Phagocytic Rate Research Articles

Optimization of Extraction Process, Structure Characterization, and Immunomodulatory Activity of Polysaccharides from Black Garlic

AbstractUltrasound assisted extraction (UAE) was used to extract polysaccharides from black garlic, and the extraction process for UAE of black garlic polysaccharides (BGPs) was optimized via RSM coupled with genetic algorithm. The optimal extraction process parameters were obtained as follows: extraction time of 30 min, liquid‐to‐solid ratio of 29 mL/g, extraction temperature of 51 ℃, and ultrasound power of 418 W, and the yield of BGPs was 10.17% ± 0.14%. Subsequently, the crude BGPs were further purified by DEAE‐52 cellulose and Sephadex G‐100 to obtain a homogeneous fraction (BGPs‐1‐SG with the molecular weight of 1.37 × 106 Da) that was comprised of mannose (Man), glucuronic acid (GlcA), rhamnose (Rha), glucose (Glc), and fucose (Fuc) with a molar ratio of 40.23:71.06:2.96:85.38:7.32. Congo red and circular dichroism spectroscopy indicate that BGPs‐1‐SG has a triple helix structure. Scanning electron microscope and atomic force microscope demonstrate that BGPs‐1‐SG showed irregular structures including sheet‐like, rod‐shaped, irregular spherical structures, and aggregated in aqueous solutions. Moreover, BGPs‐1‐SG could increase viability, phagocytic rate, and NO content of RAW264.7 cells. Furthermore, BGPs‐1‐SG could promote the secretion and mRNA expression levels of IL‐1β, IL‐6, and TNF‐α. The findings can provide an important reference for the development of functional foods.

Comparative Effects of Crude Extracts and Bioactive Compounds from Bidens pilosa and Bidens alba on Nonspecific Immune Responses and Antibacterial Activity Against Vibrio sp. in Coculture with Lactic Acid Bacteria in Hybrid Grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂).

This study investigated the effect of substances on nonspecific immune responses of head kidney leukocytes, the antimicrobial activity against Vibrio sp., as well as the time-kill of Vibrio sp. by combining the substances with lactic acid bacteria (LAB) and Pediococcus sp. The substances are B. pilosa hot water extract, B. pilosa powder extract, B. pilosa methanol extract, B. pilosa ethanol extract, B. alba hot water extract, B. alba powder extract, B. alba methanol extract, B. alba ethanol extract, and bioactive compounds, namely cytopiloyne, flavonoid, phenol, ethyl caffeate, luteolin, chlorogenic acid, butein, and linoleic acid. The results showed that some of them were nontoxic to the head kidney leukocytes, which can increase the phagocytic rate, phagocytic index, and respiratory burst. These substances were able to inhibit the growth of Vibrio sp.; they can even completely kill the pathogenic bacteria. The largest of the inhibition zone formed from the EC group at a concentration range of 5-50 µg/mL against V. parahaemolyticus, V. alginolyticus, and V. harveyi with a value of 19.7 ± 0.56, 19.3 ± 1.53, and 20.6 ± 1.53 mm. Furthermore, the time-kill studies showed that the LAB and P. acidilactici can completely kill the Vibrio sp. at 6 h incubation time, mainly in the group of combination with EC.

Biochemical and molecular responses to long-term salinity challenges in northern quahogs Mercenaria mercenaria

Salinity is a key environmental factor for aquatic organisms for survival, development, distribution, and physiological performance. Salinity fluctuation occurs often in estuary and coastal zones due to weather, tide, and freshwater inflow and thus heavily affects coastal marine aquaculture. The northern quahog Mercenaria mercenaria is an important aquaculture species along the Atlantic coast in the US, but information on the effects of salinity stress on physiological, immunological, and molecular responses is still scarce. The goal of this study was to investigate cellular and molecular responses through challenges of long-term hypo- and hyper-salinities in northern quahogs. The objectives were to: 1) measure the survival of market-sized quahogs under a three-month salinity challenge at 15 (hyposalinity), 25 (control), and 35 ppt (hypersalinity); 2) determine cellular changes of hemocytes through analysis of immune functions; 3) determine changes of the total free amino acid concentration in gills, and 4) evaluate the molecular responses in gills using RNAseq technology with qPCR verification. After a three-month salinity challenge, no mortality was observed, and increases in body weight were identified with a significantly higher increase in the hypersalinity group. Northern quahogs equilibrated their hemolymph osmolality with the ambient seawater and were verified to be osmoconformers. Significant differences were observed in total hemocyte concentration, lysosomal presence, ROS production, and phagocytic rate, but no differences were found in cell viability. The total free amino acid concentration within gills was positively correlated to water salinity, indicating amino acids were critical organic osmolytes. The transcriptome of gills using RNAseq revealed differential expression genes (DEG) encoding amino acid transporters (SLC6A3, SLC6A6, SLC6A13, SLC25A38), ion channel proteins (T38B1, GluCl, ATP2C1), and water channel protein (AQP8) in hyposalinity or/and hypersalinity groups, indicating these genes play critical roles in intracellular isosmotic regulation. Overall, the findings in this study provided new insights into osmoregulation in northern quahogs.

