Immune Phagocytosis Research Articles

Cardiac resident macrophages: The core of cardiac immune homeostasis

Cardiac resident macrophages (CRMs) are essential in maintaining the balance of the immune homeostasis in the heart. One of the main factors in the progression of cardiovascular diseases, such as myocarditis, myocardial infarction(MI), and heart failure(HF), is the imbalance in the regulatory mechanisms of CRMs. Recent studies have reported novel heterogeneity and spatiotemporal complexity of CRMs, and their role in maintaining cardiac immune homeostasis and treating cardiovascular diseases. In this review, we focus on the functions of CRMs, including immune surveillance, immune phagocytosis, and immune metabolism, and explore the impact of CRM's homeostasis imbalance on cardiac injury and cardiac repair. We also discuss the therapeutic approaches linked to CRMs. The immunomodulatory strategies targeting CRMs may be a therapeutic approach for the treatment of cardiovascular disease.

Crossing the Barrier: A Comparative Study of Listeria monocytogenes and Treponema pallidum in Placental Invasion.

Vertically transmitted infections are a significant cause of fetal morbidity and mortality during pregnancy and pose substantial risks to fetal development. These infections are primarily transmitted to the fetus through two routes: (1) direct invasion and crossing the placenta which separates maternal and fetal circulation, or (2) ascending the maternal genitourinary tact and entering the uterus. Only two bacterial species are commonly found to cross the placenta and infect the fetus: Listeria monocytogenes and Treponema pallidum subsp. pallidum. L. monocytogenes is a Gram-positive, foodborne pathogen found in soil that acutely infects a wide variety of mammalian species. T. pallidum is a sexually transmitted spirochete that causes a chronic infection exclusively in humans. We briefly review the pathogenesis of these two very distinct bacteria that have managed to overcome the placental barrier and the role placental immunity plays in resisting infection. Both organisms share characteristics which contribute to their transplacental transmission. These include the ability to disseminate broadly within the host, evade immune phagocytosis, and the need for a strong T cell response for their elimination.

Enhanced Biohomogeneous Composite Membrane-Encapsulated Nanoplatform with Podocyte Targeting for Precise and Safe Treatment of Diabetic Nephropathy.

Diabetic nephropathy (DN), associated with high mobility and disability, is the leading cause of end-stage kidney disease worldwide. Dysfunction of the mammalian target of the rapamycin (mTOR) pathway and reactive oxygen species (ROS) activation in the glomeruli is the main hypnosis for DN progression. However, the use of mTOR inhibitors for DN treatment remains controversial. In this study, we built a multifunctional selective mechanistic target of rapamycin complex 1 (mTORC1) inhibiting nanoplatform (naming as ESC-HCM-B) that targets the release of mTOR and ROS inhibitors near podocytes, aiming to confirm whether combination therapy is an alternative method for DN treatment. The results showed that ESC-HCM-B achieved high drug loading because of the core mesoporous silica nanoparticles (MSNPs), and the enhanced biohomogeneous composite membrane endowed ESC-HCM-B with the characteristics of avoiding immune phagocytosis, automatic valve-type slow-release drug, and high stability. In vitro, the nanoplatform showed high efficiency in podocyte targeting but no significant cytotoxicity or apoptotic promotion. In particular, the quantum dots carried by ESC-HCM-B further amplified the effect of "nanoenzyme"; this mechanism reduced the ROS level in podocytes induced by high glucose, protected mitochondrial damage, and restored mitochondrial energy metabolism. In vivo, the nanoplatform specifically targeted the glomerular and podocyte regions of the kidney. After treatment, the nanoplatform significantly reduced urinary protein levels and delayed glomerulosclerosis in DN rats. This nanoplatform provides a safe and effective strategy for DN treatment.

Evaluation of pharmacodynamic and patient enrichment biomarkers for SAR444881, a first-in-class anti-ILT2 monoclonal antibody for cancer immunotherapy.

