Prognosis of Performing Split-Rib Bone Graft for Cranial Bone Defects.

BACKGROUND: Frontal sinus anterior wall defects occur because of various diseases, causing not only aesthetic problems, such as forehead bulging and upper eyelid ptosis, but also exerting physical pressure on the brain or optic nerve. Therefore, this study aimed to evaluate the prognosis of performing split-rib bone graft for frontal sinus anterior wall defects. METHODS: This study included 30 patients who received a split-rib bone graft for a frontal sinus anterior wall defect. The sizes and volumes of the defects and grafts were measured using three-dimensional computed tomography before, after, and every 6 months for 2 years after the surgery. The Medical Imaging Interaction Toolkit was used for analysis. RESULTS: The average size and volume of the grafts were 27.29 cm 2 and 5.88 cm 3 , whereas they were 23.76 cm 2 and 4.80 cm 3 at 24 months after surgery, respectively. In a graft size and volume of less than 27 cm 2 and 6 cm 3 , respectively, the rate of graft take was greater than 80% during long-term observation. The younger the age, the higher the rate of graft take. No difference was found in the defect causes. Absorption occurred for up to 18 months. CONCLUSIONS: Frontal bone defect reconstruction revealed the stable results of the split-rib bone graft over a long period when the size and volume were less than 27 cm 2 and 6 cm 3 , respectively. Furthermore, bone resorption was seen in more than 20% to 30% of the patients, and the rate of resorption increased with age; thus, it is appropriate to consider overcorrection and other reconstruction methods. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Surgical Strategies for Partial Breast Reconstruction in Medial-Located Breast Cancer: A 12-Year Experience.

PURPOSE: Partial breast reconstruction is challenging in medially located breast cancer, particularly in terms of achieving satisfactory aesthetic coverage. Thus, we aimed to investigate surgical strategies for filling medial defects resulting from breast-conserving surgery to improve patient satisfaction and aesthetic outcomes. METHODS: We retrospectively evaluated 113 patients (114 cases) with medially located breast cancer between 2007 and 2018. We analysed the patient data, such as breast size, specimen weight, complications, and aesthetic results obtained using a questionnaire. RESULTS: The mean body mass index and specimen weight were 23.43 kg/m² (range, 18.5-26.8) and 83.29 g (range, 15-290 g), respectively. The tennis racket and round-block techniques were chosen for small defects (< 10%-15%) in small- and medium-sized breasts. The rotational and perforator flap techniques were used for medium-sized defects. The latissimus dorsi (LD) flap technique was used for large defects (> 30%). Hematoma was found in 1 case (0.96%), linear skin necrosis was found in 1 case (0.96%), seroma in the LD flaps was found in 8 cases (7.69%); fat necrosis in the rotational flaps was found in 2 cases (1.92%); and fat necrosis in an anterior intercostal artery perforator flap was found in 1 case (0.96%). 91 patients (87.5%) were satisfied with the aesthetic results. CONCLUSION: The techniques used in this study for medially located breast cancer can produce fine aesthetic outcomes with regard to breast size and resection volume, with few complications.

Deep Learning for the Automatic Segmentation of Extracranial Venous Malformations of the Head and Neck from MR Images Using 3D U-Net.

BACKGROUND: It is difficult to characterize extracranial venous malformations (VMs) of the head and neck region from magnetic resonance imaging (MRI) manually and one at a time. We attempted to perform the automatic segmentation of lesions from MRI of extracranial VMs using a convolutional neural network as a deep learning tool. METHODS: T2-weighted MRI from 53 patients with extracranial VMs in the head and neck region was used for annotations. Preprocessing management was performed before training. Three-dimensional U-Net was used as a segmentation model. Dice similarity coefficients were evaluated along with other indicators. RESULTS: Dice similarity coefficients in 3D U-Net were found to be 99.75% in the training set and 60.62% in the test set. The models showed overfitting, which can be resolved with a larger number of objects, i.e., MRI VM images. CONCLUSIONS: Our pilot study showed sufficient potential for the automatic segmentation of extracranial VMs through deep learning using MR images from VM patients. The overfitting phenomenon observed will be resolved with a larger number of MRI VM images.