A transcription factor ATF3 involves in the phagocytosis of granulocytes in oyster Crassostrea gigas

Phagocytosis is a major cellular mechanism for mollusk granulocytes to eliminate nonself substances and dead cells, and thus to preserve the immune homeostasis. The knowledge of the regulatory mechanisms controlling phagocytic capacity is vital to understanding the immune system. In the present study, an ATF3 homolog (CgATF3) with a typical bZIP domain was identified in the Pacific oyster Crassostrea gigas. Its highly conserved bZIP domain consisted of two structural features, a basic region for DNA binding and a leucine zipper region for dimerization. Its transcript was found to be abundantly expressed in haemocytes, which was induced by Vibrio splendidus stimulation and recombinant CgTNF-2 treatment, along with an increase of its protein content in the nucleus. Moreover, CgATF3 showed a consistent and specific high expression in granulocytes, and CgATF3+ granulocytes were characterized morphologically by the largest diameter, smaller nucleus to cytoplasmic ratio, and abundant cytoplasmic granules, and functionally by a higher capacity for phagocytosis. When CgATF3 expression was inhibited by RNAi, the expression levels of CgRab1, CgRab33 and CgCathepsin L1, as well as the phagocytic rate and index of granulocytes all decreased after V. splendidus stimulation. These results together demonstrated the involvement of CgATF3 in regulating the expressions of Rabs and Cathepsin L1, as well as the phagocytosis of granulocytes in oyster C. gigas.

Differential phagocytic expression of IC-21 macrophages and their scavenging receptors during inflammatory induction by oxysterol: A microscopic approach.

Phagocytosis by macrophages dates back to a long history in science, this present study deals with new approaches that have been analyzed and standardized towards the interesting aspects of primary and secondary macrophages. The distinct morphological differences in primary and secondary phagocytic cells were observed and the phagocytic response of secondary macrophages under the influence of 7-ketocholesterol and lipopolysaccharide was analyzed. The primary peritoneal and secondary IC-21 cells unveiled explicit differences in nuclear numbers shapes and sizes of the granules present within the cytoplasmic region. Further, potent inducers 7KCh and LPS influenced an effective activation of IC-21 macrophages and resulted in ROS generation, irregulated protein expressions of CD86, CD68, and CD206 with enhanced phagocytic responses towards goat, cow, and human RBC targets with significant phagocytic rate and index were observed. Moreover, a remarkable observation of target specificity and aggregations with IC-21 phagocytic macrophages revealed the notion that specific membrane receptors and secretory molecules (lysosomes) are primarily involved in their phagocytic mechanism. RESEARCH HIGHLIGHTS: IC-21 macrophages are peritoneal origin from mice but the primary peritoneal macrophages and cell line show distinct differences. IC-21 macrophages express target-specific phagocytosis. Phagocytosis in IC-21 macrophages is regulated by CD markers (68, 86, and 206).

Immunotoxicity and oxidative damage in Litopenaeus vannamei induced by polyethylene microplastics and copper co-exposure

This study examines the combined effects of polyethylene microplastics (PE-MP) and copper (Cu2+) on the immune and oxidative response of Litopenaeus vannamei. PE-MP adsorbed with Cu2+ at 2.3, 6.8, and 16.8 ng (g shrimp)−1) were injected into L. vannamei. Over 14 days, survival rates were monitored, and immune and oxidative stress parameters were assessed. The results showed that combined exposure to PE-MP and Cu2+ significantly reduced the survival rate and decreased total haemocyte count. Immune-related parameters (phagocytic rate, phenoloxidase and superoxide dismutase (SOD)) and antioxidant-related parameters (SOD, catalase and glutathione peroxidase mRNA and enzyme) also decreased, while respiratory burst activity significantly increased, indicating immune and antioxidant system disruption. Additionally, there was a significant increase in oxidative stress, as measured by malondialdehyde levels. Histopathological analysis revealed severe muscle, hepatopancreas, and gill damage. These results suggest that simultaneous exposure to PE-MP and Cu2+ poses greater health risks to white shrimp.

