Macrophage Phagocytosis Activity Research Articles

Naked Oat (Avena nuda L.) Oligopeptides:Immunomodulatory Effects on Innate and AdaptiveImmunity in Mice via Cytokine Secretion, AntibodyProduction, and Th Cells Stimulation.

The study aimed to investigate the immunomodulatory activity of oligopeptides derived from oat (Avena nuda L.) (OOPs). Healthy female BALB/c mice were randomly assigned to five groups, given deionized water (control) and 0.25, 0.5, 1.0, and 2.0 g/kg body weight (BW) of OOPs daily by intragastric administration. Seven assays were performed to determine the immunomodulatory effects of OOPs on immune organ ratios, cellular and humoral immune responses, macrophage phagocytosis, and natural killer (NK) cell activity. Spleen T lymphocyte subpopulations (by flow cytometry), serum cytokine and immunoglobulin levels (by multiplex sandwich immunoassays) were determined to evaluate how OOPs affected the immune system. Our results showed that OOPs could significantly improve innate and adaptive immune responses in mice through the enhancement of cell-mediated and humoral immunity, macrophage phagocytosis capacity, and NK cell activity. We concluded that the immunomodulatory effects might be attributed to increased T and Th cell percentages, serum interferon (IFN)-γ, interleukin (IL)-1 α, IL-2, IL-6, IL-10, IL-12, tumor necrosis factor (TNF)- α, and granulocyte-macrophage colony-stimulating factor (GM-CSF) secretions as well as immunoglobulin (Ig) A, IgG, and IgM productions. These results indicate that dietary OOPs could be considered as promising immunomodulators with dosages ranging from 0.25 to 2.0 g/kg BW.

Effect of alveolar macrophages phagocytosis on prognosis in patients with acute respiratory distress syndrome caused by abdominal infection: a multicenter study

To investigate the effects of alveolar macrophage phagocytosis on prognosis in patients with acute respiratory distress syndrome (ARDS) caused by abdominal infection. ARDS patients caused by severe intra-abdominal infection admitted to intensive care unit (ICU) of Tianjin Fourth Central Hospital, Tianjin Nankai Hospital, Tianjin First Central Hospital and Tianjin Fifth Central Hospital from June 2016 to March 2018 were enrolled. The gender, age, acute physiology and chronic health evaluation II (APACHE II) within 24 hours of admission, neutral red phagocytosis and alkaline phosphatase activity of macrophages in bronchoalveolar lavage fluid, the length of ICU stay, total hospitalization time, hospitalization expenses, and prognosis were recorded. According to the prognosis, the patients were divided into death group and survival group, and the parameters were compared between the two groups. Pearson test was used to analyze the correlation between neutral red phagocytosis function of macrophages and alkaline phosphatase activity and other indicators. The prognosis was analyzed by binary Logistic regression combined with neutral red phagocytosis and alkaline phosphatase activity in patients, and the predictive value of both subjects on prognosis was analyzed by the receiver operating characteristic (ROC) curve. Twenty patients were enrolled in the study, with 8 in the death group and 12 in the survival group. Compared with the survival group, the death group was older (years old: 58.50±14.86 vs. 46.67±13.40), APACHE II score was higher (21.50±3.93 vs. 13.58±4.12), neutral red phagocytosis ability and alkaline phosphatase activity of alveolar macrophages were significantly decreased (A value: 0.265±0.050 vs. 0.338±0.016; μmol/L: 12.06±1.24 vs. 17.96±3.90), and the length of ICU stay was significantly longer (days: 22.00±14.59 vs. 11.50±3.17), hospitalization cost was significantly increased (10 thousand Yuan: 24.17±11.02 vs. 13.44±3.53), the total hospitalization time was shorter (days: 25.25±15.01 vs. 35.67±8.58), and the difference was statistically significant (all P < 0.05). There was no significant difference in gender between the survival group and the death group [male (case): 8 vs. 6, P > 0.05]. The neutral red phagocytosis ability of alveolar macrophages in ARDS patients caused by abdominal infection was negatively correlated with age, APACHE II score and the length of ICU stay (r value was -0.328, -0.572, -0.809, respectively, all P < 0.05); alkaline phosphatase activity was negatively correlated with age, APACHE II score, the length of ICU stay and hospitalization expenses (r value was -0.334, -0.583, -0.470, -0.517, respectively, all P < 0.05). Binary Logistic regression analysis showed that neutral red phagocytosis [odds ratio (OR) = 0.596, 95% confidence interval (95%CI) = 0.212-0.997] and alkaline phosphatase activity (OR = 0.573, 95%CI = 0.339-0.968) were the influencing factors of prognosis (both P < 0.05). ROC curve analysis showed that the AUC of neutral red phagocytosis ability for prognosis of ARDS patients caused by abdominal infection was 0.948, and the sensitivity and specificity were 91.7% and 87.5% when the off-cut value was 0.317. The AUC of alkaline phosphatase for the prognosis of ARDS patients caused by abdominal infection was 0.813; when the cut-off value was 19.72 μmol/L, the sensitivity was 75.0%, and the specificity was 87.5%. The alveolar macrophage phagocytosis dysfunction in ARDS patients caused by severe abdominal infection was not only related to the severity of the disease, but also increased the medical burden of patients, and significantly affected the mortality of such patients.

