Killing of xenogenous and virally infected homogenous target cells by shrimp lymphocyte-like haemocytes.

Haemocytes play a crucial role in the invertebrate's immune system. In our lab, five subpopulations of shrimp haemocytes were identified in the past: hyalinocytes, granulocytes, semi-granulocytes and two subpopulations of non-phagocytic cells. In the latter two subpopulations, their characteristics such as having small cytoplasmic rims and not adhering to plastic cell-culture plates are very similar to those of mammalian lymphocytes. Therefore, they were designated lymphocyte-like haemocytes. Although little is known about their function, we hypothesize, based on their morphology, that they may have a cytotoxic activity like natural killer cells, with the ability to recognize and kill target cells. In our study, K562 cells and Sf9 cells were used as xenogenous target cells to detect the cytotoxic activity of the shrimp non-adherent lymphocyte-like haemocytes. Non-adherent haemocytes were collected and mixed with K562 cells and Sf9 cells at a 5:1 ratio and the binding activity was examined under a microscope. The binding rate of non-adherent haemocytes to K562 cells and Sf9 cells reached 6.6 % and 2.4 % after 240 min of culture, respectively. Then, the killing activity of non-adherent haemocytes was detected by an EMA staining (fluorescence microscopy), which showed 3.75 % dead K562 cells and 1.025 % dead Sf9 cells, and by Sytox® blue staining (flow cytometry), which showed 4.97 % of dead K562 cells. Next, a killing assay was developed to visualize the killing activity of shrimp non-adherent haemocytes. Non-adherent haemocytes were pre-labeled in blue (CellTracker blue) and K562/Sf9 cells in green (CFSE); dead cells were differentially stained red with ethidium bromide. The cytotoxic activity increased and reached a level of 2.59 % in K562 cells and 0.925 % in Sf9 cells at 120 min after co-culture. Furthermore, in the co-cultures of non-adherent haemocytes with K562 cells and Sf9 cells, upregulation of the gene and protein expression of the cytotoxic molecules torso-like protein and granzyme B was observed by RT-qPCR at 240 min and western blotting at 180 min. Additionally, non-adherent haemocytes were co-cultured with WSSV-inoculated shrimp ovary and lymphoid organ cells to detect the cytotoxicity to homogenous target cells. The binding activity started at 60 min in both the ovary and lymphoid organ cultures and reached at 240 min 50.62 % and 40.7 %, respectively. The killing activity was detected by EMA staining and the percentage of dead ovary and lymphoid organ cells increased respectively from 10.84 % to 6.89 % at 0 min to 13.09 % and 8.37 % at 240 min. In conclusion, we demonstrated the existence of cytotoxic activity of shrimp lymphocyte-like haemocytes against xenogenous cells from mammals and insects and against WSSV-infected homogenous shrimp cells.

Histomorphometric, Immunohistochemical, Ultrastructural Characterization of a Nano-Hydroxyapatite/Beta-Tricalcium Phosphate Composite and a Bone Xenograft in Sub-Critical Size Bone Defect in Rat Calvaria.

Nowadays, we can observe a worldwide trend towards the development of synthetic biomaterials. Several studies have been conducted to better understand the cellular mechanisms involved in the processes of inflammation and bone healing related to living tissues. The aim of this study was to evaluate tissue behaviors of two different types of biomaterials: synthetic nano-hydroxyapatite/beta-tricalcium phosphate composite and bone xenograft in sub-critical bone defects in rat calvaria. Twenty-four rats underwent experimental surgery in which two 3 mm defects in each cavity were tested. Rats were divided into two groups: Group 1 used xenogen hydroxyapatite (Bio Oss™); Group 2 used synthetic nano-hydroxyapatite/beta-tricalcium phosphate (Blue Bone™). Sixty days after surgery, calvaria bone defects were filled with biomaterial, animals were euthanized, and tissues were stained with Masson's trichrome and periodic acid-Schiff (PAS) techniques, immune-labeled with anti-TNF-α and anti-MMP-9, and electron microscopy analyses were also performed. Histomorphometric analysis indicated a greater presence of protein matrix in Group 2, in addition to higher levels of TNF-α and MMP-9. Ultrastructural analysis showed that biomaterial fibroblasts were associated with the tissue regeneration stage. Paired statistical data indicated that Blue Bone™ can improve bone formation/remodeling when compared to biomaterials of xenogenous origin.

Comparative, Histological and Histomorphometric Analysis of Three Anorganic Bovine Xenogenous Bone Substitutes: Bio-Oss, Bone-Fill and Gen-Ox Anorganic.

INTRODUCTION: Anorganic bovine xenogenous grafts show the best performance as bone substitutes in implantodontics. Bio-Oss is the world's most widely used and investigated anorganic bone substitute. This article compares two anorganic bovine bone substitutes (Bone-Fill and Gen-Ox anorganic) with Bio-Oss. MATERIALS AND METHODS: Eight New Zealand rabbits were implanted with 4 titanium cylinders randomly filled with Bio-Oss, Bone-Fill, Gen-Ox anorganic or a blood clot. Four animals were sacrificed after 8 weeks; 12 weeks later, the remaining four were sacrificed. The contents of the cylinders were removed, cut and stained with HE before they were evaluated with an optical microscope. The samples were submitted to histomorphometry for analysis. RESULTS: The bone formation with Bio-Oss at 8 weeks was 8.43 mm(2); at 12 weeks, it was 9.32 mm(2). The bone formation with Bone-Fill at 8 weeks was 7.24 mm(2); at 12 weeks, it was 9.01 mm(2). The bone formation with Gen-Ox anorganic at 8 weeks was 2.78 mm(2); at 12 weeks, it was 3.02 mm(2). The bone formation with the blood clot at 8 weeks was 0.65 mm(2); at 12 weeks, it was 0.63 mm(2). CONCLUSION: Following this model, Bone-Fill was comparable to Bio-Oss and superior to Gen-Ox and blood clot.