Knockdown of Annexin A1 induces apoptosis, causing G2/M arrest and facilitating phagocytosis activity in human leukemia cell lines

Annexin A1 (ANXA1) is an endogenous protein involved in the control of proliferation, cell cycle, phagocytosis, and apoptosis in several types of cancer. To investigate the effects of ANXA1 knockdown in leukemia cells, transfection with specific ANXA1 siRNA was performed. Cell cycle and apoptosis were analyzed using flow cytometry and a mechanism involving caspases and Bcl-2 was quantified using Western blotting. Phagocytosis activity was evaluated using hematoxylin & eosin staining. The ANXA1 expression was significantly downregulated after the knockdown and apoptosis was induced in tested cells. The expression of caspase-9 and -3 increased in U937 and Jurkat cells respectively. Bcl-2 expression was downregulated in K562 and Jurkat cells while upregulated in U937. The number of leukemic cells arrested at the G2/M phase and the phagocytosis index were significantly increased in transfected cells. This suggests that ANXA1 knockdown might be a potential approach in the therapeutic strategy for leukemia.

In vitro study of homeopathic medicines in macrophages co-cultured with Leishmania (L.) amazonensis

Background: In previous studies the effect of homeopathic medicines in murine cutaneous Leishmaniasis were described, indicating immunomodulation. The phagocytes migration and their activity increased after Thymulin 5cH and decreased after Antimonium crudum 30cH treatment. Methods: The morpho-functional features of RAW macrophages co-cultured with Leishmania (L.) amazonensis in RPMI medium were analyzed in vitro, after 24h treatment with 20% of Thymulin 5cH, 6cH and 7cH or Antimonium crudum 6cH, 30cH and 200cH. All assays were performed in blind and in three series of quadruplicate. The spreading was analyzed by the breadth and area of each macrophage, photographed in a NIKON Eclipse 200-Coolpix system and measured by Metamorph® Image analysis software. The phagocytosis was analyzed by the percentage of amastigotes incorporated into the phagocyte vacuoles. ANOVA and Tuckey Krammer were used as statistical methods. Results: A marked increase of macrophage spreading (considering breadth and area) was seen in Thymulin 5cH and Thymulin 7cH treated cells (p≤0.01), as in Antimonium crudum 30cH (p=0.05) and 200cH (p=0.001) treated cells. Only Antimonium crudum 200cH presented increase in phagocytosis index (p=0.001). Conclusions: The in vitro assay corroborates the findings obtained from in vivo studies and represents the first step to a deeper understanding of the mechanism of action.

Non-Specific Immune Response and Growth Performance of Clarias gariepinus (Burchell, 1822) Feeded with Moringa oleifera Leaf Flour Suplementation (Lamk, 1785)

Sangkuriang catfish (Clarias gariepinus) is a cultivated species favored by Indonesian and has great potential for development. However, the obstacle to the success of catfish cultivation is a disease. One alternative to overcome this problem is by giving immunostimulants from natural ingredients. The purpose of this study was to test the effectiveness of feeding with moringa leaf powder on non-specific immunity and growth performance of sangkuriang catfish. The study used a completely randomized design (CRD) with 4 treatments (100% commercial feed, commercial feed with 1.5% Moringa leaf supplementation, commercial feed with 3% Moringa leaf supplementation, and commercial feed with Moringa leaf supplementation 4.5%, and 3 replications. The results showed that the commercial feed with 4.5% moringa leaf supplementation gave the best results for total erythrocytes of 5.97x104 cells/mm3 and MCV of 92.10 fl, and increased the absolute weight growth of 46.42 g. However, it did not show a significant effect on total erythrocytes, hemoglobin, hematocrit, MCHC, MCH, MCV, total leukocytes, leukocyte differentials, phagocytic activity, phagocytosis index, absolute weight growth survival rate, and the value of the feed convention ratio (FCR).

Effects of organic mercury on Mytilus galloprovincialis hemocyte function and morphology

Filter-feeding organisms accumulate xenobiotics and other substances in their tissues. They can be useful as sentinel organisms in biomonitoring of the marine compartment. Bivalve cellular immunity is ensured by phagocytosis and cytotoxic reactions carried out by hemocytes in a network with humoral responses. These can be affected by chemical contaminants in water that can be immunosuppressors also at a low concentration increasing the sensibility to pathogens. This work is an attempt to individuate cellular markers for pollution detection, investigating the effect of methylmercury (CH3HgCl) at different concentrations on the activity and hemocyte morphology of the Mediterranean mussel, Mytilus galloprovincialis. We assessed the effect of three sub-lethal concentrations of the organometal on the cellular morphology, the efficacy of phagocytosis toward yeast cells, the alteration of the lysosomal membrane and the ability to release cytotoxic molecules. The results provide information on the alteration of hemocyte viability, modification of the morphological and cytoskeletal features and besides the cellular spreading, intrinsic ability of motile cells was used as a complementary investigation method. Exposure to the contaminant affected the percentage of phagocytosis and the phagocytosis index. Moreover, morphological and cytoskeleton alteration, caused by the pollutant, leads to reduced ability to incorporate the target and adhere to the substrate and the low ability of cells to retain neutral red could depend on the effects of methylmercury on membrane permeability. These results reinforce the use of the Mediterranean mussel as model for the evaluation of environmental quality in aquatic ecosystems integrating the novel information about hemocyte functions and morphology sensibility to organic mercury. Graphic abstract