Amino-modified polystyrene nanoplastics induced multiple response of Artemia hemocytes

Polystyrene polymers cause severe toxicity to aquatic animals. However, the process and mechanisms of innate immunity of invertebrates living at the bottom of the food chain to these pollutants remain unclear. In this study, the blood system responses of zooplankton Artemia were assessed through in vivo and in vitro exposure to amino-modified polystyrene nanoplastics (PS-NH2 NPs). The results indicated that the LC50 values of PS-NH2 NPs were 1.09 μg·mL−1 over 48 h and 0.42 μg·mL−1 over 7 d. Based on the five hemocyte subpopulations identified in Artemia, in vitro exposure assays revealed that phagocytosis was performed by plasmocytes and granulocytes with phagocytic rate of 22.64 %. TEM analysis further showed that PS-NH2 NPs caused cytoplasm vacuolization, swollen mitochondria, and lipid processing disorder. Gene expression pattern results demonstrated that Spatzle, Tollip, Hsp70, Hsp90, Casp8, API5and Pxn were significantly upregulated upon acute and chronic exposure (p < 0.05), while chronic exposure could induce significantly upregulation of ProPO (p < 0.05). Moreover, PS-NH2 NPs exposure remarkably varied the hemolymph microbiota and hemogram, particularly by increasing the proportion of adipohemocytes and phagocytes (p < 0.05). Our findings suggest that PS-NH2 NPs induce different responses in Artemia hemocyte, as primarily reflected by phagocytic processes, expression of immune and apoptosis relating genes, cell fates, hemogram and hemolymph microbiota variations. These findings support the possibility of using Artemia hemocytes as bioindicator to estimate nanoplastics pollution, thus contributing to hematological toxicity research in response to nanoplastics.

Morphologic, cytometric, quantitative transcriptomic and functional characterisation provide insights into the haemocyte immune responses of Pacific abalone (Haliotis discus hannai).

In recent years, the abalone aquaculture industry has been threatened by the bacterial pathogens. The immune responses mechanisms underlying the phagocytosis of haemocytes remain unclear in Haliotis discus hannai. It is necessary to investigate the immune mechanism in response to these bacterial pathogens challenges. In this study, the phagocytic activities of haemocytes in H. discus hannai were examined by flow cytometry combined with electron microscopy and transcriptomic analyses. The results of Vibrio parahaemolyticus, Vibrio alginolyticus and Staphylococcus aureu challenge using electron microscopy showed a process during phagosome formation in haemocytes. The phagocytic rate (PP) of S. aureus was higher than the other five foreign particles, which was about 63%. The PP of Vibrio harveyi was about 43%, the PP peak of V. alginolyticus in haemocyte was 63.7% at 1.5h. After V. parahaemolyticus and V. alginolyticus challenge, acid phosphatase, alkaline phosphatase, total superoxide dismutase, lysozyme, total antioxidant capacity, catalase, nitric oxide synthase and glutathione peroxidase activities in haemocytes were measured at different times, differentially expressed genes (DEGs) were identified by quantitative transcriptomic analysis. The identified DEGs after V. parahaemolyticus challenge included haemagglutinin/amebocyte aggregation factor-like, supervillin-like isoform X4, calmodulin-like and kyphoscoliosis peptidase-like; the identified DEGs after V. alginolyticus challenge included interleukin-6 receptor subunit beta-like, protein turtle homolog B-like, rho GTPase-activating protein 6-like isoform X2, leukocyte surface antigen CD53-like, calponin-1-like, calmodulin-like, troponin C, troponin I-like isoform X4, troponin T-like isoform X18, tumor necrosis factor ligand superfamily member 10-like, rho-related protein racA-like and haemagglutinin/amebocyte aggregation factor-like. Some immune-related KEGG pathways were significantly up-regulated or down-regulated after challenge, including thyroid hormone synthesis, Th17 cell differentiation signalling pathway, focal adhesion, melanogenesis, leukocyte transendothelial migration, inflammatory mediator regulation of TRP channels, ras signalling pathway, rap1 signalling pathway. This study is the first step towards understanding the H. discus hannai immune system by adapting several tools to gastropods and providing a first detailed morpho-functional study of their haemocytes.