In Vitro Immunomodulatory and Cytotoxic Potentials of Faloak (Sterculia quadrifida R.Br.) Bark

Faloak (Sterculia quadrifida R.Br., Sterculiaceae) is an Indonesia indigenous plant widely used as a traditional remedy for various health complaints. This study aimed to explore the immunomodulatory and cytotoxicity potentials of Faloak bark by using in vitro techniques. Barks was extracted using water, ethanol and ethanol: water (1:1) separately. Colorimetric analysis was used for the determination of the total phenolic and the total flavonoid contents. Immunomodulator activities of the extracts were evaluated by studying their effects on mice macrophages and lymphocytes using in vitro methods, while the cytotoxicity potential was evaluated against Vero and HepG2 cell lines. The active extract was further fractionated and tested using different cell lines by the MTT method. Data were analyzed using one-way ANOVA and LSD tests, with a 95% confidence level (p<0.05). All extracts significantly increased the phagocytosis of macrophages compared to that of control in a concentration-dependent manner. However, all the extracts exhibited the Stimulation Index value of < 2, which indicated no effect on lymphocyte proliferation. The ethyl acetate fraction showed the highest activity against T47D, MCF7 and HepG2 cells with IC50 9.56; 7.62; 3.24 µg/mL and the Selectivity Index (against Vero cells) as follows, 2.01; 2.52; 5.94, respectively. Faloak bark extracts can stimulate macrophage phagocytosis activity in vitro, which positively correlates to the contents of flavonoids and other phenolic compounds. The ethyl acetate fraction exhibited the highest activity, which supports its potency as cancer chemopreventive herbal medicine.

Platelet lysate outperforms FCS and human serum for co-culture of primary human macrophages and hMSCs

In vitro co-cultures of different primary human cell types are pivotal for the testing and evaluation of biomaterials under conditions that are closer to the human in vivo situation. Especially co-cultures of macrophages and mesenchymal stem cells (MSCs) are of interest, as they are both present and involved in tissue regeneration and inflammatory reactions and play crucial roles in the immediate inflammatory reactions and the onset of regenerative processes, thus reflecting the decisive early phase of biomaterial contact with the host. A co-culture system of these cell types might thus allow for the assessment of the biocompatibility of biomaterials. The establishment of such a co-culture is challenging due to the different in vitro cell culture conditions. For human macrophages, medium is usually supplemented with human serum (hS), whereas hMSC culture is mostly performed using fetal calf serum (FCS), and these conditions are disadvantageous for the respective other cell type. We demonstrate that human platelet lysate (hPL) can replace hS in macrophage cultivation and appears to be the best option for co-cultivation of human macrophages with hMSCs. In contrast to FCS and hS, hPL maintained the phenotype of both cell types, comparable to that of their respective standard culture serum, as well as the percentage of each cell population. Moreover, the expression profile and phagocytosis activity of macrophages was similar to hS.