2571 Background: Leukocyte Ig-like receptor B1 [LILRB1; ILT2] is an inhibitory receptor expressed on various immune cells. ILT2 binds to classical and nonclassical MHC class I molecules, with highest affinity to HLA-G. ILT2-mediated inhibition leads to impairment of immune cell proliferation, differentiation, phagocytosis, cytotoxicity and cytokine secretion. Antagonism of ILT2 signaling may serve as a novel target for anti-cancer immunotherapy. SAR444881 (BND-22) is a novel humanized IgG4 monoclonal antagonist antibody which selectively binds to ILT2 and blocks its interaction with MHC I molecules. SAR444881 induces robust macrophage and lymphocyte-driven anti-tumor activity in in vitro and in vivo models. To overcome the limitation that LILRB proteins are not expressed in rodents, we conducted a series of in vivo studies using humanized mouse models, cancer patient biopsies and ex vivo co-culture systems to interrogate the pharmacodynamic (PD) response of ILT2 antagonism as well as inform the combinatorial and patient enrichment strategies for SAR444881. Methods: SAR444881-induced modulation of PD biomarkers was evaluated in humanized xenograft models. Ex vivo co-culture system has been established using patient tumor tissues and isolated PBMC or other immune cells. Biomarker expression on immune cells and secreted soluble proteins were monitored by flow cytometry and ELISA, respectively. Procured tumor samples of patients with various advanced solid tumors, including Head and neck squamous cell carcinoma (HNSCC), Gastric Cancer (GC), Non-Small Cell Lung Cancer (NSCLC) and Colorectal Cancer (CRC) were utilized to evaluate protein or gene expression levels of potential predictive biomarkers (ILT2, HLA = G, PD-L1) using immunohistochemistry (IHC) and whole transcriptomic analysis (RNAseq). Results: 1) PD biomarkers: in murine models, SAR444881 modulates intra-tumoral sub-populations of CD8+ T cells, NK cells and Macrophage polarization; 2) Combination strategy: functional study in an ex vivo system showed that addition of an anti-PD-1 antibody induced an increased production of pro-inflammatory cytokines, including IFNγ and TNFα, compared to SAR444881 or anti-PD-1 alone. Concomitantly, combining SAR444881 with cetuximab resulted in increased phagocytosis compared to isotype control. 3) Predictive biomarkers: protein and gene profiling results suggest enriched expression of ILT2 in majority of biopsies from several tumor types including NSCLC, HNSCC and CRC. Conclusions: These data inform the PD response biomarkers, combination, and patient enrichment strategies for the clinical development of SAR444881 to maximize its benefits for cancer patients. An ongoing phase 1/2 trial of SAR444881 mono- and combination therapy in patients with advanced solid tumors is testing these concepts in the clinic (NCT04717375).

Клінічна ефективність фітоентеросорбенту в комплексному лікуванні хворих із вперше діагностованим деструктивним туберкульозом легень

Актуальність. Пошук патогенетичних методів лікування, що спрямовані на нормалізацію імунного статусу хворого на туберкульоз без призначення специфічних імуномодуляторів, залишається актуальним. Це стало основою для планування нашого дослідження. Ентеросорбенти є безпечними препаратами, що мають імуномодулюючі властивості, і ці властивості забезпечуються не прямим, а опосередкованим їх впливом на стан системного імунітету завдяки детоксикаційній дії. Мета дослідження — вивчити вплив фітоентеросорбенту на показники системного імунітету та ефективність застосування його в комплексному лікуванні хворих на вперше діагностований деструктивний туберкульоз легень. Матеріали та методи. У рандомізованому контрольованому дослідженні брали участь 125 пацієнтів. Це були хворі на лікарсько-чутливий туберкульоз органів дихання з розпадом легеневої тканини та без розпаду і 25 здорових добровольців. Вивчали стан гуморальної, клітинної ланок імунітету, нейтрофільного та моноцитарного фагоцитозу. А також проводили дослідження впливу використання фітоентеросорбенту в комплексній терапії хворих на туберкульоз на концентрацію протитуберкульозного препарату ізоніазиду в крові хворих. Результати. Використання в комплексній терапії хворих на лікарсько-чутливий вперше діагностований деструктивний туберкульоз легень фітоентеросорбенту не вплинуло на показники клітинного імунітету, але сприяло нормалізації показників його В-ланки — вірогідному зниженню кількості CD22+-лімфоцитів, рівня IgG, IgA та циркулюючих імунних комплексів у крові хворих, нормалізації фагоцитарної активності фагоцитуючих клітин. Використання фітоентеросорбенту в комплексному лікуванні хворих на туберкульоз не призвело до зниження концентрації протитуберкульозного препарату ізониазіду в крові хворих, а, навпаки, підвищило її и не сприяло появі побічних реакцій від прийому ізоніазиду. Висновки. Комплексне лікування хворих на лікарсько-чутливий вперше діагностований деструктивний туберкульоз легень з використанням фітоентеросорбенту призвело до підвищення ефективності лікування за темпами загоєння деструкцій на 21,4 %.

