With Socioeconomic Deprivation in Hepatocellular Carcinoma.

INTRODUCTION: Pernicious health disparities have been reported in patients with hepatocellular carcinoma (HCC). Few tools exist to screen patients in order to facilitate educational and outreach initiatives. We hypothesize that neighborhood-level socioeconomic metrics such as the Area Deprivation Index (ADI) can predict inferior outcomes in patients with early-stage HCC. METHODS: A single institution's retrospective review of patients with Surveillance, Epidemiology, and End Results Stage I HCC between 2000 and 2020 was conducted. Univariate and multivariate models were constructed to identify clinical and socioeconomic variables correlated with treatment-specific survival. Kaplan-Meier analysis was performed to compare survival differences between cohorts. RESULTS: A total of 558 patients were included in this study with newly diagnosed Surveillance, Epidemiology, and End Results Stage I HCC. Multivariate models demonstrated native model of end-stage liver disease, largest tumor size, insurance type, the distance to our transplant center, and the ADI score, a validated metric for a patient's socioeconomic status, are independent risk factors for worse overall survival (all P-values < 0.05). Concerningly, despite similar maximal tumor size, number of tumors, and native model of end-stage liver disease scores, patients from high ADI neighborhoods are 20% less likely to receive surgical treatment, especially liver transplantation. CONCLUSIONS: The ADI is a useful tool for identifying patients at the time of presentation who are at risk for inferior treatment for early stage HCC, and the ADI should be incorporated as a social vital sign.

GM-CSF drives myelopoiesis, recruitment and polarisation of tumour-associated macrophages in cholangiocarcinoma and systemic blockade facilitates antitumour immunity.

OBJECTIVE: Intrahepatic cholangiocarcinoma (iCCA) is rising in incidence, and at present, there are limited effective systemic therapies. iCCA tumours are infiltrated by stromal cells, with high prevalence of suppressive myeloid populations including tumour-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). Here, we show that tumour-derived granulocyte-macrophage colony-stimulating factor (GM-CSF) and the host bone marrow is central for monopoiesis and potentiation of TAMs, and abrogation of this signalling axis facilitates antitumour immunity in a novel model of iCCA. METHODS: Blood and tumours were analysed from iCCA patients and controls. Treatment and correlative studies were performed in mice with autochthonous and established orthotopic iCCA tumours treated with anti-GM-CSF monoclonal antibody. RESULTS: Systemic elevation in circulating myeloid cells correlates with poor prognosis in patients with iCCA, and patients who undergo resection have a worse overall survival if tumours are more infiltrated with CD68+ TAMs. Mice with spontaneous iCCA demonstrate significant elevation of monocytic myeloid cells in the tumour microenvironment and immune compartments, and tumours overexpress GM-CSF. Blockade of GM-CSF with a monoclonal antibody decreased tumour growth and spread. Mice bearing orthotopic tumours treated with anti-GM-CSF demonstrate repolarisation of immunosuppressive TAMs and MDSCs, facilitating T cell response and tumour regression. GM-CSF blockade dampened inflammatory gene networks in tumours and TAMs. Human tumours with decreased GM-CSF expression exhibit improved overall survival after resection. CONCLUSIONS: iCCA uses the GM-CSF-bone marrow axis to establish an immunosuppressive tumour microenvironment. Blockade of the GM-CSF axis promotes antitumour T cell immunity.

Investigation of structure and dynamics in a photochromic molecular crystal by NMR crystallography.

A photochromic anil, N-(3,5-di-t-butylsalicylidene)-4-amino-pyridine, has been studied by single-crystal X-ray diffraction, multinuclear magic-angle spinning NMR, and first-principles density functional theory (DFT) calculations. Interpretation of the solid-state NMR data on the basis of calculated chemical shifts confirms the structure is primarily composed of molecules in the ground-state enol tautomer, whereas thermally activated cis-keto and photoisomerised trans-keto states exist as low-level defects with populations that are too low to detect experimentally. Variable temperature 13 C NMR data reveal evidence for solid-state dynamics, which is found to be associated with fast rotational motion of t-butyl groups and 180° flips of the pyridine ring, contrasting the time-averaged structure obtained by X-ray diffraction. Comparison of calculated chemical shifts for the full crystal structure and an isolated molecule also reveals evidence for an intermolecular hydrogen bond involving the pyridine ring and an adjacent imine carbon, which facilitates the flipping motion. The DFT calculations also reveal that the molecular conformation in the crystal structure is very close to the energetic minimum for an isolated molecule, indicating that the ring dynamics arise as a result of considerable steric freedom of the pyridine ring and which also allows the molecule to adopt a favourable conformation for photochromism.