Abstract A051: Melanoma displays an evolutionarily conserved resistance to upregulation of prophagocytic signals and to CD47 blockade

Abstract Therapeutic activation of macrophage phagocytosis has the ability to restrain tumor growth through phagocytic clearance of tumor cells and activation of the adaptive immune response. Our objective for this study was to evaluate the effects of modulating pro- and anti-phagocytic pathways in malignant melanoma. In order to identify evolutionarily conserved mechanisms of resistance that may be important for melanoma cell survival, we utilized a multispecies approach and examined the phagocytosis of human, mouse, and dog melanoma cells in vitro. We blocked the interaction between CD47 on tumor cells and SIRPα on macrophages using anti-CD47 monoclonal antibodies, SIRPα mimotopes, and a soluble fusion protein of the SIRPα extracellular domain. In all cases, we confirmed that these reagents blocked &amp;gt;85% of the maximum binding of CD47 to recombinant SIRPα. We observed that melanoma cells from all three species displayed unexpected resistance to phagocytosis that could not be fully mitigated by blockade of the “don’t eat me” signal CD47 or by chemotherapeutic enhancement of known “eat me” signals. In vitro, CD47 blockade minimally enhanced phagocytosis of melanoma cells, and loss of CD47 expression did not increase sensitivity to phagocytosis. In vivo, CD47 blockade enhanced the proliferation of tumor-specific CD8+ T-cells, but this response failed to inhibit tumor growth. Resistance to phagocytosis was not mediated by soluble factors, as phagocytosis of melanoma cells was not enhanced by inhibition of secretory pathways, and phagocytosis of sensitive lymphoma tumor cells was not impaired in the presence of melanoma cells or melanoma culture supernatants. siRNA-mediated knockdown of 47 prospective “don’t eat me” signals similarly did not enhance melanoma cell phagocytosis. Interestingly, in lymphoma cells, CD47 knockout alone did not enhance phagocytosis, suggesting that at least part of the pro-phagocytic effect of CD47 blockade is due to Fc receptor engagement. Restoration of CD47 expression in lymphoma cells re-established sensitivity to CD47 blockade. We conclude that melanoma cells possess an evolutionarily conserved resistance to macrophage phagocytosis. Further investigation will be needed to define and overcome the mechanisms that mediate melanoma cell resistance to innate immunity. Citation Format: Katie L. Anderson, Kristin M. Snyder, Daisuke Ito, Debra C. Lins, Lauren J. Mills, Kipp Weiskopf, Nan G. Ring, Aaron M Ring, Yoji Shimizu, Matthew F. Mescher, Irving L. Weissman, Jaime F. Modiano. Melanoma displays an evolutionarily conserved resistance to upregulation of prophagocytic signals and to CD47 blockade [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A051.

TNF May Negatively Regulate Phagocytosis of Devil Facial Tumour Disease Cells by Activated Macrophages

ABSTRACTIntroduction: Macrophage phagocytosis of pathogens and tumour cells is an important early event in protection against infectious disease and cancer. As tumour necrosis factor α (TNF) is an important cytokine in macrophage activation, we investigated the involvement of TNF in macrophage phagocytosis of tumour cells.Methods: We used Devil Facial Tumour Disease (DFTD) cancer cells as the target tumour cells. The Tasmanian devil (Sarcophilus harrisii) population is threatened by the transmissible DFTD. Using DFTD cells provided the opportunity to determine if these cells can be phagocytosed and investigate requirement for TNF. As effector cells, bone marrow derived macrophages (BMDMs), generated from C57BL/6 wild type (B6.WT) and C57BL/6 TNF-/- (B6.TNF-/-) mice were used. Phagocytosis of DFTD cells was investigated by confocal microscopy and flow cytometry.Results: DFTD cells were consistently phagocytosed by B6.WT and B6.TNF-/- BMDMs with similar efficiency in vitro. Consequently the DFTD cells are not resistant to phagocytosis. Following activation by exposure to IFNγ and LPS or LPS alone, B6.TNF-/- BMDMs had higher phagocytic efficiency and lower nitric oxide (NO) production compared to wild-type controls. In addition, NO seems to be unlikely to be the involved in phagocytosis efficiency in IFNγ and LPS activated B6.TNF-/- macrophages and consequences thereof.Conclusion: Our results indicate that TNF is not required for IFNγ and LPS or LPS alone activation of macrophage phagocytosis. TNF may negatively regulate macrophage phagocytosis of tumour cells.