Norepinephrine regulates TNF expression via the A1AR-p38 MAPK-Relish pathway in granulocytes of oyster Crassostrea gigas

Norepinephrine (NE) is involved in regulating cytokine expression and phagocytosis of immune cells in the innate immunity of vertebrates. In the present study, the modulation mechanism of NE on the biosynthesis of TNFs in oyster granulocytes was explored. The transcripts of CgTNF-1, CgTNF-2 and CgTNF-3 were highly expressed in granulocytes, and they were significantly up-regulated after LPS stimulation, while down-regulated after NE treatment. The phagocytic rate and apoptosis index of oyster granulocytes were also triggered by LPS stimulation and suppressed by NE treatment. The mRNA expressions of CgMAPK14 and CgRelish were significantly induced after NE treatment, and the translocation of CgRelish from cytoplasm to nucleus was observed. The concentration of intracellular Ca2+ in granulocytes was significantly up-regulated upon NE incubation, and this trend reverted after the treatment with DOX (specific antagonist for NE receptor, CgA1AR-1). No obvious significance was observed in intracellular cAMP concentrations in the PBS, NE and NE + DOX groups. Once CgA1AR-1 was blocked by DOX, the mRNA expressions of CgMAPK14 and CgRelish were significantly inhibited, and the translocation of CgRelish from cytoplasm to nucleus was also dramatically suppressed, while the mRNA expression of CgTNF-1 and the apoptosis index increased significantly to the same level with those in LPS group, respectively. These results collectively suggested that NE modulated TNF expression in oyster granulocyte through A1AR-p38 MAPK-Relish signaling pathway.

Assessment of fish protein hydrolysate as a substitute for fish meal in white shrimp diets: Impact on growth, immune response, and resistance against Vibrio parahaemolyticus infection

This study investigated the effects of fish protein hydrolysate derived from barramundi on growth performance, muscle composition, immune response, disease resistance, histology and gene expression in white shrimp (Penaeus vannamei). In vitro studies demonstrated FPH enhanced mRNA expressions of key immune-related genes and stimulated reactive oxygen species (ROS) production and phagocytic activity in shrimp hemocytes. To evaluate the effects of substituting fish meal with FPH in vivo, four isoproteic (43 %), isolipidic (6 %), and isoenergetic diets (489 kcal/100 g) were formulated with fish meal substitution levels of 0 % (control), 30 % (FPH30), 65 % (FPH65), and 100 % (FPH100). After 8-week feeding, the growth performance of FPH65 and FPH100 were significantly lower than that of control and FPH30 (p < 0.05). Similarly, the midgut histological examination revealed the wall thickness and villi height of FPH100 were significantly lower than those of control (p < 0.05). The shrimps were received the challenge of AHPND + Vibrio parahaemolyticus at week 4 and 8. All FPH-fed groups significantly enhanced resistance against Vibrio parahaemolyticus at week 4 (p < 0.05). However, this protective effect diminished after long-period feeding. No significant difference of survival rate was observed among all groups at week 8 (p > 0.05). The expressions of immune-related genes were analyzed at week 4 before and after challenge. In control group, V. parahaemolyticus significantly elevated SOD in hepatopancreas and Muc 19, trypsin, Midline-fas, and GPx in foregut (p < 0.05). Moreover, hepatopancreatic SOD of FPH65 and FPH100 were significantly higher than that of control before challenge (p < 0.05). Immune parameters were measured at week 8. Compared with control, the phagocytic index of FPH 30 was significantly higher (p < 0.05). However, dietary FPH did not alter ROS production, phenoloxidase activity, phagocytic rate, and total hemocyte count (p > 0.05). These findings suggest that FPH30 holds promise as a feed without adverse impacts on growth performance while enhancing the immunological response of white shrimp.

Regulation of mouse digestive function, intestinal mucosal barrier function, and inflammatory reaction by lycium barbarum polysaccharide pathway through myosin light chain kinase

This research investigated the impacts of lycium barbarum polysaccharide (LBP) on the digestive function, intestinal mucosal barrier function, inflammatory response, and myosin light chain kinase (MLCK) signaling pathway in immunosuppressed mice. 70 mg/kg cyclophosphamide was injected into abdomen for the preparation of immune suppression model. Healthy BALB/c mice served as control for the analysis of the differences in gastrointestinal motility and absorptive capacity, intestinal mucosal barrier function, the phagocytic ability of abdominal macrophages, serum immune factor and inflammatory factor levels, and the activation status of the MLCK signaling pathway after continuous gavage with 100 mg/kg LBP. Results revealed a decrease in d-xylose content, phagocytic rate, index of abdominal macrophages, and spleen index in the serum and urine of model mice compared to those of controls. In addition, levels of IgA, IgG, IgM, IL-6 (interleukin-6), IL-12, and interferon-γ (IFN-γ) decreased, while MLCK and myosin light chain (MLC) levels rose (P < 0.01). Versus those in Model group, urine d-xylose content, phagocytic rate, index of abdominal macrophages, spleen index, and the levels of IgA, IgG, IgM, IL-6, IL-12, and IFN-γ of mice undergoing the gavage with LBP increased, while MLCK and p-MLC levels declined (P < 0.05). In conclusion, LBP improved digestive absorption and immune function of immunosuppressed mice and regulated intestinal mucosal barrier immune system by inhibiting MLCK signaling pathway activation.