Immune response and phagocytosis of children with streptococcal tonsillitis and infected with the herpes virus type 6.

The aim of the work is to study the indicators of cellular and humoral immune response and FAN of patients with tonsillitis in children infected with HHV-6 type.
 Materials and methods of research. In 72 children aged 3–15 years (mean age 8.42±1.44 years), patients with streptococcal tonsillitis of moderate severity, the levels of the main subpopulations of blood lymphocytes were determined using monoclonal antibodies by solid-phase enzyme-linked immunosorbent assay, phagocytic activity of neutrophils (FAN) using the nitrosine tetrazolium reduction test (HCT test) and serum IgA, IgM, IgG levels by radial immunodiffusion. The studies were carried out in the acute period and in the period of convalescence. Mathematical and statistical processing of the data obtained was carried out using Microsoft Excel 2003 and Statistica 6.0 programs. The significance of differences between the mean values was determined using the Student's test (t).
 Results. Different mechanisms of formation of pathological process at streptococcal tonsillitis at children depending on the concomitant HHV-6 type infection are revealed. In patients with mixed infection in the acute period of tonsillitis determine the signs of deficiency of the cellular immune system with the maximum deviation of CD-lymphocytes (t=3.13 vs. t=2.16 in mono-infection), the tension in the humoral link of the immune response Ig deviation (t=1.63 against t=0.91 in mono-infection) on the background of phagocytosis violation in terms of FAN deviation (t=3.22 against t=2.01 in mono-infection). By the period of convalescence, the restoration of the imbalance of specific and non-specific protection factors does not occur. Increased IgG content in the acute period is one of the features of streptococcal tonsillitis in children on the background of HHV-6t infection.
 Conclusions. Concomitant VGL-6 type infection in children with streptococcal tonsillitis affects the mechanisms of formation of the inflammatory process and the reactivity of patients, forming the preconditions for adverse outcomes of the disease. The obtained results can be the basis for early detection of herpes virus infection in patients with tonsillitis, will help to optimize the management of patients in different periods of the disease.

Investigation of the Initial Host Response of Naïve Atlantic Salmon (Salmo salar) Inoculated with Paramoeba perurans.

Amoebic Gill Disease (AGD), caused by the ectoparasite Paramoeba perurans is characterised by hyperplasia of the gill epithelium and lamellar fusion. In this study, the initial host response of naïve Atlantic salmon (Salmo salar) inoculated with P. perurans was investigated. Using gel-free proteomic techniques and mass spectrometry gill and serum samples were analysed at 7 timepoints (2, 3, 4, 7, 9, 11 and 14 days) post-inoculation with P. perurans. Differential expression of immune related proteins was assessed by comparison of protein expression from each time point against naïve controls. Few host immune molecules associated with innate immunity showed increased expression in response to gill colonisation by amoebae. Furthermore, many proteins with roles in immune signalling, phagocytosis and T-cell proliferation were found to be inhibited upon disease progression. Initially, various immune factors demonstrated the anticipated increase in expression in response to infection in the serum while some immune inhibition became apparent at the later stages of disease progression. Taken together, the pro-immune trend observed in serum, the lack of a robust early immune response in the gill and the diversity of those proteins in the gill whose altered expression negatively impact the immune response, support the concept of a pathogen-derived suppression of the host response.

A Study on Morphometric Dimensions of Human Fetal Spleen at Different Gestational Ages

Introduction:The spleen is the largest hemolymphatic organ of the human body that plays a significant role in Hematopoiesis and immune phagocytosis. Recent studies have emphasized the importance of spleen as hematopoietic organ in fetal period, role in synthesis of immunoglobulins & defense of human body against infections. A thorough knowledge of splenic morphometry and dimensions is vital in comprehending the development of spleen in fetal period.Aim:The aim of the study is to study detailed morphometric analysis human fetal splenic specimens of various gestational ages and compare the findings with earlier studiesSubjects and Methods:The present study included 40 fetal cadaveric spleen and morphometric features i.e.,length, breadth, width and weight of spleen were measured.Results:The average weight of the fetal spleen was 1.98 gm and varied from 0.1 to 5.1 gm. The average length, breadth and width of spleen in prenatal group were 2.21 cm, 1.39 cm and 0.645 cm respectively. In the present study the average weight of the fetal spleen was 1.98 gm and varied from 0.1 to 5.1 gm. There was statistically significant increase in splenic weight till 33 weeks.Conclusion:Knowledge of spleen size at different gestational ages will help in early detection and diagnosis of splenic anomalies, identifying congenital malformations, early diagnosis and prompt treatment of intrauterine infections.