Intelectin mediated phagocytosis and killing activity of macrophages in blunt snout bream (Megalobrama amblycephala)

Intelectin, a lectin discovered recently, has been identified in various vertebrate species, such as fish, amphibians, and mammals. In one of our previous studies, the efficient bacteria binding and agglutinating activity of the recombinant Megalobrama amblycephala intelectin protein (rMamINTL) and the enhanced immunopositive localization have been observed in the hepatic macrophage-like cells (kupffer cells) post Aeromonas hydrophila infection. Thus, the present study primarily focuses on the regulatory effects of rMamINTL on M. amblycephala macrophages. This study revealed a prominent LPS-binding activity of rMamINTL and a significantly increased phagocytosis of rMamINTL-treated A. hydrophila by M. amblycephala macrophages. However, the rMamINTL-treated M. amblycephala macrophages exhibited no evident regulatory effect on phagocytosis, whereas the enhanced killing activity of the rMamINTL-treated macrophages was observed, which may be attributed to the induced respiratory burst activity and the expression of inflammatory cytokines. In addition, the anti-proliferation effect of rMamINTL on two tumor cells was observed. However, its mechanism remains to be further studied. In short, these results show that MamINTL is a multifunctional immune protein with effective immunomodulatory activity.

Immunomodulatory and Antioxidant Activity of Green Grass Jelly Leaf Extract (Cyclea barbata Miers.) In Vitro

Green grass jelly (Cyclea barbata Miers.) is known for its benefit to human health especially in supporting body’s immune system and wellness. This research aimed to determine immunomodulatory and antioxidant activity of green grass jelly leaf extracts in vitro. Old leaves were collected as sample then dried and ground to powder. The extraction was done with sohxletation using three different solvents, chloroform, ethyl acetate, and ethanol. The immunomodulatory activity was evaluated by treating the crude extracts at concentrations of 50, 100, and 500 mg/mL on macrophages of rat in vitro. Macrophage cells separated form peritoneal fluid used RPMI medium. Phagocytosis activity and phagocytosis capacity of macrophages were performed in vitro using latex beads that suspended in phosphate buffered saline (PBS). The antioxidant activity was measured by spectrophotometry technique with 1,1-diphenyl-2-picrylhydrazyl (DPPH) solution. All treatments were done three replicates. Detection of the bioactive groups of the extracts was done by Thin Layer Chromatography (TLC). The results showed that ethyl acetate extract has the highest phagocytosis activity followed by chloroform extract and ethanol extract, respectively. Optimum concentration was reached at 100 mg/mL of ethyl acetate extract. The ethyl acetate extract was also the highest antioxidant activity index 7.7 followed by both extracts of chloroform and ethanol similar index value of 6.25 and 6.3, respectively. The ethyl acetate extract has a high immunomodulatory activity and antioxidant activity which contained phenolics, flavonoids, tannins, and terpenoids.

The Potential of Wild Mango Leaves from Sumatera as the Immunostimulant Agent

Wild Mango from Sumatera is potentially new immunostimulant. It contains mangiferin, which is potentially become the immunostimulant candidate. This study aimed to prove the immunostimulatory effect of wild mango leaves extract on white rat peritoneum fluid induced by Staphylococcus aureus bacteria. The immunostimulatory effect was determined based on the activity and capacity of macrophage phagocytosis. This study used 54 male white rats about 130 g - 290 g. Based on the results,the highest phagocytosis activity of macrophage cells was found in ethanol extract of Mangga Hutan leaves dose 2 with a percentage of phagocytosis activity was 84%. The highest phagocytosis capacity of the macrophage cells was also found in ethanol extract of Mangga Hutan leaves dose 2 with an average phagocytic capacity of 171.67 from 50 active macrophages. Results of this study indicated that wild mango leaves from Sumatera has the potential immunostimulant activity. This indicates that the wild mango have the potential for therapeutic efficacy for the prevention of degenerative diseases caused by immune deficiencies.

Increased susceptibility against Cryptococcus neoformans of lupus mouse models (pristane-induction and FcGRIIb deficiency) is associated with activated macrophage, regardless of genetic background.