A trace amine associated receptor mediates antimicrobial immune response in the oyster Crassostrea gigas

Trace amine-associated receptors (TAARs) are a class of G protein-coupled receptors, playing an immunomodulatory function in the neuroinflammatory responses. In the present study, a TAAR homologue with a 7tm_classA_rhodopsin-like domain (designated as CgTAAR1L) was identified in oyster Crassostrea gigas. The abundant CgTAAR1L transcripts were detected in visceral ganglia and haemocytes compared to other tissues, which were 55.35-fold and 32.95-fold (p < 0.01) of those in adductor muscle, respectively. The mRNA expression level of CgTAAR1L in haemocytes significantly increased and reached the peak level at 3 h after LPS or Poly (I:C) stimulation, which was 4.55-fold and 12.35-fold of that in control group, respectively (p < 0.01). After the expression of CgTAAR1L was inhibited by the injection of its targeted siRNA, the mRNA expression levels of interleukin17s (CgIL17-1, CgIL17-5 and CgIL17-6), and defensin (Cgdefh1) significantly decreased at 3 h after LPS stimulation, which was 0.51-fold (p < 0.001), 0.39-fold (p < 0.01), 0.48-fold (p < 0.05) and 0.41-fold (p < 0.05) of that in the control group, respectively. The nuclear translocation of Cgp65 protein was suppressed in the CgTAAR1L-RNAi oysters. Furthermore, the number of Vibrio splendidus in the haemolymph of CgTAAR1L-RNAi oysters significantly increased (4.11-fold, p < 0.001) compared with that in the control group. In contrast, there was no significant difference in phagocytic rate of haemocytes to V. splendidus in the CgTAAR1L-RNAi oysters. These results indicated that CgTAAR1L played an important role in the immune defense against bacterial infection by inducing the expressions of interleukin and defensin.

Abstract 5246: Therapeutic efficacy of glypican-3 peptide-linked chimeric antigen receptor-macrophages in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the third most lethal cancer and sixth most diagnosed cancer worldwide. Recently, chimeric antigen receptor-T cell (CAR-T) therapy has been demonstrated to be a promising immunotherapy for the treatment of hematologic malignancies such as leukemia. However, its application in solid tumors has proven to be challenging. To overcome this limitation, chimeric antigen receptor-macrophages (CAR-Ms) have emerged as a new immunotherapy against solid cancers, capitalizing on the unique attributes of macrophages in penetrating the tumor microenvironment, promoting antigen presentation, and priming T cells. Traditionally, CAR-M therapy involves the transfer of an edited CAR gene into macrophages via viral infection. However, this method is expensive, involves tedious procedures, and has long-term safety concerns. In the present study, we employed peptides to mimic the genetic approach for producing CAR-Ms to reduce the above uncertainties. To achieve this, we developed a novel bioconjugation method called the phthalaldehydeamine capture (PAC) reaction to produce peptidic CAR-Ms (pCAR-Ms) targeting Glypican-3 (GPC3) in HCC cells. This was achieved by linking GPC3 specific peptides to Raw264.7 murine macrophages. Using an in vitro co-culture system, we demonstrated the specificity and efficacy of pCAR-Ms against GPC3, as evidenced by a significant increase in the number of phagocytosed cells with high GPC3 expression when compared to their low-expression counterparts, by flow cytometry and confocal microscopy. The specificity of pCAR-Ms was further proven in GPC3-knockdown HCC cells, with a significant decrease in the phagocytic rate. Using an HCC xenograft model derived from MHCC97L cells, we confirmed the specific homing of pCAR-Ms to tumor sites via intravenous injection of lipopolysaccharide (LPS)-differentiated pCAR-Ms. Strikingly, pCAR-Ms suppressed HCC tumor growth when compared with untagged macrophages and PBS control. In conclusion, we provided evidence of the potency and specificity of non-viral-based pCAR-Ms in targeting HCC with simple and short production time and increased flexibility. Citation Format: Wing Hei Leung, Oi Ning Leung, Tsun Ting Wong, Kin Wah Lee. Therapeutic efficacy of glypican-3 peptide-linked chimeric antigen receptor-macrophages in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5246.