Identification of the differentially expressed protein biomarkers in rat blood plasma in response to gamma irradiation

Purpose: Simple, rapid and high-throughput dose assessment is critical for clinical diagnosis, treatment and emergency intervention in a large-scale radiological accident. The goal of this study is to screen and identify new ionizing radiation-responsive protein biomarkers in rat plasma.Materials and methods: Sprague-Dawley rats were exposed to single doses of 0, 1, 3, 5 Gy of Cobalt-60 γ-rays total body irradiation at a dose rate of 1 Gy/min. The tandem mass tag labeling (TMT) combined with liquid chromatography mass spectrometry (LC-MS/MS) approach was used to screen the differentially expressed proteins in rat plasma collected at 1, 3, 5 and 7 days post-irradiation. Bioinformatics analysis was conducted to explore the biological functions of these proteins. The expression levels of candidate radiation-sensitive protein biomarkers were confirmed using enzyme-linked immune-sorbent assay (ELISA).Results: A total of 503 differentially expressed proteins were identified. Most of these proteins were implicated in immune response, phagocytosis and signal transduction following ionizing radiation. Five up-regulated proteins including alpha-2-macroglobulin (A2m), chromogranin-A (CHGA), glutathione pertidase 3 (GPX3), clusterin (Clu) and ceruloplasmin (Cp) were selected for ELISA analysis. It was found that the expression levels of A2m, CHGA and GPX3 protein were increased in a dose-dependent manner at 1, 3 and 5 days after irradiation.Conclusion: Proteomics analysis revealed radiation-induced differentially expressed proteins in rat plasma. Our results suggested that A2m, CHGA, GPX3 protein expressions alterations in rat plasma may have potential as biomarkers to evaluate radiation exposure.

Whey milk proteomics from Schistosoma mansoni-infected mice reveals proteins involved in immunomodulation of the offspring.

Milk from schistosomotic mothers can modulate the immune response of their offspring. However, its characterization and potential of modulating immunity has not yet been fully elucidated. Thus, the aim of this study was to evaluate whey proteins from the milk of Schistosoma mansoni-infected mice in order to identify the fractions which can act as potential immunomodulatory tools. For this, we did a mass spectrometry (nanoUPLC-MSE) analysis to characterize the proteomic profile of milk from infected (MIM) and non-infected mice (MNIM). It was possible to identify 29 differentially expressed proteins: 15 were only found in MIM, 10 only found in MNIM, and 4 were downregulated in MIM group. Gene Ontology (GO), pathway enrichment analysis, and protein-protein interaction (PPI) analyses indicated differentially expressed proteins linked to biological processes and pathways in MIM group such as the following: fructose 1,6-biphosphate metabolic and glycolytic processes, glucose metabolism, and neutrophil degranulation pathways. The downregulated and unique proteins identified in MNIM group were involved in the positive regulation of B cell activation and receptor signaling pathway, in the innate immune response, complement activation, and phagocytosis. The present findings revealed a protein profile that may be involved in the activation and deactivation of the offspring's immune system in the long term, conferring a protective character due to the previous contact with milk from infected mothers.

Role of brain-specific angiogenesis inhibitor 1 in synapse and cognitive impairment

Adhesion G protein-coupled receptors(aGPCRs) play a significant role in cognitive impairment related diseases. As an important member of aGPCRs, brain-specific angiogenesis inhibitor 1(BAI1) has a prominent impact on anti-angiogenesis, anti-tumor and participating in immune phagocytosis. Recent research found out that BAI1 exerts a great influence on synaptogenesis and synaptic plasticity, but few studying concerning BAI1 in nervous system. Nowadays, the aging of population aggravates the occurrence of cognitive impairment. The pathogenesis of Alzheimer's disease and vascular cognitive impairment remains elusive, and identification of cognitive impairment at an early stage faces challenges. In the stage of mild cognitive impairment, synaptic damage is evident. BAI1 can regulate the function of postsynaptic membrane, synaptogenesis, synaptic signal transmission and the morphological development of dendritic spines. Therefore, it may potentially act as an early-warning index and intervention target for cognitive impairment. Key words: Brain-specific angiogenesis inhibitor 1; Cognitive impairment; Adhesion G protein-coupled receptors; Synaptogenesis; Mild cognitive impairment