The severity of cryptococcosis in lupus from varying genetic-backgrounds might be different due to the heterogeneity of lupus-pathogenesis. This study explored cryptococcosis in lupus mouse models of pristane-induction (normal genetic-background) and FcGRIIb deficiency (genetic defect). Because the severity of lupus nephritis, as determined by proteinuria and serum creatinine, between pristane and FcGRIIb-/- mice were similar at 6-month-old, Cryptococcus neoformans was intravenously administered in 6-month-old mice and were age-matched with wild-type. Indeed, the cryptococcosis disease severity, as evaluated by mortality rate, internal-organ fungal burdens and serum cytokines, between pristane and FcGRIIb-/- mice was not different. However, the severity of cryptococcosis in wild-type was less severe than the lupus mice. On the other hand, phagocytosis activity of peritoneal macrophages from lupus mice (pristane and FcGRIIb-/-) was more predominant than the wild-type without the difference in macrophage killing-activity among these groups. In addition, the number of active T helper cells (Th-cell) in the spleen, including Th-cells with intracellular IFN-γ, from lupus mice (pristane and FcGRIIb-/-) was higher than wildtype. Moreover, these active Th-cells were even higher after 2 weeks of cryptococcal infection. These data support enhanced macrophage activation through prominent Th-cells in both lupus models. In conclusion, an increased susceptibility of cryptococcosis in both lupus models was independent to genetic background. This might due to Th-cell enhanced macrophage phagocytosis with the interference of macrophage killing activity from Cryptococcal immune-evasion properties.

Immunomodulatory activity of a novel polysaccharide from Lonicera japonica in immunosuppressed mice induced by cyclophosphamide.

Lonicera japonica is a typical Chinese herbal medicine. We previously reported a method to isolate polysaccharides from Lonicera japonica (LJP). In this study, we first performed a qualitative analysis of LJP using the Fourier Transform Infrared Spectrometer (FT-IR) and explored the monosaccharide composition of LJP using the pre-column derivatization high performance liquid chromatography (HPLC) method. We then investigated the immunomodulatory function of LJP in cyclophosphamide (CTX)-induced immunosuppressed mouse models. The results showed that LJP had the characteristic absorption of typical polysaccharides consisting of 6 types of monosaccharides. In addition, LJP can increase significantly the organ index, splenic lymphocyte proliferation, macrophage phagocytosis, and natural killer (NK) cell activity in CTX-treated mice. LJP could also restore the levels of serum cytokines interleukin (IL-2), tumor necrosis factor (TNF-α) and Interferon-γ (IFN-γ) in the CTX-treated mice. Finally, the results on measuring the T-lymphocytes subsets of spleen also confirmed LJP-induced immunomodulatory activity in immunosuppressed mice from another perspective. Therefore, LJP could be used as a potential immunomodulatory agent.

Conjugation of chitosan oligosaccharides enhances immune response to porcine circovirus vaccine by activating macrophages

Porcine circovirus type 2 (PCV2)-associated diseases have led to great economic losses to the pig industry. Our lab previously found that conjugation of chitosan oligosaccharides (COS) or via a carrier protein enhanced the immunogenicity of PCV2 vaccine against infectious pathogens. However, precise mechanisms and signal transduction pathways underlying the efficacy of COS conjugation remains poorly defined. In this study, to better understand the effects and mechanism of COS conjugates maintain the adjuvant potential in vivo, we investigated its augmentation of macrophage function, including cell activation, NO production, cytokine production and phagocytosis. Additionally, the role of Toll-like receptors (TLR) proteins in this process was also assessed. The results indicate that, as compared to the PCV and PCV/COS, conjugation of COS effectively enhanced the NO production, cytokines generation and phagocytosis activity of macrophages. Noticeably, the generation of NO and proinflammatory cytokines was closely related to the TLR2/4 signaling pathways, strongly suggesting that conjugation of COS regulates innate and adaptive immunity by activation of macrophages, resulting in immune enhancement. In summary, the present study provides a potential mechanism of COS conjugation as a novel adjuvant to improve immune responses against various diseases.