Pathogenicity Levels of Colombian Strains of Candida auris and Brazilian Strains of Candida haemulonii Species Complex in Both Murine and Galleria mellonella Experimental Models

Candida auris and Candida haemulonii complex (C. haemulonii, C. haemulonii var. vulnera and C. duobushaemulonii) are phylogenetically related species that share some physiological features and habits. In the present study, we compared the virulence of these yeast species using two different experimental models: (i) Galleria mellonella larvae to evaluate the survival rate, fungal burden, histopathology and phagocytosis index and (ii) BALB/c mice to evaluate the survival. In addition, the fungal capacity to form biofilm over an inert surface was analyzed. Our results showed that in both experimental models, the animal survival rate was lower when infected with C. auris strains than the C. haemulonii species complex. The hemocytes of G. mellonella showed a significantly reduced ability to phagocytize the most virulent strains forming the C. haemulonii species complex. Interestingly, for C. auris, it was impossible to measure the phagocytosis index due to a general lysis of the hemocytes. Moreover, it was observed a greater capability of biofilm formation by C. auris compared to C. haemulonii species complex. In conclusion, we observed that C. auris and C. haemulonii complex have different levels of pathogenicity in the experimental models employed in the present study.

CHITOSAN NANOPARTICLE AS A DELIVERY SYSTEM FOR POLYPHENOLS FROM MENIRAN EXTRACT (PHYLLANTHUS NIRURI L.): FORMULATION, OPTIMIZATION, AND IMMUNOMODULATORY ACTIVITY

Objective: This study aims to formulate meniran extract into polymeric nanoparticles. Better stability of active substances in formulas compared to unformulated extracts is expected to increase immunomodulatory activity. Methods: Nanoparticles were formulated using ionic gelation method with chitosan and tripolyphosphate polymers. Optimize the mixture of nanoparticles using simplex lattice design (SLD) with the help of Design-Expert (DX) software. Evaluation of particle size and potential zeta using dynamic light scattering (DLS). Interactions between components were analyzed using Fourier transform infrared spectrophotometry-attenuated total reflectance (FTIR-ATR) and morphology of the lyophilization results observed using scanning electron microscopy (SEM). Immunomodulatory tests using the latex assay method. The parameters tested included phagocytosis index, phagocytic activity, and nitric oxide secretion. Results: The optimum mixture of the formulation process was obtained in the composition of chitosan 0.270 %, extract 0.626 %, and tripolyphosphate 0.074 % with desirability value of 0.841. Optimal response with particle size 434.7±3.90 d. nm, polydispersity index 0.285±0.03 and entrapment efficiency 62.98±0.65 %. The zeta potential value in the optimum formula is 11.9±0.1 mV with a positive charge. Phagocytosis index and phagocytic activity of nanoparticles differed significantly (p<0.05) compared with unformulated extracts. Conclusion: Meniran extract was successfully formulated into polymeric nanoparticles using chitosan-tripolyphosphate polymer. The developed nanoparticles have the immunomodulatory activity that is better than unformulated extract.

Biological Actions, Electrical Conductance and Silicon-Containing Microparticles of Arsenicum Album Prepared in Plastic and Glass Vials

Introduction According to the “silica hypothesis” formulated to explain homeopathy, the information of starting materials would be transferred to cells by silica nanoparticles detached from the glassware walls by serial dilution and agitation through epitaxy. We compared the biological activity, electrical current and silicon microparticle content (by means of scanning electron microscopy/energy-dispersive X-ray spectroscopy) of high dilutions (HDs) of arsenic prepared in plastic and glass vials to investigate the role of silica in their biological effects in vitro. Materials and Methods Co-cultures of macrophages and yeast (Saccharomyces cerevisiae) were treated with different HDs of arsenic prepared in plastic and glass vials. Macrophage morphology, phagocytosis index, nitric oxide (NO), and cytokine production were evaluated. Results Measurable amounts of silicon microparticles were detected only in the HDs prepared in glass vials, but ultra-centrifugation eliminated them. Specific and non-specific results were observed. Non-specific pro-inflammatory effects were seen in all dilutions prepared in plastic vials, including elevation of pro-inflammatory cytokines, NO and macrophage phagocytic index. Only the 200th centesimal dilution of arsenic produced specific decrease in interleukin-6 production in macrophages, and it was independent of the vial type or the presence of microparticles of silica in the medicine samples. The nature of the vials had an impact on the electric flow in the respective fluids. Conclusion The non-specific, pro-inflammatory effects might be attributed to organic residuals detached from the vials' plastic walls during manipulation. Instead, specific silica-independent effects of the homeopathic medicine can be attributed to the decrease of interleukin-6 after treatment with the 200th centesimal dilution of arsenic.