Prepectoral breast reconstruction with complete anterior implant coverage using a single, large, square-shaped acellular dermal matrix.

BACKGROUND: Several studies have discussed various methods of prepectoral direct-to-implant (DTI) breast reconstruction using an acellular dermal matrix (ADM) prosthesis to achieve full coverage. However, methods for anterior coverage have rarely been reported. In this study, prepectoral DTI breast reconstruction with complete anterior implant coverage was performed using a square piece of ADM. This study aimed to introduce our prepectoral DTI technique and determine its functional and cosmetic outcomes as well as compare them with those of existing subpectoral DTI techniques. METHODS: This prospective comparative study focused on 29 patients (35 breasts) and 34 patients (35 breasts) who underwent breast reconstruction via subpectoral implant insertion (control group) and anterior coverage prepectoral implant insertion (anterior coverage group), respectively. Postoperative complications were noted, and breast symmetry was evaluated using the Vectra H2 three-dimensional scanner. The modified Kyungpook National University Hospital Breast-Q (KNUH Breast-Q) scale was used to assess the patient's subjective satisfaction with the reconstruction and postoperative quality of life. RESULTS: No remarkable differences in terms of complications (seroma, skin necrosis, nipple-areola complex necrosis, hematoma, capsular contracture, and infection) were noted in both groups. Compared with controls, considerably better results were observed among those in the anterior coverage group in terms of the mean drain removal period. Furthermore, those in the anterior coverage group showed greater symmetry on three-dimensional scans than the controls; however, this was not statistically significant. Subjective satisfaction and postoperative quality of life measured using the KNUH Breast-Q scale were not significantly different between both groups. CONCLUSIONS: Considering its stability, faster recovery time, and cosmetic benefit, prepectoral breast reconstruction with anterior implant coverage using a single, large ADM is a good choice to perform breast reconstruction with implant insertion in patients who have undergone mastectomy. LEVEL OF EVIDENCE: II.

Flow cytometric characterization of the hemocytes of sea hares from tidal pools in Jeju Island off the south coast of Korea.

The members in the family Aplysidae known as sea hares play a crucial role as a grazer in small tidal pools or shallow subtidal hard bottoms. Like other marine gastropods, hemocyte types and functions of sea hares are limitedly known. This study identified and characterized the hemocytes of common Aplysia species Aplysia kurodai, A. juliana, and A. oculifera in rocky tidal pools in Jeju Island off the south coast of Korea using flow cytometry and light microscopy. The flow cytometry identified three different hemocytes in the hemolymph of the three sea hare species: granulocytes, hyalinocytes, and blast-like cells. The granulocytes exhibited pseudopodia on the cell surface and granules in the cytoplasm. Morphology of the hyalinocyte was similar to that of the granulocytes, while they lack cytoplasmic granules. The blast-like cells were small and round, with very thin cytoplasm. The hyalinocytes were the most abundant in the hemolymph, accounting for 89.8-92.6% of the total hemocytes. Flow cytometry indicated that the granulocytes and blast-like cells were less than 5.6% and 5.4% of the total hemocyte populations. Flow cytometry also revealed that the granulocytes and hyalinocytes are engaged in cellular defensive activities such as intra-cellular lysosomal content, phagocytosis, and ROS production. The mean lysosomal contents of the granulocytes (0.4 × 105-0.2 × 105 A U.) were 2-3 times higher than that of hyalinocytes (0.2 × 105-0.6 × 105 A U.). In addition, the ROS production of the granulocytes (0.98 × 106-1.95 × 106 A U.) was about twice higher than that of the hyalinocytes (0.62 × 106-1.14 × 106 A U.). Of the three species of sea hares, the granulocytes showed comparatively higher phagocytosis capacity (70.4-92.3%) than that of the hyalinocytes (34.8-46.0%). Flow cytometry and microscopy indicated that the hemocyte types and their functions were identical, regardless of the species.