Correlation Between Total Flavonoid Contents and Macrophage Phagocytosis Activity of Fractions From Faloak (Sterculia quadrifida R.Br.) Barks Ethanolic Extract In Vitro

On Timor island, Nusa Tenggara Timur, faloak barks (Sterculia quadrifida R.Br.) has been used empirically to restore stamina. Faloak bark ethanolic extract proved to have immunomodulatory activity in vitro, which can increase macrophage phagocytosis activity. This research aimed: (i) to determine the immunomodulatory active fraction of faloak bark ethanolic extract, (ii) to determine the total flavonoid contents of faloak extract and fractions, and (iii) to evaluate the correlation of the total flavonoid contents of those extract and fractions with their macrophage phagocytosis activity. The simplisia powder is macerated with 96% ethanol. The extract was dissolved in methanol:water (9:1v/v) was then subsequently partitioned with n-hexane, ethyl acetate, and water to obtain n-hexane fraction, ethyl acetate fraction, water fraction, and insoluble fraction. Faloak extract and fractions at concentration 62,5; 125; 250; 500μg/mL were tested for their effect on the peritoneal macrophage phagocytosis of Balb/c mice in vitro by the latex beads method. Phagocytosis capacity and phagocytosis index were analyzed using one-way anova and post hoc Tukey HSD test with 95% confidence level. The results showed that ethyl acetate fraction had the highest macrophage phagocytosis capacity and the highest total flavonoid content compared to other fractions. The highest macrophage phagocytosis capacity of ethyl acetate fraction at concentration of 250 μg/mL was 51,94±4,67%, this value was significantly different from cell control (7,50±1,29%), negative controls of 0,0625% dimethylsulphoxide (6,25±0,36%), as well as positive control of 200 μg/mL echinaceae extract syrup® (9,97±0,33%). The total flavonoid content of ethyl acetate fraction determined by aluminum chloride method was 4,290±0.029 mg of quercetin equivalent/g fraction. There was a positive and strong correlation between the total flavonoid content of these extract and fractions with their macrophage phagocytosis capacity (Pearson correlation coefficient of 0,781) and showing linear relationship y=4,721x+19,663; R2=0,61.

Effects of dietary toxic cyanobacteria and ammonia exposure on immune function of blunt snout bream (Megalabrama amblycephala)

Cyanobacterial blooms caused by water eutrophication have become a worldwide problem. During the degradation of toxic cyanobacterial blooms, elevated ammonia and microcystins concentrations co-occur and exert toxicity on fish. Up to now, the combined effect of microcystins and ammonia on fish immunotoxicity has not been reported. The present study investigated immune responses of blunt snout bream (Megalabrama amblycephala) to dietary toxic cyanobacteria and ammonia exposure. Megalobrama amblycephala were exposed to solutions with different concentrations of NH3–N (0, 0.06, 0.12 mg/L) and fed with diets containing 15% and 30% of toxic cyanobacteria lyophilized powder for 30 d. The microcystins concentration in different organs of Megalobrama amblycephala was in the following sequence: head kidney > liver > intestine > gonad > spleen > gill > trunk kidney > brain > muscle > heart. In both head kidney and spleen, the MC-LR and MC-RR concentration increased significantly with increasing NH3–N concentration. It indicates that NH3–N maybe promote the accumulation of microcystins in immune organs of Megalobrama amblycephala. Meanwhile, broadened peripheral interspace of lymphocytes, nucleus shrivel and edematous mitochondria were observed in head kidney lymphocyte of toxic treatment fish. Moreover, there were significant interactions between dietary toxic cyanobacteria and ammonia exposure on head kidney macrophage phagocytosis activity, respiratory burst activities, total number of white blood cells and the transcriptional levels of sIgM, mIgD and sIgZ genes. Our data clearly demonstrated that dietary toxic cyanobacteria combined with ammonia exposure showed a synergistic effect on Megalobrama amblycephala immunotoxicity.

Anti-human SIRPα antibody is a new tool for cancer immunotherapy.