Exacerbation of Streptococcus Equi (Strangles) by Overly Nutritious Diets in Horses: A Model for Infectious Bacterial Diseases of Horses and Other Livestock

Strangles is a highly pathogenic and contagious infection of horses and other equids caused by Streptococcus equi. Reports in the literature summarize the severe complications of strangles, but provide little information on the factors enhancing the pathogenicity and complications that result in high mortalities. However, there are reports suggesting exceptionally nutritious diets will exacerbate strangles infections and promote complications, including bacteremia and metastases to lymph nodes throughout the body and sequalae like purpura hemorrhagic and myositis. Dietary ingredients may enhance S. equi capsular polysaccharide (CPS) which protects bacteria from the immune system and phagocytosis. Low-virulent bacteria may become highly virulent pathogenic bacteria. Overly nutritious diets enhance the pathogenicity of S. equi and efficiency of bacterins, vaccines and antibiotics intended for controlling and treating strangles in horses. The effect of diet on the pathogenesis of S. equi in horses may be an ideal model for other bacterial diseases of horses as well as bacterial diseases like Pasturella multocida respiratory infections in cattle and other livestock.

Abstract 3677: Early effects of CSF-470 melanoma vaccine plus adjuvants in immune cell stimulation

Abstract Background: In a Phase II/III clinical trial (CASVAC-0401, NCT01729663), cutaneous melanoma (CM) patients (pts) stages II/III were randomized to receive CSF-470 vaccine or IFN-α (2:1). Vaccine CSF-470 is composed of four lethally-irradiated allogeneic CM cell lines; BCG (Bacillus Calmette-Guérin) and rhGM-CSF were used as adjuvants. At the end of the Phase II part of the study (30 pts), a higher distant-metastases-free-survival was observed in vaccinated pts. All vaccinated pts developed inflammation at the vaccination site. Therefore, in an attempt to understand these first steps of immune reaction, leukocytes migration, cytokines synthesis and monocyte phagocytosis promoted by the vaccine components were studied in vitro. Methods: Cell migration of healthy donors’ leukocytes or mononuclear cells (MN) towards vaccine components was assayed in 0,5 μm transwells. Cytokines were measured by ELISA in supernatants of CSF-470 vaccine and MN (1:1, 0,5x106 each) cocultures. For phagocytosis assays, CM cell lines were labeled with PKH67 lipophilic dye, irradiated, and cocultured with purified monocytes. After 24 hs, cultures were stained with CD14 and double staining (CD14+PKH67+) was analyzed by flow cytometry. Results: In migration assays, CSF-470 vaccine induced higher attraction of leukocytes as compared to culture medium (p=0.003, unpaired-T-test). GM-CSF exerted little attraction, although combined with BCG it increased the migration triggered by CSF-470 (vaccine vs vaccine plus adjuvants, p=0.034, unpaired-T-test). When using MN, CSF-470 alone or plus adjuvants, highly attracted monocytes, which represented 10%-65% of the migrating CD45+ population (before migration 6-15%) (p<0.0001, paired-T-test). In the lymphocyte population, natural killer cells were mainly attracted towards the vaccine either with or without adjuvants, representing 12%-33% of total migrated lymphocyte population (before migration 4-25% ) (p=0.002, paired-T-test). Regarding cytokine production, 6 hs cocultures of CSF-470 vaccine plus adjuvants and MN, promoted TNFα production (385±217 pg/ml). IFNγ release was detected in CSF-470:MN cocultures after 48 hs (109,2 pg/ml). BCG appears to be the most important adjuvant for production of these proinflammatory cytokines. IL-10, but not IL-12p70, was also detected. Interestingly, approximately 60% of monocytes phagocytosed CSF-470 cells after 24 hs of coculture, either in the presence or absence of adjuvants. Conclusions: Our in vitro results suggest that recruitment of leukocytes, mostly from the innate immune system, is an early event after CSF-470 vaccine + BCG + GM-CSF interaction with immune cells. Also, early release of TNFα and IFNγ proinflammatory cytokines and efficient vaccine phagocytosis by monocytes would favor subsequent antigen processing and presentation. Further experiments will be performed to address the initial steps triggered after CSF-470 vaccination. Citation Format: Maria B. Pampena, Estefanía P. Juliá, Yamila S. Rocca, Paula A. Blanco, María M. Barrio, José Mordoh, Estrella M. Levy. Early effects of CSF-470 melanoma vaccine plus adjuvants in immune cell stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3677. doi:10.1158/1538-7445.AM2017-3677