Mechanisms of enhanced HIV spread through T-cell virological synapses.

An elaborate network of cell-cell interactions in the immune system is essential for vertebrates to mount adaptive immune responses against invading pathogens. For lymphotropic viruses such as the human immunodeficiency virus type 1 (HIV-1), these immune cell interactions can also promote the spread of the virus within the host. The main target of HIV-1 infection is the CD4(+) helper T lymphocyte, a cell type that is responsible for coordinating immune responses and modulating effector responses to foreign antigens. As part of their normal immune surveillance duties, these cells migrate actively within lymphoid tissues and can travel from inductive sites to effector sites in search of their cognate antigen. For CD4(+) T cells, there is an ongoing search for a unique peptide antigen presented in the context of class II MHC that can activate a proliferative or tolerogenic response. This iterative and continual probing and interrogation of other cells determine the outcome of immune responses. Recent studies in vitro have revealed that the viral infection program induces cell-cell interactions called virological synapses between infected and uninfected CD4(+) T cells. These long-lived, virally induced adhesive contacts greatly enhance the rate of productive infection and may be central to the spread of the virus in vivo. Here, we review aspects of this efficient mode of cell-to-cell infection and the implications for our understanding of HIV-1 pathogenesis.

Cannabinoid receptor 2-mediated attenuation of CXCR4-tropic HIV infection in primary CD4+ T cells.

Agents that activate cannabinoid receptor pathways have been tested as treatments for cachexia, nausea or neuropathic pain in HIV-1/AIDS patients. The cannabinoid receptors (CB(1)R and CB(2)R) and the HIV-1 co-receptors, CCR5 and CXCR4, all signal via Gαi-coupled pathways. We hypothesized that drugs targeting cannabinoid receptors modulate chemokine co-receptor function and regulate HIV-1 infectivity. We found that agonism of CB(2)R, but not CB(1)R, reduced infection in primary CD4+ T cells following cell-free and cell-to-cell transmission of CXCR4-tropic virus. As this change in viral permissiveness was most pronounced in unstimulated T cells, we investigated the effect of CB(2)R agonism on to CXCR4-induced signaling following binding of chemokine or virus to the co-receptor. We found that CB(2)R agonism decreased CXCR4-activation mediated G-protein activity and MAPK phosphorylation. Furthermore, CB(2)R agonism altered the cytoskeletal architecture of resting CD4+ T cells by decreasing F-actin levels. Our findings suggest that CB(2)R activation in CD4+ T cells can inhibit actin reorganization and impair productive infection following cell-free or cell-associated viral acquisition of CXCR4-tropic HIV-1 in resting cells. Therefore, the clinical use of CB(2)R agonists in the treatment of AIDS symptoms may also exert beneficial adjunctive antiviral effects against CXCR4-tropic viruses in late stages of HIV-1 infection.

Cell-to-cell transfer of HIV-1 via virological synapses leads to endosomal virion maturation that activates viral membrane fusion.

HIV-1 can infect T cells by cell-free virus or by direct virion transfer between cells through cell contact-induced structures called virological synapses (VS). During VS-mediated infection, virions accumulate within target cell endosomes. We show that after crossing the VS, the transferred virus undergoes both maturation and viral membrane fusion. Following VS transfer, viral membrane fusion occurs with delayed kinetics and transferred virions display reduced sensitivity to patient antisera compared to mature, cell-free virus. Furthermore, particle fusion requires that the transferred virions undergo proteolytic maturation within acceptor cell endosomes, which occurs over several hours. Rapid, live cell confocal microscopy demonstrated that viral fusion can occur in compartments that have moved away from the VS. Thus, HIV particle maturation activates viral fusion in target CD4+ T cell endosomes following transfer across the VS and may represent a pathway by which HIV evades antibody neutralization.