Effects of spawning stress on the immune capacity of blood cockle Tegillarca granosa occurring on the south coast of Korea.

Spawning in marine bivalves is a great energy-demanding process, and it often results in lethal and sublethal stresses during the post-spawning period, including depressed immune capacity. The blood cockle Tegillarca granosa (Linnaeus, 1758) distributes widely in silty-mud tidal flats on the south coast of Korea, and they spawn in late summer. To understand the impacts of spawning on immune parameters, we analyzed the total hemocyte count (THC), hemocyte mortality, phagocytosis capacity, and reactive oxygen species (ROS) production of T. granosa in pre-, and post-spawning condition using a flow cytometer. Histology indicated that the blood cockles occurring on the south coast of Korea ripe and ready to spawn in July, and they spawned in August and September. The THC in the blood cockle hemolymph declined from pre-spawning (1.2 × 108 cell mL-1) to post-spawning (0.9 × 108 cell mL-1), possibly due to the spawning stress and the massive infiltration of hemocytes in the gonad to phagocytose and resorb the residual gametes during the post-spawning period. The hemocyte mortality increased linearly from August (4.1%) to November (9.1%), as the histology revealed that the blood cockle completed spawning, and they resorbed the relict gametes. The granulocyte phagocytosis capacity declined dramatically from July (12.7%) to September (6.0%), when the cockles were engaged in active spawning. The flow cytometry revealed that the production of reactive oxygen species (ROS) from the granulocytes and the erythrocytes type II increased linearly from August (0.8-0.9 × 105 A U.) to December (2.1-2.8 × 105 A U.), which may cause stresses at a cellular level during this period. As the data indicated, spawning is a stressful activity inducing depressed immunological capacities in the blood cockles.

Nasoethmoid orbital fracture reconstruction using a three-dimensional printing-based craniofacial plate.

The face is one of the most important parts of the body. Untreated facial fractures can result in deformities that can be harmful to patients. Three-dimensional (3D) printing is a rapidly evolving technology that has recently been widely applied in the medical field as it can potentially improve patient treatment. Although 3D printing technology is mostly used for craniofacial surgery, some studies have proved that it can be used to treat nasoethmoid orbital fractures. In this study, a patient-customized plate was constructed using a 3D printer and applied in a simulated surgery for the treatment of nasoethmoid orbital fracture.

Delayed unilateral hematoma after reconstructive and aesthetic breast surgery with implants in Asian patients: two case reports.

Hematomas represent one of the postoperative complications in patients undergoing reconstructive or aesthetic breast surgery with a silicone implant. Although there are few reports of intracapsular hematoma, those presenting late hematoma after reconstructive and aesthetic augmentation surgeries are rarer. This study reported two Asian patients with late hematoma after reconstruction and aesthetic breast surgery. A 54-year-old female patient underwent bilateral nipple-sparing mastectomy with immediate breast reconstruction using anatomically shaped textured implant for intraductal carcinoma in August 2019. Contralateral nipple-sparing mastectomy was performed for the BRCA gene mutation on the left breast, which was immediately reconstructed with an anatomically shaped textured implant. In a 1-year postoperative magnetic resonance imaging evaluation, an extracapsular hematoma was found on the right side, which was removed following the removal of both implants. Another case was a 63-year-old female patient who underwent augmentation of both breasts with smooth round implants and experienced right unilateral swelling and painless firmness about 30 years postoperatively. A preoperative magnetic resonance imaging evaluation showed both intracapsular and extracapsular ruptures on the right breast and a bulging implant herniation on the left breast. During the operation, hematoma, implants, and capsule were all removed. The excised capsule was sent for histological evaluation. Slightly dark colored blood was emptied before removing the semisolid-state intracapsular hematoma. In both cases, the patients responded well postoperatively and were discharged to their homes with no postsurgical complications, including seroma, or additional hematoma on the breasts. The etiology of late hematoma following breast augmentation or reconstruction has been poorly characterized. Further reports are needed to clearly establish the reasons for this increase in late hematoma formation.