IMMUNOMODULATORY EFFECT TEST FROM MORINGA LEAF EXTRACT (MORINGA OLEIFERA L.) WITH CARBON CLEARANCE METHOD IN MALE WHITE MICE

Objective: Moringa oleifera leaf has chemical compounds that have been utilized by the community to cure health problems. One of its activities is an immunomodulator. The aim of this study is to determine the immunomodulatory effect from M. oleifera leaf using a carbon clearance method to measure the activity of phagocytic cells in exterminating pathogens that enter the body then followed by calculating the total leukocyte cells. The parameters of this test are phagocytosis index and total leukocyte cells.Methods: Twenty white male mice were divided into four groups. Group I (vehicle control) was treated with sodium-carboxymethyl cellulose (NaCMC) 0.5%, Group II-IV were treated by M. oleifera leaf extract given to the mice for six consecutive days orally in doses of 10, 30, 100 mg/kg. On the seventh day, white male mice were given with intravenous carbon suspension through their tails. The value of phagocytosis index (PI > 1) indicated immunostimulant activity. The data were analyzed by using one-way analysis of variance and Duncan test.Results: The analysis of variance results showed that the groups treated with Moringa leaf extract are significantly different with the vehicle groups (NaCMC 0.5%) (p<0.05). Increased doses of Moringa leaf extract are effective to improve the immunomodulator effect. It was included that Moringa leaf extract had the immunomodulatory capabilities as an immunostimulant.Conclusion: Immunomodulatory effect test of M. oleifera Lam. Based on the result of the research about immunomodulatory effect test from Moringa leaf extract (Moringa Oleifera L.) with carbon clearance method in male white mice, it can be concluded that Moringa leaf extract (Moringa Oleifera L.) has effect as an Immunomodulator.

UJI AKTIVITAS FAGOSITOSIS MAKROFAG EKSTRAK ETANOL DAUN SUJI (Dracaena angustifolia (Medik.)Roxb. ) SECARA IN VITRO

UJI AKTIVITAS FAGOSITOSIS MAKROFAG EKSTRAK ETANOL DAUN SUJI (Dracaena angustifolia (Medik.)Roxb. ) SECARA IN VITRONestri Handayani 1,2*), Subagus Wahyuono2), Triana Hertiani2), Retno Murwanti2)1)Program Studi Farmasi, FMIPA, Universitas Sebelas Maret Surakarta2)Fakultas Farmasi Universitas Gadjah Mada Yogyakarta*Email : [email protected] ABSTRACT Immunomodulatory activity has been carried out on ethanol extract of suji leaf (Dracaena angustifolia (Medik.) Roxb with in vitro macrofag phagocytosis method. . Suji is a plant that is often used as a food coloring and has long been used medicinally. This study aimed to test in vitro macrophage phagocytosis on ethanol extract of suji leaf. The suji leaves are extracted by maceration method. The obtained viscous extract was then tested for Thin Layer Chromatography (TLC) and in vitro macrophage phagocytosis test. The parameters used are Phagocytosis Index and Macrophage Capacity. The results of in vitro macrophage phagocytosis test with 4 concentrations of 10.25,50 and 100 microgram / ml, showed for the largest average Fagocytosis (IF) Index shown at concentration 10 mcg / ml, while concentrations 25 and 50 mcg / ml showed values The same phagocytic (KF) capacity. Keywords : Suji leaf, Phagocytic Macrophage Activity, ethanolic extract ABSTRAKUji aktivitas imunomodulator telah dilakukan terhadap ekstrak etanol daun suji (Dracaena angustifolia (Medik.)Roxb.. Suji merupakan tanaman yang sering digunakan sebagai pewarna makanan dan telah lama digunakan untuk obat. Penelitian ini bertujuan untuk melakukan uji fagositosis makrofag in vitro terhadap ekstrak etanol daun suji. Daun suji diekstraksi dengan metode maserasi. Ekstrak kental yang diperoleh kemudian dilakukan uji Kromatografi Lapis Tipis (KLT) dan uji fagositosis makrofag in vitro. Parameter yang digunakan adalah Indeks fagositosis dan Kapasitas makrofag. Hasil uji fagositosis makrofag in vitro dengan 4 konsentrasi sebesar 10,25,50 dan 100 mikrogram/ml, menunjukkan untuk Indeks Fagositosis (IF) rata-rata terbesar ditunjukkan pada konsentrasi 10 mcg/ml, sedangkan konsentrasi 25 dan 50 mcg/ml menunjukkan nilai Kapasitas Fagositosis (KF) yang sama besar. Kata kunci : Daun suji, fagositosis makrofag, ekstrak etanol