A requirement for the p85 PI3K adapter protein BCAP in the protection of macrophages from apoptosis induced by endoplasmic reticulum stress.

Macrophages are innate immune cells that play key roles in regulation of the immune response and in tissue injury and repair. In response to specific innate immune stimuli, macrophages may exhibit signs of endoplasmic reticulum (ER) stress and progress to apoptosis. Factors that regulate macrophage survival under these conditions are poorly understood. In this study, we identified B cell adapter protein (BCAP), a p85 PI3K-binding adapter protein, in promoting survival in response to the combined challenge of LPS and ER stress. BCAP was unique among nine PI3K adapter proteins in being induced >10-fold in response to LPS. LPS-stimulated macrophages incubated with thapsigargin, a sarcoplasmic/endoplasmic reticulum calcium ATPase inhibitor that induces ER stress, underwent caspase-3 activation and apoptosis. Macrophages from BCAP(-/-) mice exhibited increased apoptosis in response to these stimuli. BCAP-deficient macrophages demonstrated decreased activation of Akt, but not ERK, and, unlike BCAP-deficient B cells, expressed normal amounts of the NF-κB subunits, c-Rel and RelA. Retroviral transduction of BCAP-deficient macrophages with wild-type BCAP, but not a Y4F BCAP mutant defective in binding the SH2 domain of p85 PI3K, reversed the proapoptotic phenotype observed in BCAP-deficient macrophages. We conclude that BCAP is a nonredundant PI3K adapter protein in macrophages that is required for maximal cell survival in response to ER stress. We suggest that as macrophages engage their pathogenic targets, innate immune receptors trigger increased expression of BCAP, which endows them with the capacity to withstand further challenges from ongoing cellular insults, such as ER stress.

Tracking and quantitation of fluorescent HIV during cell-to-cell transmission.

The green fluorescent protein (GFP) is a powerful genetic marking tool that has enabled virologists to monitor and track viral proteins during HIV infection. Expression-optimized Gag-GFP constructs have been used to study virus-like particle (VLP) assembly and localization in cell types that are easily transfected. The development of HIV-1 variants carrying GFP within the context of the viral genome has facilitated the study of infection and has been particularly useful in monitoring the transfer of virus between cells following virological synapse formation. HIV Gag-iGFP, a viral clone that contains GFP inserted between the matrix (MA) and capsid (CA) domains of Gag, is the first replication competent molecular clone that generates fluorescent infectious particles. Here, we discuss some methods that exploit HIV Gag-iGFP to quantify cell-to-cell transmission of virus by flow cytometry and to track the proteins during assembly and transmission using live-cell imaging.

Visualizing cell-to-cell transfer of HIV using fluorescent clones of HIV and live confocal microscopy.

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

Phagocytosis in macrophages lacking Cbl reveals an unsuspected role for Fc gamma receptor signaling and actin assembly in target binding.

Fc gamma receptor (Fc gammaR)-mediated phagocytosis is known to require tyrosine kinases (TKs). We identified c-Cbl and Cbl-b as proteins that undergo tyrosine phosphorylation during phagocytosis. Cbl-deficient macrophages displayed enhanced Fc gammaR-mediated signaling and phagocytosis. Surprisingly, binding of IgG-coated targets (EIgG) was also enhanced. c-Cbl-deficient macrophages expressed less Fc gammaRIIb, the inhibitory Fc gamma receptor; however, this did not account for enhanced target binding. We isolated the function of one Fc receptor isoform, Fc gammaRI, using IgG2a-coated targets (EIgG2a). Cbl-deficient macrophages demonstrated a disproportionate increase in binding EIgG2a, suggesting that signal strength regulates binding efficiency toward opsonized targets. In resting cells, Fc gammaRI colocalized with the Src family TK Hck in F-actin-rich structures, which was enhanced in Cbl-deficient macrophages. Target binding was sensitive to TK inhibitors, profoundly inhibited following depletion of cholesterol, and ablated at 4 degrees C or in the presence of inhibitors of actin polymerization. Sensitivity of EIgG binding to cytoskeletal disruption was inversely proportional to opsonin density. These findings challenge the view that Fc gammaR-mediated binding is a passive event. They suggest that dynamic engagement of TKs and the cytoskeleton enables macrophages to serve as cellular "Venus fly traps", with the capacity to capture phagocytic targets under conditions of limiting opsonin density.