Interaction of signal regulatory protein α (SIRPα) expressed on the surface of macrophages with its ligand CD47 expressed on target cells negatively regulates phagocytosis of the latter cells by the former. We recently showed that blocking Abs to mouse SIRPα enhanced both the Ab‐dependent cellular phagocytosis (ADCP) activity of mouse macrophages for Burkitt's lymphoma Raji cells opsonized with an Ab to CD20 (rituximab) in vitro as well as the inhibitory effect of rituximab on the growth of tumors formed by Raji cells in nonobese diabetic (NOD)/SCID mice. However, the effects of blocking Abs to human SIRPα in preclinical cancer models have remained unclear given that such Abs have failed to interact with endogenous SIRPα expressed on macrophages of immunodeficient mice. With the use of Rag2−/−γc −/− mice harboring a transgene for human SIRPα under the control of human regulatory elements (hSIRPα‐DKO mice), we here show that a blocking Ab to human SIRPα significantly enhanced the ADCP activity of macrophages derived from these mice for human cancer cells. The anti‐human SIRPα Ab also markedly enhanced the inhibitory effect of rituximab on the growth of tumors formed by Raji cells in hSIRPα‐DKO mice. Our results thus suggest that the combination of Abs to human SIRPα with therapeutic Abs specific for tumor antigens warrants further investigation for potential application to cancer immunotherapy. In addition, humanized mice, such as hSIRPα‐DKO mice, should prove useful for validation of the antitumor effects of checkpoint inhibitors before testing in clinical trials.

Protective effects of chicken egg yolk immunoglobulins (IgY) against experimental Aeromonas hydrophila infection in blunt snout bream (Megalobrama amblycephala)

The emergence of multi antibiotic resistance by the pathogens and toxic impacts on host metabolism has opened new perspectives to rational novel vaccine techniques. Outbreaks of Aeromonas hydrophila in aquaculture caused high mortality throughout the world and resulted in the extensive economic loss in the aquaculture industry. In this study, we report the efficacy of anti-A. hydrophila IgY antibodies by passive vaccination and its prophylactic or therapeutic effects against A. hydrophila in blunt snout bream. Inactivated A. hydrophila immunized hens produced effective IgY antibodies that were stable at temperatures less than 60 °C or the pH value was >4. The specific IgY can be bound directly to A. hydrophila that efficiently agglutinated and inhibited the bacterial growth in a dose-dependent manner. The specific IgY had significantly enhanced the phagocytosis activity of macrophages and resulted in rapid bacterial clearance. Anti-A. hydrophila IgY antibodies significantly increased macrophage mediated respiratory burst, including nitric oxide and superoxide anion production and subsequently killed the pathogen. Histopathological studies of intestine and spleen from vaccinated blunt-snout bream challenged with A. hydrophila showed the structural integrity of the organs was maintained intact from the bacterial injury. In addition, the prophylactic and therapeutic immunization, protected the blunt snout bream and the survival is approximately about 60% and 50%, respectively. These data suggest that specific IgY has the potential for protecting blunt snout bream against A. hydrophila infection and show promise for the future development of harmless vaccines.

Anti-inflammatory effect of lysozyme from hen egg white on mouse peritoneal macrophages.

Lysozyme from hen egg has been reported to possess an anti-inflammatory effect. However, little is known about its detailed mechanism. The mechanism of anti-inflammatory effect of lysozyme was examined in this study. When mouse macrophage-like cell line RAW264.7 cells and mouse peritoneal macrophages were activated with lipopolysaccharide (LPS) and then treated with lysozyme, the production of tumor necrosis factor-α and interleukin-6 was significantly suppressed. The effect was induced by suppressing the gene expression levels of both cytokines. Phagocytosis activity of peritoneal macrophages was not altered by the treatment with lysozyme, suggesting that lysozyme shows the anti-inflammatory effect without inhibiting the phagocytotic response of macrophages. In addition, lysozyme inhibited phosphorylation of c-jun N-terminal kinase (JNK) and was taken up by macrophages within 1h after treatment of the cells with lysozyme. Overall results suggest that lysozyme is taken up intracellularly and suppresses LPS-induced inflammatory responses by inhibiting JNK phosphorylation.

Apoptosis Activity of the Mouse Macrophage Cell Line J774A.1 Infected with a Recombinant BCG consisting the C-Terminus of Merozoite Surface Protein-1 of Plasmodium falciparum.