Effect of probiotic feed supplementation on skin ulceration syndrome in Apostichopus japonicus

Skin ulceration syndrome is among the most harmful diseases in sea cucumber (Apostichopus japonicus). The annual loss due to this disease is more than 1 billion dollars, which seriously affected the healthy and sustainable development of A. japonicus. In this paper, animals were divided into 4 experimental groups: a control group given basic feed, experimental groups with 1% Lactobacillus, 1% Bacillus, 1% mixture with 0.5% Lactobacillus and 0.5% Bacillus added to the basic feed. After feeding for 60 days, the samples were taken to analyze the growth performance, digestive enzyme activity, and immune indexes of A. japonicus. Furthermore, the resistance to the skin ulceration syndrome of A. japonicus was analyzed. The results showed that the probiotics promoted the growth performance of A. japonicus, the weight gain rate of the control group was 120.08±12.07%, the weight gain rate of the Lactobacillus group was 152.11±10.06%, the weight gain rate of the Bacillus group was 148.03±9.88%, and the weight gain rate of the mixed bacteria group was as high as 210.11±19.41%. The digestive enzyme activities in the intestinal tract of A. japonicus increased significantly in the probiotic-added groups, and the mixed bacteria were more effective in enhancing the digestive enzyme activities than single Bacillus and Lactobacillus. In addition, the probiotics could significantly increase the phagocytosis rate of the body cavity cells of A. japonicus. The phagocytic rate of Lactobacillus and Bacillus subtilis was comparable; the mixed probiotics were significantly higher than those of single probiotics, and this phagocytic rate gradually increased over time. After Vibrio splendidus and Pseudoalteromonas infection, the mortality rate of A. japonicus in the probiotic-added group was reduced, which indicated that probiotics could improve the immunity of A. japonicus, and the ability to resist the infection of Vibrio splendidus and Pseudoalteromonas was enhanced. Bacillus exhibits superior antimicrobial ability compared to Lactobacillus, and the mixed strain displays the most effective antimicrobial properties. This study provided a scientific basis for effectively preventing and controlling skin ulceration syndrome in the aquaculture of A. japonicus.

Application of nZnO supported with nanoclay for improving shrimp immunity

This study discloses the nanoscale silicate platelet-supported nZnO (ZnONSP) applied as novel feed additives in aquaculture. The preparation of the nanohybrid (ZnO/NSP = 15/85, w/w) was characterized by UV–visible spectroscopy, powder X-ray diffraction and transmission electron microscope. The effects of ZnONSP on growth, zinc accumulation, stress response, immunity and resistance to Vibrio alginolyticus in white shrimp (Penaeus vannamei) were \\demonstrated. To evaluate the safety of ZnONSP, shrimps (2.0 ± 0.3 g) were fed with ZnONSP containing diets (200, 400 and 800 mg/kg) for 56 days. Dietary ZnONSP did not affect the weight gain, specific growth rate, feed conversion ratio, survival rate, zinc accumulation, and the expression of heat shock protein 70 in tested shrimps. To examine the immunomodulatory effect of ZnONSP, shrimps (16.6 ± 2.4 g) were fed with the same experimental diets for 28 days. Dietary ZnONSP improved the immune responses of haemocyte in tested shrimps, including phagocytic rate, phagocytic index, respiratory burst, and phenoloxidase activity, and upregulated the expression of several genes, including lipopolysaccharide, β-1,3-glucan binding protein, peroxinectin, penaeidin 2/3/4, lysozyme, crustin, anti-lipopolysaccharide factor, superoxide dismutase, glutathione peroxidase, clotting protein and α-2-macroglobulin. In the challenge experiment, shrimps (17.2 ± 1.8 g) were fed with ZnONSP containing diets (400 and 800 mg/kg) for 7 days and then infected with Vibrio alginolyticus. Notably, white shrimps that received ZnONSP (800 mg/kg) showed significantly improved Vibrio resistance, with a survival rate of 71.4 % at the end of 7-day observation. In conclusion, this study discovers that ZnONSP is a new type of immunomodulatory supplement that are effective on enhancing innate cellular and humoral immunities, and disease resistance in white shrimp.