Flow cytometric characterization of the hemocytes of blood cockles Anadara broughtonii (Schrenck, 1867), Anadara kagoshimensis (Lischke, 1869), and Tegillarca granosa (Linnaeus, 1758) as a biomarker for coastal environmental monitoring.

Marine bivalves are often used as a sentinel species in coastal environmental monitoring since changes in the environmental quality are often well preserved in their cells and tissues. Anadara and Tegillarca species of Arcidae, the blood cockles, are considered to be good sentinel species in monitoring coastal pollution and ecosystem health because they are distributed widely in the subsurface of intertidal mudflats. Internal cellular defense of the blood cockles to physical and biological stresses is mediated by the circulating hemocytes, while their hemocyte types and functions are poorly studied. In this study, we first characterized morphology and immune-related activities of hemocytes of three common blood cockles Anadara broughtonii, A. kagoshimensis, and Tegillarca granosa using flow cytometry. Based on cell morphology and immunological functions, we described five types of hemocytes identically in the three blood cockles: erythrocytes type-I (erythrocytes-I), erythrocytes type-II (erythrocytes-II), granulocytes, hyalinocytes, and blast-like cells. Erythrocytes were round cells containing hemoglobin with numerous granules in the cytoplasm and these cells consist of two central populations. Erythrocytes-I were the most abundant cells accounting for 80-89% of the total circulating hemocytes and exhibited a certain level of lysosome and oxidative capacity. Erythrocytes-II were the largest cells and displayed high lysosome content and the most active oxidative capacity. Both erythrocytes-I and erythrocytes-II did not show phagocytosis capacity. Granulocytes were intermediated-sized hemocytes characterized by granules in the cytoplasm and long pseudopodia on the cell surface, and these cells were mainly engaged in the cellular defense exhibiting the largest lysosome content, the most active phagocytosis, and high oxidative capacity. Contrary to granulocytes, hyalinocytes were comparatively small and round cells and exhibited no granules in the cytoplasm. Hyalinocytes displayed a certain level of lysosome and phagocytosis and oxidative capacities. Blast-like cells characterized by the smallest size and small quantity of cytoplasm and exhibited an absence of phagocytosis and extremely low oxidative capacity, suggesting that this population is not directly involved in the cell-mediated immune activities. In conclusion, flow cytometry indicated that three blood cockles had five types of hemocytes, and the erythrocytes and granulocytes were mainly involved in the immunological activities.

Effect of Epigallocatechin Gallate on Viability of Kudoa septempunctata.

Kudoa septempunctata have been reported as a causative agent for acute transient gastrointestinal troubles after eating raw olive flounder (Paralichthys olivaceus). It raised public health concerns and quarantine control in several countries. Quantitative evaluation on viability of K. septempunctata is crucial to develop effective chemotherapeutics against it. A cytometry using fluorescent stains was employed to assess effect of three compounds on viability of K. septempunctata. Epigallocatechin gallate reduced markedly viability of K. septempunctata at 0.5 mM or more, and damaged K. septempunctata spores by producing cracks.

Temporal changes in hemocyte functions of the oyster Saccostrea kegaki (Torigoe & Inaba, 1981) on Jeju Island off the south coast of Korea are closely associated with annual gametogenesis.