Pathway-gene identification for pancreatic cancer survival via doubly regularized Cox regression

BackgroundRecent global genomic analyses identified 69 gene sets and 12 core signaling pathways genetically altered in pancreatic cancer, which is a highly malignant disease. A comprehensive understanding of the genetic signatures and signaling pathways that are directly correlated to pancreatic cancer survival will help cancer researchers to develop effective multi-gene targeted, personalized therapies for the pancreatic cancer patients at different stages. A previous work that applied a LASSO penalized regression method, which only considered individual genetic effects, identified 12 genes associated with pancreatic cancer survival.ResultsIn this work, we integrate pathway information into pancreatic cancer survival analysis. We introduce and apply a doubly regularized Cox regression model to identify both genes and signaling pathways related to pancreatic cancer survival.ConclusionsFour signaling pathways, including Ion transport, immune phagocytosis, TGFβ (spermatogenesis), regulation of DNA-dependent transcription pathways, and 15 genes within the four pathways are identified and verified to be directly correlated to pancreatic cancer survival. Our findings can help cancer researchers design new strategies for the early detection and diagnosis of pancreatic cancer.

ENHANCEMENT OF IMMUNE RESPONSES BY PROBIOTIC PROPERTIES OF LACTOBACILLUS ACIDOPHILUS NCDC 195

Probiotics are microbial food supplements with beneficial effects on human health. Along with the dietary supplement, probiotics also have immunomodulatory properties. Present study was carried out using the in-vivo utilization of albino mice for the study of immunomodulatory effect of Lactobacillus acidophilus NCDC 195. To estimate the increase immune responses Nitroblue Tetrazolium reduction test, (Inducible Nitric Oxide synthase) test and Phagocytosis (% age) have been studied. Results were compared with the control value and. It was observed that feeding the Swiss albino mice for 15 days with Probiotic dosage (Yogurt), causes 1.98 times increase in Nitroblue Tetrazolium reduction test as compared to control, whereas Inducible Nitric Oxide synthase and phagocytosis shows 1.71 and 1.82 times increase in immune responses respectively. Cell mediated immune response was also calculated, where there was increase of 1.04, 1.07 and 1.02 times in Delayed Type Hypersensitivity (DTH) as compared to control was observed. Keywords: Immunomodulation, Nitroblue Tetrazolium (NBT) reduction, Probiotic, Phagocytosis, Cell mediated immune response, Inducible Nitric Oxide synthetase.

Macrophages at work: phagocytosis of urinary fungi.

A 56-year-old woman who underwent a lung transplant followed 2 years later by a deceased donor kidney transplant presented with pyuria. Enterococcus bacteremia was previously treated with amoxicillin/sulbactam for 14 days. Subsequent to this therapy, urinalysis was carried out, which revealed the following: specific gravity 1.010; pH 7.5; nitrite negative; albumin 1+; hemoglobin 3+. Urine microscopy revealed one to two squamous epithelial cells/high power field (HPF); ∼20 white blood cells/HPF; >50 red blood cells/HPF; 0–2 hyaline casts/low power field; and blastoconidia and pseudomycelia (Candida sp.). Interestingly, numerous macrophages phagocytosing blastoconidia were noted throughout the specimen (Figure 1). Fig. 1. (A) Candida blastoconidia and pseudomycelia are seen on urine microscopy. Several macrophages have phagocytosed blastoconidia (arrows). (B) Several macrophages containing blastoconidia are noted (arrows). Candida sp. is a human fungal pathogen. The major host defense against systemic Candida infection is ingestion of fungi by cells of the immune system, in particular macrophages and neutrophils. The fungal cell membrane is the initial contact with the immune system and plays an important role in host immune cell recognition and phagocytosis [1]. It is a dynamic organelle that determines both the shape of the fungus and its viability. Depending on the environment, these organisms are able to undergo reversible morphological changes moving between yeast, pseudohyphal and hyphal forms [2]. This plasticity allows fungi to successfully infect different anatomical sites. Hyphae have invasive properties that allow tissue penetration and escape from immune cells [3], whereas yeast forms are suited to disseminate via the bloodstream [4]. Phagocytic clearance of fungal pathogens consists of four stages: (i) accumulation of phagocytes where fungi reside; (ii) recognition of fungal molecular patterns; (iii) ingestion of fungi attached to phagocyte cell membranes and (iv) phagocyte processing of engulfed fungi by fusion [5]. The urinary findings of macrophages ingesting fungi in our patient support the importance of this host defense against such invading microorganisms.