Impaired polymorphonuclear neutrophil functions in diabetics

Background and objectives: Polymorphonuclear neutrophils (PMN) are the first line of host resistance against infections. Diabetics are prone to both bacterial and fungal infections. The present study evaluated the phagocytic and killing activity of PMN in diabetics. Material and methods: Females aged 30 to 50 years with and without diabetes mellitus were enrolled. Functions of PMN were assessed by determining the phagocytic rate, phagocytic index and killing of C. albicans by PMN. Results: A total of 36 diabetic patients and 15 age matched non-diabetic healthy individuals were enrolled. Phagocytosis and killing of C. albicans by PMN were significantly (p&lt;0.05) lower in patients with diabetes mellitus compared to non-diabetic healthy individuals (86.5±14.6 vs. 94.5±4.2; 56.7±23.8 vs. 81.5±24.2). Conclusion: Phagocytic and killing functions of PMN were significantly reduced in patients with diabetes mellitus. IMC J Med Sci. 2024; 18(1):008. DOI: https://doi.org/10.55010/imcjms.18.008 *Correspondence:Tanzinah Nasrin, Microbiologist, Quality Control Laboratory, Department of Fisheries, Ministry of Fisheries and Livestock, Dhaka, Bangladesh. Email: tanzinahn8@gmail.com

Customizing biomimetic surface attributes of dendritic lipopeptide nanoplatforms for extended circulation

The pressing demand for innovative approaches to create delivery systems with heightened drug loading and prolonged circulation has spurred numerous efforts, yielding some successes but accompanied by constraints. Our study proposes employing dendritic lipopeptide with precisely balanced opposing charges to extend blood residency for biomimetic nanoplatforms. Neutrally mixed-charged zwitterionic nanoparticles (NNPs) achieved a notable 19 % simvastatin loading content and kept stable even after one-month storage at 4 °C. These nanoplatforms demonstrated low cytotoxicity in NIH-3T3 and L02 cells and negligible hemolysis (<5 %). NNPs inhibited protein adhesion (>95 %) from positively and negatively charged sources through surface hydration. In comparison to positively charged CNPs, NNPs demonstrated an 86 % decrease in phagocytic rate by BMDMs, highlighting their efficacy. Importantly, NNPs showed prolonged circulation compared to CNPs and free simvastatin. These findings highlight the potential of this biomimetic nanoplatform for future therapeutic applications with enhanced drug loading and circulation traits.

Ultrasound assisted aqueous two-phase extraction of polysaccharides from corn stigma: Process optimization, structure characterization, and immunomodulatory activity

The two models of ultrasound assisted aqueous two-phase extraction (UATPE) of polysaccharides from corn stigma were established by response surface methodology (RSM) and artificial neural network (ANN), and compared the prediction ability of RSM and ANN models. Subsequently, the extraction process of corn stigma polysaccharides (CSPs) was optimized by the combination of genetic algorithm (GA) and ANN. The results show that two models could precisely predict the CSPs yield, whereas the prediction ability of ANN was better than RSM. The optimum combination of process parameters was as follows: the ultrasound power of 466 W, extraction temperature of 54 °C, liquid-to-solid ratio of 47 mL/g, and extraction time of 31 min. The highest CSPs yield was 9.89% ± 0.14% under the above parameters, and then the crude CSPs were further purified by DEAE-52 and Sephadex G − 100 chromatography to obtain a homogenous fraction (CSPs-2-SG with a molecular weight of 2.86 × 106 Da) that consisted of Man, GlcA, GalA, Glc, Gal, and Xyl with a molar ratio of 9.14:16.35:11.27:91.08:6.49:4.71. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) demonstrated that CSPs-2-SG showed irregular structures including uneven fragments, some holes, and spherical aggregates on the surface. Finally, the immunomodulatory activity of CSPs-2-SG was evaluated by RAW264.7 cells. CSPs-2-SG could improve viability, phagocytic rate, and NO content of RAW264.7 cells. Moreover, CSPs-2-SG could increase the levels of IL − 1β, IL-6, and TNF-α in RAW264.7 cells. The findings provide important references for utilizing corn stigma as a potential natural immune regulator.

Targeting CD24 “Don't Eat Me” Signal Restores Phagocytic Activity of Nurse-like Cells from Patients with Chronic Lymphocytic Leukemia