Hemocyte parameters have been used as a proxy to characterize the health condition of marine bivalves, as the effects of external and internal stresses are reflected well in these parameters. Marine bivalve hemocyte functions are often depressed during the post-spawning period due to physiological stress and energy depletion. In this study, we analyzed temporal changes in hemocyte parameters of the oyster Saccostrea kegaki on Jeju Island, off the south coast of Korea, using flow cytometry. Total hemocyte count (THC), hemocyte types, hemocyte mortality, and phagocytosis capacity were analyzed. S. kegaki spawned during June and August, when the sea surface temperature increased from 18 to 23 °C. Most of the oysters were in the spent and resting phases from September to January. THC dropped dramatically from September to October, when most oysters completed spawning. Histology revealed that the residual eggs or sperm were actively resorbed through phagocytosis by hemocytes during the spent stage. Hemocyte mortality also showed its annual peak in October, possibly due to increased resorbing activities. The phagocytosis capacities of the granulocytes decreased dramatically from September to February. The level of energy reserves (glycogen) in post-spawning tissues was significantly lower than that prior to spawning. The low energy level reserve during the post-spawning period likely reduced the THC and immune capacities, as oysters may have been unable to acquire sufficient food from the ambient environment.

Surgical Intervention for Refractory Donor-Site Seroma After Immediate Breast Reconstruction with Latissimus Dorsi Flap.

Donor-site seroma persisting for > 3 weeks is the most common complication of latissimus dorsi (LD) flaps for breast reconstruction after mastectomy. Conservative treatment is insufficient for refractory seroma resolution. We report the results of surgical intervention for refractory donor-site seroma. Between January 2012 and April 2018, 319 patients undergoing breast reconstruction using an LD flap at our hospital were treated with conservative therapy if seroma was found. Refractory seroma of > 3 months' duration was observed in five patients (1.6%). Before surgery, the precise location and extent of the capsule were determined using chest computed tomography, and surgical intervention was planned. An en bloc capsulectomy was performed after confirming the internal capsule. Simultaneously, a quilting suture, bolster suture, and fibrin sealant were applied to prevent recurrence. The mean age of the patients with refractory seroma was 45.4 ( ± 7.3) years, and their mean body mass index was 31.0 ( ± 6.2) kg/m2. All patients underwent breast reconstruction with an extended LD flap or extended LD flap with a silicone implant after total mastectomy. During the follow-up period of ≥ 10.4 months, a mean of 34.4 ( ± 10.4) needle aspiration procedures were performed, and a mean of 12.8 ( ± 2.2) cc of seroma fluid was removed each time. Intraoperatively, the formation of a capsule with a well-defined border was confirmed; all patients showed complete resolution of donor-site seroma within 4 weeks after surgical intervention. Refractory donor-site seroma occurrence was rare in the cases assessed. Surgical intervention was successful in cases of refractory donor-site seroma unsuccessfully treated with conservative therapy and is thus one of the most effective methods for complete resolution of refractory seroma.Level of Evidence IV This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Flow cytometric characterization of hemocytes of the solitary ascidian, Halocynthia roretzi.

Internal defense of ascidians relies, at least partially, on cells circulating in body fluids and infiltrating in tissues, referred to as hemocytes, although structure and composition of ascidian hemocytes still remain unclear. In the present study, we investigated hemocyte types and their functions of the solitary ascidian Halocynthia roretzi using flow cytometry. Based on morphology, cellular activities and intracellular parameters from the flow cytometry, we identified eight hemocyte types including, three granulocytes (Gr-1, Gr-2, and Gr-3), 4 hyalinocytes (Hy-1, Hy-1', Hy-2, and Hy-3) and lymphocyte-like (Ly-like) cells. The granulocyte Gr-1 accounted for 30% of the total circulating hemocytes and exhibited highest density of lysosomes and oxidative activity. Gr-1 was deeply involved in phagocytosis and degradation of foreign material. Hyalinocytes consist of two main populations, Hy-1 and Hy-2, and each accounted for 30% of the circulating hemocyte. Hy-1 displayed lysosomal content, an inducible oxidative activity, and no proteases, while Hy-2 expressed highest density of intracellular proteases, no lysosomes and a low oxidative activity. It was believed that Hy-2 may represent an important link between cellular and humoral immune reactions. Hy-1 did not show phagocytosis activity. Hy-3 and the Ly-like cells presented a similar profile except for their size and complexity, and Hy-3 may represent an intermediate differentiation/maturation step between Ly-like cells and other hemocyte populations. This first characterization of the hemocyte populations of H. roretzi provides a solid basis to investigate further their respective roles and functions in physiological and pathological contexts.