Interaction between M-Like Protein and Macrophage Thioredoxin Facilitates Antiphagocytosis for Streptococcus equi ssp. zooepidemicus

Streptococcus equi ssp. zooepidemicus (S. zooepidemicus, S.z) is one of the common pathogens that can cause septicemia, meningitis, and mammitis in domesticated species. M-like protein (SzP) is an important virulence factor of S. zooepidemicus and contributes to bacterial infection and antiphagocytosis. The interaction between SzP of S. zooepidemicus and porcine thioredoxin (TRX) was identified by the yeast two-hybrid and further confirmed by co-immunoprecipitation. SzP interacted with both reduced and the oxidized forms of TRX without inhibiting TRX activity. Membrane anchored SzP was able to recruit TRX to the surface, which would facilitate the antiphagocytosis of the bacteria. Further experiments revealed that TRX regulated the alternative complement pathway by inhibiting C3 convertase activity and associating with factor H (FH). TRX alone inhibited C3 cleavage and C3a production, and the inhibitory effect was additive when FH was also present. TRX inhibited C3 deposition on the bacterial surface when it was recruited by SzP. These new findings indicated that S. zooepidemicus used SzP to recruit TRX and regulated the alternative complement pathways to evade the host immune phagocytosis.

Recombinant expression and functional characterization of a C-type lectin(Fclectin)from the Chinese shrimp(Fenneropenaeus chinensis)

Chinese shrimp(Fenneropenaeus chinensis)is distributed mainly along Chinese inshore areas,and is one of the most important farmed shrimp in China.The studies on innate immune responses of shrimps,especially on immune defense against the main crustacean pathogens,will provide more knowledge of shrimp immunity to prevent infectious diseases.Invertebrates do not possess an adaptive immune system based on highly specific antibodies and antigen receptors.They must rely on efficient immune defenses capable of protecting them against invading microorganisms.The chief issue of crustacean immunity should concern non-self-recognition mechanisms.Proteins that specifically bind to certain carbohydrate components on the surface of microorganisms play an important role in non-self-recognition and cleaning up of the invading microorganisms.Such proteins are known as pattern recognition receptors(PRRs).Lectins exist in almost all living organisms.Due to their ability of binding to terminal sugars on glycoproteins and glycolipids,lectins are primary candidates for pattern recognition receptors in innate immunity.C type Lectin is regarded as a potential molecule involved in immune recognition and phagocytosis through opsonization in crustacean.In the preliminary study,a novel C-type lectin was cloned from hemocytes of Chinese shrimp,(Fenneropenaeus chinensis).It contains two tandem carbohydrate recognition domains(CRDs)/C-type lectin-like domains.Both of the CRDs contain a QPD(Gln-Pro-Asp)motif that has a predicted binding specificity for galactose-type sugar.In this research,two recombinant target proteins(rFclectin-CRD1 and rFclectin-CRD2)were expressed by prokaryotic expression system.The result showed that fusion protein was expressed in the form of inclusion bodies.The LC-ESI-MS analysis showed that two peptide fragments of rFclectin-CRD1 and rFclectin-CRD2 were identical with the corresponding sequence of F.chinensis C-type lectin.Recombinant protein was purified by immobilized-metal affinity chromatography and Ni-NTA technology.The concentrations of purified target proteins were 0.4 g/L.rFclectin-CRD1 and rFclectin-CRD2 had agglutinating and antimicrobial activity against main pathogens in aquaculture in a calcium-dependent manner.The agglutinating activity can be inhibited by multiple carbohydrates,such as galactose,peptidoglycan and lipopolysaccharide.These results suggest that Fclectin,as a Ca2+dependent carbohydrate-recognition protein,is one of the important PRRs.It might play a crucial role in the innate immunity of the shrimp and is expected to be applied in disease control.