Quantitative 3D video microscopy of HIV transfer across T cell virological synapses.

The spread of HIV between immune cells is greatly enhanced by cell-cell adhesions called virological synapses, although the underlying mechanisms have been unclear. With use of an infectious, fluorescent clone of HIV, we tracked the movement of Gag in live CD4 T cells and captured the direct translocation of HIV across the virological synapse. Quantitative, high-speed three-dimensional (3D) video microscopy revealed the rapid formation of micrometer-sized "buttons" containing oligomerized viral Gag protein. Electron microscopy showed that these buttons were packed with budding viral crescents. Viral transfer events were observed to form virus-laden internal compartments within target cells. Continuous time-lapse monitoring showed preferential infection through synapses. Thus, HIV dissemination may be enhanced by virological synapse-mediated cell adhesion coupled to viral endocytosis.

Regulatory T cells inhibit dendritic cells by lymphocyte activation gene-3 engagement of MHC class II.

Lymphocyte activation gene-3 (LAG-3) is a CD4-related transmembrane protein expressed by regulatory T cells that binds MHC II on APCs. It is shown in this study that during Treg:DC interactions, LAG-3 engagement with MHC class II inhibits DC activation. MHC II cross-linking by agonistic Abs induces an ITAM-mediated inhibitory signaling pathway, involving FcgammaRgamma and ERK-mediated recruitment of SHP-1 that suppresses dendritic cell maturation and immunostimulatory capacity. These data reveal a novel ITAM-mediated inhibitory signaling pathway in DCs triggered by MHC II engagement of LAG-3, providing a molecular mechanism in which regulatory T cells may suppress via modulating DC function.

ATP-binding cassette transporter A7 enhances phagocytosis of apoptotic cells and associated ERK signaling in macrophages.

The mammalian ATP-binding cassette transporters A1 and A7 (ABCA1 and -A7) show sequence similarity to CED-7, a Caenorhabditis elegans gene that mediates the clearance of apoptotic cells. Using RNA interference or gene targeting, we show that knock down of macrophage ABCA7 but not -A1 results in defective engulfment of apoptotic cells. In response to apoptotic cells, ABCA7 moves to the macrophage cell surface and colocalizes with the low-density lipoprotein receptor-related protein 1 (LRP1) in phagocytic cups. The cell surface localization of ABCA7 and LRP1 is defective in ABCA7-deficient cells. C1q is an opsonin of apoptotic cells that acts via phagocyte LRP1 to induce extracellular signal-regulated kinase (ERK) signaling. We show that ERK signaling is required for phagocytosis of apoptotic cells and that ERK phosphorylation in response to apoptotic cells or C1q is defective in ABCA7-deficient cells. These studies reveal a major role of ABCA7 and not -A1 in the clearance of apoptotic cells and therefore suggest that ABCA7 is an authentic orthologue of CED-7.

Myosin X is a downstream effector of PI(3)K during phagocytosis.

Phagocytosis is a phosphatidylinositol-3-OH-kinase (PI(3)K)-dependent process in macrophages. We identified Myo10 (Myosin-X), an unconventional myosin with pleckstrin homology (PH) domains, as a potential downstream target of PI(3)K. Myo10 was recruited to phagocytic cups in a wortmannin-sensitive manner. Expression of a truncation construct of Myo10 (Myo10 tail) in a macrophage cell line or cytosolic loading of anti-Myo10 antibodies in bovine alveolar macrophages inhibited phagocytosis. In contrast, expression of a Myo10 tail construct containing a point mutation in one of its PH domains failed to inhibit phagocytosis. Expression of Myo10 tail inhibited spreading, but not adhesion, on IgG-coated substrates, consistent with a function for Myo10 in pseudopod extension. We propose that Myo10 provides a molecular link between PI(3)K and pseudopod extension during phagocytosis.