First report of Perkinsus honshuensis in the variegated carpet shell clam Ruditapes variegatus in Korea.

The recent discovery of Perkinsus honshuensis, a new Perkinsus species infecting Manila clams Ruditapes philippinarum (Sowerby, 1852), in Japan, suggested that, based on proximity, P. honshuensis could also be in Korean waters, where to date, P. olseni was believed to be the only Perkinsus species present. Perkinsus sp. infections consistently occurred among Ruditapes variegatus clams on a pebble beach on Jeju Island, off the south coast of Korea. The typical 'signet ring' morphology of the parasite was observed in the connective tissue of the digestive gland, and infection intensity was comparatively low (3.3 × 103 ± 1.2 × 104 to 1.3 × 104 ± 6.1 × 104 cells g-1 gill weight). Further DNA analyses of internal transcribed spacer (ITS-1, 5.8S and ITS-2) and non-transcribed spacer (NTS) regions of the parasite showed 98.9-99.8 and 98.5-99.5% similarity to those of P. honshuensis from Japan, respectively. Phylogenetic analyses using ITS and NTS sequences indicated that Perkinsus sp. from Jeju formed a highly supported clade with P. honshuensis. This is the first report of P. honshuensis infections in clams in Korean waters and the first report of R. variegatus as a host for that parasite.

Assessment of the fitness of the mussel Mytilus galloprovincialis two years after the Hebei Spirit oil spill.

In December 2007, >150km of the West coast of Korea were heavily polluted by crude oil leaked from the oil tanker Hebei Spirit, leading to mass mortality of bivalve mollusks on the intertidal areas. Two years after, mussels Mytilus galloprovincialis were collected from two impacted sites to investigate sub-lethal effects of the oil spill. Tissue content in polycyclic aromatic hydrocarbons (PAHs), hemocyte parameters, reproductive status and energetic reserves were analyzed. PAHs in tissues of mussels as well as hemocyte parameters were not different between impacted and control sites. Energetic reserves were altered in mussels from the impacted sites. Glycogen content remained low at polluted sites, whatever the season. Two years after the Hebei Spirit oil spill, mussels then presented altered energetic metabolism. Further investigations are thus warranted to monitor the sustainability of mussel populations on the oil spilled West coast of Korea.

Substantial changes in hemocyte parameters of Manila clam Ruditapes philippinarum two years after the Hebei Spirit oil spill off the west coast of Korea.

Two years after the Hebei Spirit oil spill occurred off the west coast of Korea, we determined sub-lethal effects of the spilled oil on hemocyte parameters of Ruditapes philippinarum in the damaged areas. Clams in the spilled sites displayed unusually high proportion of granulocytes, which may result in higher phagocytosis capacity and reactive oxygen species production. Hemocytes in clams from the polluted sites also displayed less DNA damage and mortality than in the control site, possibly due to a faster phagocytosis of the impaired cells. Glycogen, the major energetic reserve, was depleted in clams from the spilled sites, potentially due to energetic consumption for maintenance of a large pool of granulocytes, detoxification processes and oxidative stress. Modified hemocyte parameters in clams in the spilled area, may reflect sub-lethal physiological stresses caused by the residual oils in the sediment, in conjunction with environmental modifications such as food availability and pathogens pattern.

First report of Urosporidium sp., a haplosporidian hyperparasite infecting digenean trematode Parvatrema duboisi in Manila clam, Ruditapes philippinarum on the west coast of Korea.