Abstract 2440: CD47 is a prognostic factor and an antibody target that synergizes with rituximab to eradicate non-Hodgkin lymphoma

Abstract Monoclonal antibodies are standard therapeutics for several cancers including the anti-CD20 antibody rituximab for B cell non-Hodgkin lymphoma (NHL). Rituximab and other antibodies are not curative, and must be combined with cytotoxic chemotherapy for clinical benefit. Here we identify CD47 as an attractive monoclonal antibody target in NHL. A major function of CD47 is to inhibit phagocytosis, through binding its receptor, SIRP-alpha, on phagocytes. We hypothesize that NHL cells over-express CD47 to evade immune phagocytosis and that blockade of CD47 signaling by a monoclonal antibody can eliminate NHL cells by enabling phagocytic engulfment. Here we investigate the therapeutic potential of an anti-CD47 antibody alone and in combination with rituximab for the treatment of NHL. We predict that the combination of anti-CD47 antibody and rituximab through utilization of two independent antibody mechanisms will result in synergistic phagocytosis and elimination of NHL cells. We found that CD47 protein is highly expressed on primary human NHL cells compared to normal B cell counterparts. Higher CD47 gene expression predicted an inferior outcome in several cohorts of NHL patients, providing independent prognostic value. Blocking anti-CD47 antibodies preferentially enabled phagocytosis of NHL cells but not normal cell counterparts in vitro while the combination of anti-CD47 antibody and rituximab resulted in synergistic phagocytosis of NHL cells. In vivo, anti-CD47 antibody treatment of human NHL-engrafted mice reduced lymphoma burden and prolonged survival compared to IgG control treatment. Furthermore, anti-CD47 antibody treatment in combination with rituximab led to elimination of lymphoma and cure of NHL-engrafted mice compared to either antibody treatment as a single agent. The above in vivo results were observed in both a disseminated and localized human NHL cell line mouse model as well as in primary human lymphoma xenotransplants. These antibodies synergized through a novel mechanism combining Fc receptor (FcR)-dependent and FcR-independent stimulation of phagocytosis that might be applicable to many other cancers. Together, these data provide the rationale for utilizing an anti-CD47 antibody either alone or in combination with rituximab in treating human NHL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2440.

Therapeutic Antibody Targeting of CD47 Synergizes with Rituximab to Completely Eradicate Human B-Cell Lymphoma Xenografts.

Abstract 2716Poster Board II-692The anti-CD20 antibody, rituximab, is standard therapy for many CD20 positive B-cell lymphomas, significantly improving long-term survival in combination with chemotherapy. However, rituximab alone is not curative in many non-Hodgkin lymphoma (NHL) patients with observation of primary and acquired resistance, arguing for a need for additional targeted therapies. We identified the cell surface protein CD47 as a potential therapeutic antibody target in human NHL. A major function of CD47 is to inhibit phagocytosis through binding its receptor SIRPα, on phagocytes. We hypothesize that NHL cells over-express CD47 to evade immune phagocytosis and that blockade of CD47 signaling with a monoclonal antibody can eliminate NHL cells by enabling phagocytic engulfment. We have previously shown that an anti-CD47 antibody enables phagocytosis of acute myeloid leukemia (AML) stem cells and eliminates AML in vivo. Here we investigate the therapeutic potential of an anti-CD47 antibody alone and in combination with rituximab for the treatment of NHL. We predict that the combination of anti-CD47 antibody and rituximab will result in synergistic elimination of NHL cells by both blocking an inhibitory signal and delivering a positive signal for phagocytosis.We found that CD47 protein is highly expressed on primary human B-cell NHL cells compared to normal peripheral blood B-cells. Higher CD47 gene expression independently predicted a worse clinical outcome in several cohorts of NHL patients including diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, and chronic lymphocytic leukemia. Blocking anti-CD47 antibodies preferentially enabled phagocytosis of NHL cells but not normal cell counterparts in vitro. Using isobologram analyses, the combination of anti-CD47 antibody and rituximab resulted in synergistic phagocytosis of NHL cells at higher levels compared to either anti-CD47 antibody or rituximab alone. In addition, anti-CD47 antibody and rituximab mediated their therapeutic effects through Fc-receptor-independent and Fc-receptor-dependent effector mechanisms, respectively. In vivo, anti-CD47 antibody treatment of NHL-engrafted mice reduced lymphoma burden and prolonged survival compared to IgG control. Furthermore, combination treatment with anti-CD47 antibody and rituximab led to elimination of lymphoma and cure of NHL-engrafted mice. These in vivo results were observed in both a disseminated and localized human NHL cell line mouse model as well as in primary human DLBCL mouse xenotransplants. Together, these data provide the rationale for utilizing an anti-CD47 antibody either alone or in combination with rituximab in treating human NHL. Disclosures:Weissman:U.S. Patent Application 11/528,890 entitled “Methods for Diagnosing and Evaluating Treatment of Blood Disorders.”: Patents & Royalties; Stem Cells Inc.: Cofounder and director; Cellerant Inc.: cofounder; Amgen: Equity Ownership.