Macrophage apoptosis exerts an efficient mechanism in controlling intracellular infection during innate immune response against various pathogens including malaria parasites. This study was carried out to determine the apoptosis activity in mouse macrophage cell line J774A.1 infected with a Mycobacterium bovis bacille Calmette-Guerin (BCG) clone and a recombinant BCG clone expressing the C-terminus of merozoite surface protein-1 (BCG-MSP1C) of Plasmodium falciparum for 48 h. In this study, a parent BCG cells was used as a control. The nuclear staining with Hoechst 33342 showed that the BCG-MSP1C cells was capable of increasing the nuclear condensation and morphological stages of apoptosis in the infected cells compared to the BCG-infected cells and the lipopolysaccharide (LPS)-stimulated cells. The flow cytometric analysis using Annexin-V and Propidium iodide (PI) staining confirmed that the BCG-MSP1C cells significantly increased the percentage of early apoptotic activity in the infected macrophage higher than the one stimulated by the parent BCG cells and LPS. This apoptotic response corresponded with the reduction of the anti-apoptotic Bcl-2 protein expression and higher p53 expression. The colorimetric assay demonstrated that the BCG cells capable of stimulating higher production of caspase-1, –3, –8 and –9 while the BCG-MSP1C cells stimulated the expression of caspase-1 and -9 in the infected macrophages, suggesting the involvement of mitochondrial-mediated (intrinsic) pathway of apoptosis. In conclusion, both the BCG and BCG-MSP1C cells are capable of inducing macrophage apoptosis activity in the mouse macrophage cell line J774A.1. This mechanism is important for the elimination of pathogens such as malaria parasite during the phagocytosis activity of macrophage. However, the BCG-MSP1C cells showed higher apoptosis activity than those produced by the parent BCG cells.

Loss of Function of P2X7 Receptor Scavenger Activity in Aging Mice: A Novel Model for Investigating the Early Pathogenesis of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a leading cause of irreversible, severe vision loss in Western countries. Recently, we identified a novel pathway involving P2X7 receptor scavenger function expressed on ocular immune cells as a risk factor for advanced AMD. In this study, we investigate the effect of loss of P2X7 receptor function on retinal structure and function during aging. P2X7-null and wild-type C57bl6J mice were investigated at 4, 12, and 18 months of age for macrophage phagocytosis activity, ocular histological changes, and retinal function. Phagocytosis activity of blood-borne macrophages decreased with age at 18 months in the wild-type mouse. Lack of P2X7 receptor function reduced phagocytosis at all ages compared to wild-type mice. At 12 months of age, P2X7-null mice had thickening of Bruchs membrane and retinal pigment epithelium dysfunction. By 18 months of age, P2X7-null mice displayed phenotypic characteristics consistent with early AMD, including Bruchs membrane thickening, retinal pigment epithelium cell loss, retinal functional deficits, and signs of subretinal inflammation. Our present study shows that loss of function of the P2X7 receptor in mice induces retinal changes representing characteristics of early AMD, providing a valuable model for investigating the role of scavenger receptor function and the immune system in the development of this age-related disease.

Immune profiles vary seasonally, but are not significantly related to migration distance or natal dispersal, in a migratory songbird.

A central tenet of ecoimmunology is that an organism's environment shapes its optimal investment in immunity. For example, the benefits of acquired (relatively pathogen specific) versus innate (nonspecific) immune defenses are thought to vary with the risk of encountering familiar versus unfamiliar pathogens. Because pathogen communities vary geographically, individuals that travel farther during seasonal migration or natal dispersal are predicted to have higher exposure to novel pathogens, and lower exposure to familiar pathogens, potentially favoring investment in innate immunity. During the breeding season, migratory animals' exposure to familiar pathogens should increase, potentially favoring investment in acquired immunity. We hypothesized that song sparrows Melospiza melodia adjust their constitutive immune profiles in response to risk of encountering novel versus familiar pathogens. We predicted that individuals migrating longer distances (inferred from stable hydrogen isotope analysis of claws) and less philopatric individuals (inferred from microsatellite assignment testing) would rely more heavily on acquired than innate defenses. We also predicted that reliance on acquired defenses would increase throughout the early breeding season. Consistent with trade-offs between acquired and innate defenses, levels of immunoglobulin Y (acquired) varied negatively with macrophage phagocytosis activity (innate). Levels of acquired relative to innate immunity did not vary significantly with migration distance or philopatry, but increased throughout the early breeding season. Macrophage phagocytosis was not significantly repeatable between years. Song sparrows appear to shift from innate defenses immediately after migration to acquired defenses with increasing time at the breeding grounds. These patterns highlight the plasticity of constitutive immune defenses in migratory animals.