Introduction Chronic Lymphocytic Leukemia (CLL) is an indolent B-cell lymphoproliferative disease that carries the risk of evolving into a life-threatening aggressive histology, known as Richter's syndrome. Despite the advent of new agents such as ibrutinib and venetoclax that have dramatically changed the clinical fate of CLL, young patients still experience refractory and resistant disease, with few therapeutic options available over time. Therefore, the need for new strategies is pivotal for this chronic and insidious condition. Drug resistance might be triggered by the tumor microenvironment (TME), which provides survival signals to CLL tumor cells. Their interaction is mainly established with monocyte-derived nurse-like cells (NLCs). NLCs resemble the immunosuppressive M2-like tumor-associated macrophages (TAMs) and can create a profitable connection with CLL in terms of increasing proliferation and survival (Filip, Blood Cells Mol Dis. 2013). The possibility of targeting the TME by blocking the “Don't Eat Me” signal (DEMs) CD47 has provided restoration of phagocytosis within TME and interesting clinical results in heavily pretreated cases of lymphoma (Advani, NEJM. 2019). The use of agents targeting the DEMs within TME could be a promising approach in CLL as well. CD24 is another DEMs that was first validated in a preclinical model of solid cancer (Barkal, Nature. 2019). It was subsequently found to be upregulated in Mantle-cell lymphoma (MCL) and involved in DEMs as well, since CD24 blockade provided an increase in phagocytosis of MCL cells in vitro (Aroldi, Blood. 2021). This effect was particularly evident in association with anti-CD47 monoclonal antibody (mAb) and Rituximab (Aroldi, Blood. 2022). Although MCL has different pathogenesis compared to CLL, these two entities share some features, particularly regarding the B-cell of origin and the vital interaction with non-cancerous cells within TME. In this study, we explore the possibility of targeting CD24 in human primary samples of CLL (huCLL), redirecting the activity of NLCs against CLL cells using anti-CD24 mAb alone or in combination with other agents (anti-CD47, Rituximab). Methods We analyzed a microarray dataset of patients with CLL to check for any correlation between CD24 expression and survival (GSE22762). We also checked the CD24 antigen expression of huCLL, isolated and differentiated monocytes into M2-like TAMs in vitro, as well as conducted co-culture assays using flow cytometry. These methods were previously described (Aroldi, Blood. 2021; Aroldi, Blood. 2022). Unstimulated autologous CD14+ cell population - defined as precursors of NLCs - were isolated from patients with CLL and used for co-culture analysis without any previous cytokine stimulation for differentiation. Results In a cohort of CLL patients, higher CD24 levels correlated with a lower survival rate compared to lower expression ones (Figure 1). We also checked the CD24 surface in an extended cohort of huCLL and compared it to MEC-1 and PCL-12. huCLL showed higher CD24 surface levels than MEC-1 and PCL-12 and demonstrated better phagocytosis when co-cultured with M2-like TAMs and anti-CD24 mAb (Figure 2A). We then isolated precursors of circulating NLCs from 5 huCLL patients and found higher levels of Siglec-10 (CD24 ligand) than in healthy controls. In this setting, blocking CD24 redirected the phagocytic activity of NLCs against huCLL, showing higher phagocytic rate in cases of multiple combinations (anti-CD24, anti-CD47, anti-CD20, Figure 2B). Conclusions CLL could be another blood cancer subtype sensitive to CD24/Siglec-10 DEMs blocking agents, and CD24 expression negatively impacts survival. Anti-CD24 mAb, in particular, showed the ability to redirect patient-derived NLCs' phagocytic activity to cognate huCLL cancer cells, resulting in higher clearance in cases of multiple combination. Boosting the autologous innate immune system might, therefore, provide further therapeutic options in the setting of CD24 + CLL, particularly for those patients who have failed several lines of treatment and have limited options available.

Differential responses of monocyte-derived macrophages from Theileria orientalis infected carrier cattle to Pasteruella multocida B:2 infection and latex beads: A preliminary study

This study aims to evaluate the responses of peripheral blood monocyte-derived macrophages (PBMDMs) from Theileria orientalis carrier cattle following exposure to Pasteruella multocida B:2 (PM B:2) and latex beads. Twenty-six male crossbred Kedah-Kelantan (KK) cattle were sampled for this study and quantitative PCR (qPCR) was employed in the detection of T. orientalis MPSP gene. Bactericidal assay using a 10:1 multiplicity of infection was performed to measure the phagocytosis and intracellular killing of PM B:2 by PBMDMs. The cell cultures were inoculated with 107 cfu/mL of PM B:2 and incubated in a humidified incubator. The absence of clinical signs, previous history of T. orientalis infection and an MPSP gene copy number below 15,000 GC/μL suggest that the cattle were asymptomatic chronic carriers. A non-significant phagocytic and mean cell death rates were observed in the PBMDMs of T. orientalis positive cattle relative to clinically healthy cattle (CHC) (p > 0.05). The PBMDMs of T. orientalis positive cattle had the lowest mean rate of intracellular killing relative to the CHC at the 30th minute post-infection only (p < 0.05). Exposure to latex beads caused an increase in the appearance of multinucleated macrophages following incubation of PBMDMs from T. orientalis positive cattle. Furthermore, the phagocytic index of PBMDMs of T. orientalis positive cattle were low or poor compared to that of CHC (p = 0.000). Therefore, our findings suggest that PBMDMs from cattle with chronic T. orientalis infection can efficiently phagocytise and kill PM: B2 but exhibited poor phagocytosis ability for foreign bodies despite appearance of multinucleated macrophages.