In this study, we first report on the occurrence of Urosporidium sp., a haplosporidian hyperparasite infecting the trematode, Parvatrema duboisi, which parasitizes Manila clams, Ruditapes philippinarum on the west and south coasts of Korea. The larval P. duboisi infected by the sporocyst stage of Urosporidium sp. demonstrated numerous small yellowish spores in their tissues. The heavily infected metacercariae exhibited degenerate bodies and the larvae were often motionless. Clams heavily infected by the metacercariae of P. duboisi also displayed abnormal golden spots on the mantle tissue. In histology, different life stages of Urosporidium sp. could be identified, including the uni-nucleate, plasmodial, sporogonic stages and the acid fast mature spores released from the cyst. In scanning electron microscopy (SEM), the mature spore exhibited a semi-circular rim around the apical end and the orifice was covered internally with a flap. Loop-like filaments ornamentation was also identified from Urosporidium sp. in SEM, suggesting that Urosporidium sp. found in this study is a new member in the genus. Prevalence of Urosporidium sp.-infected trematodes in this study ranged from 2.5% to 24.0% in April 2010 and the infection was observed from 8 sampling sites out of the 26 sites surveyed on the west and south coasts.

The known and unknown sources of reactive oxygen and nitrogen species in haemocytes of marine bivalve molluscs.

Reactive oxygen and nitrogen species (ROS and RNS) are naturally produced in all cells and organisms. Modifications of standard conditions alter reactive species generation and may result in oxidative stress. Because of the degradation of marine ecosystems, massive aquaculture productions, global change and pathogenic infections, oxidative stress is highly prevalent in marine bivalve molluscs. Haemocytes of bivalve molluscs produce ROS and RNS as part of their basal metabolism as well as in response to endogenous and exogenous stimuli. However, sources and pathways of reactive species production are currently poorly deciphered in marine bivalves, potentially leading to misinterpretations. Although sources and pathways of ROS and RNS productions are highly conserved between vertebrates and invertebrates, some uncommon pathways seem to only exist in marine bivalves. To understand the biology and pathobiology of ROS and RNS in haemocytes of marine bivalves, it is necessary to characterise their sources and pathways of production. The aims of the present review are to discuss the currently known and unknown intracellular sources of reactive oxygen and nitrogen species in marine bivalve molluscs, in light of terrestrial vertebrates, and to expose principal pitfalls usually encountered.

Comparative study on the hemocytes of subtropical oysters Saccostrea kegaki (Torigoe & Inaba, 1981), Ostrea circumpicta (Pilsbry, 1904), and Hyotissa hyotis (Linnaeus, 1758) in Jeju Island, Korea: morphology and functional aspects.

We first characterized the morphology and immune-related activities of hemocytes in the subtropical oysters Saccostrea kegaki, Ostrea circumpicta, and Hyotissa hyotis using light microscopy and flow cytometry. Hemocytes of these three oyster species were classified into three main types: 1) granulocytes containing numerous granules in the cytoplasm, 2) hyalinocytes with no or fewer granules, and 3) blast-like cells characterized by the smallest size and very thin cytoplasm. The percentage of each hemocyte population was similar in all species; hyalinocytes were the most abundant cell in the hemolymph accounting for more than 59%, followed by granulocytes (23-31%) and blast-like cells (3-5%). The size of granulocytes of S. kegaki was smaller (P < 0.05) than those of O. circumpicta and H. hyotis. Light microscopy also allowed the description of vacuolated cells characterized by large vacuoles in the cytoplasm. Flow cytometry analysis confirmed that the granulocytes of the three oyster species were the major hemocytes engaged in cellular defense with the largest lysosome content, and the most active phagocytosis activity and oxidative activity, as was previously reported in several marine bivalves. Phagocytic activity was the lowest in S. kegaki hemocytes, and PMA-stimulated oxidative activity was the lowest in H. hyotis hemocytes. Our results provide the basic information of hemocytes population of three subtropical oysters for further investigations associated with various environmental disease stresses.